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2. Call for Papers: Exercise and the kidneys in health and disease.
- Author
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Kirkman, Danielle L. and Sequeira-Lopez, Maria Luisa S.
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NUTRITIONALLY induced diseases , *EXERCISE physiology , *KIDNEY physiology , *SYMPTOMS , *CHRONIC kidney failure - Published
- 2023
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3. Renal-specific loss of ferroportin disrupts iron homeostasis and attenuates recovery from acute kidney injury.
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Soofi, Abdul, Li, Vivie, Beamish, Jeffrey A., Abdrabh, Sham, Hamad, Mawieh, Das, Nupur K., Shah, Yatrik M., and Dressler, Gregory R.
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IRON in the body ,ACUTE kidney failure ,POOR communities ,CHRONIC kidney failure ,IRON ,OXYGEN consumption ,NEPHROLOGY - Abstract
Chronic kidney disease is increasing at an alarming rate and correlates with the increase in diabetes, obesity, and hypertension that disproportionately impact socioeconomically disadvantaged communities. Iron plays essential roles in many biological processes including oxygen transport, mitochondrial function, cell proliferation, and regeneration. However, excess iron induces the generation and propagation of reactive oxygen species, which lead to oxidative stress, cellular damage, and ferroptosis. Iron homeostasis is regulated in part by the kidney through iron resorption from the glomerular filtrate and exports into the plasma by ferroportin (FPN). Yet, the impact of iron overload in the kidney has not been addressed. To test more directly whether excess iron accumulation is toxic to kidneys, we generated a kidney proximal tubule-specific knockout of FPN. Despite significant intracellular iron accumulation in FPN mutant tubules, basal kidney function was not measurably different from wild type kidneys. However, upon induction of acute kidney injury (AKI), FPN mutant kidneys exhibited significantly more damage and failed recovery, evidence for ferroptosis, and increased fibrosis. Thus, disruption of iron export in proximal tubules, leading to iron overload, can significantly impair recovery from AKI and can contribute to progressive renal damage indicative of chronic kidney disease. Understanding the mechanisms that regulate iron homeostasis in the kidney may provide new therapeutic strategies for progressive kidney disease and other ferroptosis-associated disorders. NEW & NOTEWORTHY Physiological iron homeostasis depends in part on renal resorption and export into the plasma. We show that specific deletion of iron exporters in the proximal tubules sensitizes cells to injury and inhibits recovery. This can promote a chronic kidney disease phenotype. Our paper demonstrates the need for iron balance in the proximal tubules to maintain and promote healthy recovery after acute kidney injury. [ABSTRACT FROM AUTHOR]
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- 2024
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4. Evidence that methylglyoxal and receptor for advanced glycation end products are implicated in bladder dysfunction of obese diabetic ob/ob mice.
- Author
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Oliveira, Akila L., Medeiros, Matheus L., Ghezzi, Ana Carolina, Alonso dos Santos, Gabriel, Mello, Glaucia Coelho, Monica, Fabíola Z., and Antunes, Edson
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ADVANCED glycation end-products ,BLADDER diseases ,PYRUVALDEHYDE ,MICE - Abstract
Glycolytic overload in diabetes causes large accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO) and overproduction of advanced glycation end products (AGEs), which interact with their receptors (RAGE), leading to diabetes-associated macrovascular complications. The bladder is an organ that stays most in contact with dicarbonyl species, but little is known about the importance of the MGO-AGEs-RAGE pathway to diabetes-associated bladder dysfunction. Here, we aimed to investigate the role of the MGO-AGEs-RAGE pathway in bladder dysfunction of diabetic male and female ob/ ob mice compared with wild-type (WT) lean mice. Diabetic ob/ob mice were treated with the AGE breaker alagebrium (ALT711, 1 mg/kg) for 8 wk in drinking water. Compared with WT animals, male and female ob/ob mice showed marked hyperglycemia and insulin resistance, whereas fluid intake remained unaltered. Levels of total AGEs, MGO-derived hydroimidazolone 1, and RAGE in bladder tissues, as well as fluorescent AGEs in serum, were significantly elevated in ob/ob mice of either sex. Collagen content was also markedly elevated in the bladders of ob/ob mice. Void spot assays in filter paper in conscious mice revealed significant increases in total void volume and volume per void in ob/ob mice with no alterations of spot number. Treatment with ALT-711 significantly reduced the levels of MGO, AGEs, RAGE, and collagen content in ob/ob mice. In addition, ALT-711 treatment normalized the volume per void and increased the number of spots in ob/ob mice. Activation of AGEs-RAGE pathways by MGO in the bladder wall may contribute to the pathogenesis of diabetes-associated bladder dysfunction. NEW & NOTEWORTHY The involvement of methylglyoxal (MGO) and advanced glycation end products (AGEs) in bladder dysfunction of diabetic ob/ob mice treated with the AGE breaker ALT-711 was investigated here. Diabetic mice exhibited high levels of MGO, AGEs, receptor for AGEs (RAGE), and collagen in serum and/or bladder tissues along with increased volume per void, all of which were reduced by ALT-711. Activation of the MGO-AGEs-RAGE pathway in the bladder wall contributes to the pathogenesis of diabetes-associated bladder dysfunction. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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5. Practical notes on popular statistical tests in renal physiology.
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Mamenko, Mykola, Lysikova, Daria V., Spires, Denisha R., Tarima, Sergey S., and Ilatovskaya, Daria V.
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KIDNEY physiology ,INTRACELLULAR calcium ,STATISTICS ,REPRODUCIBLE research ,CELL physiology - Abstract
Competent statistical analysis is essential to maintain rigor and reproducibility in physiological research. Unfortunately, the benefits offered by statistics are often negated by misuse or inadequate reporting of statistical methods. To address the need for improved quality of statistical analysis in papers, the American Physiological Society released guidelines for reporting statistics in journals published by the society. The guidelines reinforce high standards for the presentation of statistical data in physiology but focus on the conceptual challenges and, thus, may be of limited use to an unprepared reader. Experimental scientists working in the renal field may benefit from putting the existing guidelines in a practical context. This paper discusses the application of widespread hypothesis tests in a confirmatory study. We simulated pharmacological experiments assessing intracellular calcium in cultured renal cells and kidney function at the systemic level to review best practices for data analysis, graphical presentation, and reporting. Such experiments are ubiquitously used in renal physiology and could be easily translated to other practical applications to fit the reader's specific needs. We provide step-by-step guidelines for using the most common types of t tests and ANOVA and discuss typical mistakes associated with them. We also briefly consider normality tests, exclusion criteria, and identification of technical and experimental replicates. This review is supposed to help the reader analyze, illustrate, and report the findings correctly and will hopefully serve as a gauge for a level of design complexity when it might be time to consult a biostatistician. [ABSTRACT FROM AUTHOR]
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- 2022
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6. Guidelines for microbiome studies in renal physiology.
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Muralitharan, Rikeish R., Snelson, Matthew, Meric, Guillaume, Coughlan, Melinda T., and Marques, Francine Z.
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Gut microbiome research has increased dramatically in the last decade, including in renal health and disease. The field is moving from experiments showing mere association to causation using both forward and reverse microbiome approaches, leveraging tools such as germ-free animals, treatment with antibiotics, and fecal microbiota transplantations. However, we are still seeing a gap between discovery and translation that needs to be addressed, so that patients can benefit from microbiome-based therapies. In this guideline paper, we discuss the key considerations that affect the gut microbiome of animals and clinical studies assessing renal function, many of which are often overlooked, resulting in false-positive results. For animal studies, these include suppliers, acclimatization, baseline microbiota and its normalization, littermates and cohort/cage effects, diet, sex differences, age, circadian differences, antibiotics and sweeteners, and models used. Clinical studies have some unique considerations, which include sampling, gut transit time, dietary records, medication, and renal phenotypes. We provide best-practice guidance on sampling, storage, DNA extraction, and methods for microbial DNA sequencing (both 16S rRNA and shotgun metagenome). Finally, we discuss follow-up analyses, including tools available, metrics, and their interpretation, and the key challenges ahead in the microbiome field. By standardizing study designs, methods, and reporting, we will accelerate the findings from discovery to translation and result in new microbiome-based therapies that may improve renal health. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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7. CORRIGENDUM.
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DIGESTIVE system diseases ,KIDNEY diseases ,CANCER prevention - Abstract
This document is a corrigendum for an article titled "Podocytes from hypertensive and obese mice acquire an inflammatory, senescent, and aged phenotype" published in the American Journal of Physiology: Renal Physiology. The corrigendum adds Dr. Thomas Carroll as an author based on their contribution to the study. The author line and affiliation line have been updated accordingly. Additionally, the grants section of the article reflects additional funding received for the research. The corrigendum also includes copyright information and a link to access the article online. [Extracted from the article]
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- 2024
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8. Control of ENaC ubiquitination.
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Shi, Shujie, Frindt, Gustavo, Whelan, Sarah Christine M., and Palmer, Lawrence G.
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UBIQUITINATION ,GOLGI apparatus ,PEPTIDES ,AMILORIDE ,ANGIOTENSIN I - Abstract
Ubiquitination influences the expression of the epithelial Na
+ channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na+ could stimulate ubiquitination. Administration of amiloride to block Na+ entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na+ produced by monensin in FRT cells or acute Na+ repletion in rats increased ubiquitination and decreased overall ENaC expression. NEW & NOTEWORTHY: We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na+ channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na+ can stimulate ENaC ubiquitination. [ABSTRACT FROM AUTHOR]- Published
- 2024
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9. Diffusion tensor MRI is sensitive to fibrotic injury in a mouse model of oxalate-induced chronic kidney disease.
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Virgincar, Rohan S., Wong, Aaron K., Barck, Kai H., Webster, Joshua D., Hung, Jeffrey, Caplazi, Patrick, Choy, Man Kin, Forrest, William F., Bell, Laura C., Crespigny, Alex J. de, Dunlap, Debra, Jones, Charles, Kim, Dong Eun, Weimer, Robby M., Shaw, Andrey S., Brightbill, Hans D., and Xie, Luke
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DIFFUSION magnetic resonance imaging ,CHRONIC kidney failure ,RENAL fibrosis ,DIFFUSION tensor imaging ,LABORATORY mice - Abstract
Chronic kidney disease (CKD) is characterized by inflammation and fibrosis in the kidney. Renal biopsies and estimated glomerular filtration rate (eGFR) remain the standard of care, but these endpoints have limitations in detecting the stage, progression, and spatial distribution of fibrotic pathology in the kidney. MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo both in clinical and preclinical studies. However, these imaging studies have not systematically identified fibrosis particularly deeper in the kidney where biopsy sampling is limited, or completed an extensive analysis of whole organ histology, blood biomarkers, and gene expression to evaluate the relative strengths and weaknesses of MRI for evaluating renal fibrosis. In this study, we performed DTI in the sodium oxalate mouse model of CKD. The DTI parameters fractional anisotropy, apparent diffusion coefficient, and axial diffusivity were compared between the control and oxalate groups with region of interest (ROI) analysis to determine changes in the cortex and medulla. In addition, voxel-based analysis (VBA) was implemented to systematically identify local regions of injury over the whole kidney. DTI parameters were found to be significantly different in the medulla by both ROI analysis and VBA, which also spatially matched with collagen III immunohistochemistry (IHC). The DTI parameters in this medullary region exhibited moderate to strong correlations with histology, blood biomarkers, hydroxyproline, and gene expression. Our results thus highlight the sensitivity of DTI to the heterogeneity of renal fibrosis and importance of whole kidney noninvasive imaging. NEW & NOTEWORTHY: Chronic kidney disease (CKD) can be characterized by inflammation and fibrosis of the kidney. Although standard of care methods have been limited in scope, safety, and spatial distribution, MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo. In this study, we performed DTI in an oxalate mouse model of CKD to systematically identify local kidney injury. DTI parameters strongly correlated with histology, blood biomarkers, hydroxyproline, and gene expression. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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10. Cilia-deficient renal tubule cells are primed for injury with mitochondrial defects and aberrant tryptophan metabolism.
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Xiaofeng Zuo, Winkler, Brennan, Lerner, Kasey, Ilatovskaya, Daria V., Zamaro, Aleksandra S., Yujing Dang, Yanhui Su, Peifeng Deng, Wayne Fitzgibbon, Hartman, Jessica, Kwon Moo Park, and Lipschutz, Joshua H.
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KIDNEY tubules ,TRYPTOPHAN ,METABOLISM ,ACUTE kidney failure ,MITOCHONDRIA - Abstract
The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism. NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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11. Validation of an organ mapping antibody panel for cyclical immunofluorescence microscopy on normal human kidneys.
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Brewer, Maya, Migas, Lukasz G., Clouthier, Kelly A., Allen, Jamie L., Anderson, David M., Pingry, Ellie, Farrow, Melissa, Quardokus, Ellen M., Spraggins, Jeffrey M., Van de Plas, Raf, and de Caestecker, Mark P.
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IMMUNOFLUORESCENCE ,MICROSCOPY ,IMAGE registration ,FLUORESCENCE microscopy ,KIDNEYS - Abstract
The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel of antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for two-dimensional (2-D) and three-dimensional (3-D) cyclical IF (CyCIF) to provide a more detailed method for evaluating tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using opensourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types, and how these are altered in disease. NEW & NOTEWORTHY We describe here validation criteria used to define on organ mapping panel of antibodies that can be used to define 18 cell types and five extracellular matrix compartments using cyclical immunofluorescence (CyCIF) microscopy. As CyCIF does not require specialized instrumentation, and image registration required to assemble CyCIF images can be performed by any laboratory without specialized computational skills, this technology is accessible to any laboratory with access to a fluorescence microscope and digital scanner. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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12. Long-term follow-up of TREK-1 KO mice reveals the development of bladder hypertrophy and impaired bladder smooth muscle contractility with age.
- Author
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Xie, Alison Xiaoqiao, Iguchi, Nao, and Malykhina, Anna P.
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SMOOTH muscle ,SMOOTH muscle physiology ,MUSCLE aging ,BLADDER ,URINATION disorders - Abstract
Stretch-activated two-pore domain K
+ (K2P ) channels play important roles in many visceral organs, including the urinary bladder. The TWIK-related K+ channel TREK-1 is the predominantly expressed K2P channel in the urinary bladder of humans and rodents. Downregulation of TREK-1 channels was observed in the urinary bladder of patients with detrusor overactivity, suggesting their involvement in the pathogenesis of voiding dysfunction. This study aimed to characterize the long-term effects of TREK-1 on bladder function with global and smooth muscle-specific TREK-1 knockout (KO) mice. Bladder morphology, bladder smooth muscle (BSM) contractility, and voiding patterns were evaluated up to 12 mo of age. Both sexes were included in this study to probe the potential sex differences. Smooth muscle-specific TREK-1 KO mice were used to distinguish the effects of TREK-1 downregulation in BSM from the neural pathways involved in the control of bladder contraction and relaxation. TREK-1 KO mice developed enlarged urinary bladders (by 60.0% for males and by 45.1% for females at 6 mo; P < 0.001 compared with the age-matched control group) and had a significantly increased bladder capacity (by 137.7% at 12 mo; P < 0.0001) and compliance (by 73.4% at 12 mo; P < 0.0001). Bladder strips isolated from TREK-1 KO mice exhibited decreased contractility (peak force after KCl at 6 mo was 1.6 ± 0.7 N/g compared with 3.4 ± 2.0 N/g in the control group; P = 0.0005). The lack of TREK-1 channels exclusively in BSM did not replicate the bladder phenotype observed in TREK-1 KO mice, suggesting a strong neurogenic origin of TREK-1-related bladder dysfunction. NEW & NOTEWORTHY: This study compared voiding function and bladder phenotypes in global and smooth muscle-specific TREK-1 KO mice. We found significant age-related changes in bladder contractility, suggesting that the lack of TREK-1 channel activity might contribute to age-related changes in bladder smooth muscle physiology. [ABSTRACT FROM AUTHOR]- Published
- 2024
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13. In chronic kidney disease altered cardiac metabolism precedes cardiac hypertrophy.
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Williams, Matthew J., Halabi, Carmen M., Patel, Hiral M., Joseph, Zachary, McCommis, Kyle, Weinheimer, Carla, Kovacs, Attila, Lima, Florence, Finck, Brian, Malluche, Hartmut, and Hruska, Keith A.
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CHRONIC kidney failure ,HEART metabolism ,CARDIAC hypertrophy ,VASCULAR smooth muscle ,ARTERIAL diseases - Abstract
Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
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14. Renal vascular control during normothermia and passive heat stress does not differ between healthy younger men and women.
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Freemas, Jessica A., Worley, Morgan L., Gabler, Mikaela C., Hess, Hayden W., Goss, Curtis S., Baker, Tyler B., Johnson, Blair D., Chapman, Christopher L., and Schlader, Zachary J.
- Subjects
YOUNG women ,YOUNG men ,ACUTE kidney failure ,DOPPLER ultrasonography ,VASCULAR resistance - Abstract
Men are likely at greater risk for heat-induced acute kidney injury compared with women, possibly due to differences in vascular control. We tested the hypothesis that the renal vasoconstrictor and vasodilator responses will be greater in younger women compared with men during passive heat stress. Twenty-five healthy adults [12 women (early follicular phase) and 13 men] completed two experimental visits, heat stress or normothermic time-control, assigned in a block-randomized crossover design. During heat stress, participants wore a water-perfused suit perfused with 50°C water. Core temperature was increased by ∼0.8°C in the first hour before commencing a 2-min cold pressor test (CPT). Core temperature remained clamped and at 1-h post-CPT, subjects ingested a whey protein shake (1.2 g of protein/kg body wt), and measurements were taken pre-, 75 min, and 150 min post-protein. Beat-to-beat blood pressure (Penaz method) was measured and segmental artery vascular resistance (VR, Doppler ultrasound) was calculated as segmental artery blood velocity ÷ mean arterial pressure. CPT-induced increases in segmental artery VR did not differ between trials (trial effect: P = 0.142) nor between men (heat stress: 1.5-±-1.0 mmHg/cm/s, normothermia: 1.4-±-1.0 mmHg/cm/s) and women (heat stress: 1.4-±-1.2 mmHg/cm/s, normothermia: 2.1-±-1.1 mmHg/cm/s) (group effect: P = 0.429). Reductions in segmental artery VR following oral protein loading did not differ between trials (trial effect: P = 0.080) nor between men (heat stress: −0.6-±-0.8 mmHg/cm/s, normothermia: −0.6-±-0.6 mmHg/cm/s) and women (heat stress: −0.5-±-0.5 mmHg/cm/s, normothermia: −1.1-±-0.6 mmHg/cm/s) (group effect: P = 0.204). Renal vasoconstrictor responses to the cold pressor test and vasodilator responses following an oral protein load during heat stress or normothermia do not differ between younger men and younger women in the early follicular phase of the menstrual cycle. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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15. Perspective on G protein-coupled receptors in renal physiology.
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Pluznick, Jennifer L. and Fenton, Robert A.
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G protein coupled receptors ,KIDNEY physiology ,G proteins ,OLFACTORY receptors - Published
- 2023
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16. Collecting duct water permeability inhibition by EGF is associated with decreased cAMP, PKA activity, and AQP2 phosphorylation at Ser269.
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Chung-Lin Chou, Limbutara, Kavee, Kao, Anika R., Clark, Jevin Z., Nein, Ellen H., Raghuram, Viswanathan, and Knepper, Mark A.
- Abstract
Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-D-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser
264 and Ser269 . This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-a subunit increased phosphorylation at Ser256, Ser264 , and Ser269 . None of the other kinases tested phosphorylated Ser269 . In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269 . These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269 , a site previously shown to regulate AQP2 endocytosis. [ABSTRACT FROM AUTHOR]- Published
- 2024
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17. Guidelines on antibody use in physiology research.
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Brooks, Heddwen L., de Castro Brás, Lisandra E., Brunt, Keith R., Sylvester, Megan A., Parvatiyar, Michelle S., Sirish, Padmini, Bansal, Shyam S., Sule, Rasheed, Eadie, Ashley L., Knepper, Mark A., Fenton, Robert A., Lindsey, Merry L., DeLeon-Pennell, Kristine Y., and Gomes, Aldrin V.
- Abstract
Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
18. Current perspective on circadian function of the kidney.
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Benjamin, Jazmine I. and Pollock, David M.
- Abstract
Behavior and function of living systems are synchronized by the 24-h rotation of the Earth that guides physiology according to time of day. However, when behavior becomes misaligned from the light-dark cycle, such as in rotating shift work, jet lag, and even unusual eating patterns, adverse health consequences such as cardiovascular or cardiometabolic disease can arise. The discovery of cell-autonomous molecular clocks expanded interest in regulatory systems that control circadian physiology including within the kidney, where function varies along a 24-h cycle. Our understanding of the mechanisms for circadian control of physiology is in the early stages, and so the present review provides an overview of what is known and the many gaps in our current understanding. We include a particular focus on the impact of eating behaviors, especially meal timing. A better understanding of the mechanisms guiding circadian function of the kidney is expected to reveal new insights into causes and consequences of a wide range of disorders involving the kidney, including hypertension, obesity, and chronic kidney disease. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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19. Podocyte injury at young age causes premature senescence and worsens glomerular aging.
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Veloso Pereira, Beatriz Maria, Yuting Zeng, Maggiore, Joseph C., Schweickart, Robert Allen, Eng, Diana G., Kaverina, Natalya, McKinzie, Sierra R., Chang, Anthony, Loretz, Carol J., Thieme, Karina, Hukriede, Neil A., Pippin, Jeffrey W., Wessely, Oliver, and Shankland, Stuart J.
- Subjects
FOCAL segmental glomerulosclerosis ,AGING ,MIDDLE age ,KIDNEY glomerulus diseases ,AGE - Abstract
As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan blackstaining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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20. Impaired hemodynamic renal reserve response following recovery from established acute kidney injury and improvement by hydrodynamic isotonic fluid delivery.
- Author
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Ullah, Md Mahbub, Collett, Jason A., Bacallao, Robert L., and Basile, David P.
- Subjects
ACUTE kidney failure ,NEPHRECTOMY ,T helper cells ,HEMODYNAMICS ,VENAE cavae ,RENAL veins - Abstract
Renal reserve capacity may be compromised following recovery from acute kidney injury (AKI) and could be used to identify impaired renal function in the face of restored glomerular filtration rate (GFR) or plasma creatinine. To investigate the loss of hemodynamic renal reserve responses following recovery in a model of AKI, rats were subjected to left unilateral renal ischemia-reperfusion (I/R) injury and contralateral nephrectomy and allowed to recover for 5 wk. Some rats were treated 24 h post-I/R by hydrodynamic isotonic fluid delivery (AKI-HIFD) of saline through the renal vein, previously shown to improve recovery and inflammation relative to control rats that received saline through the vena cava (AKI-VC). At 5 wk after surgery, plasma creatinine and GFR recovered to levels observed in uninephrectomized sham controls. Baseline renal blood flow (RBF) was not different between AKI or sham groups, but infusion of L-arginine (7.5 mg/kg/min) significantly increased RBF in sham controls, whereas the RBF response to L-arginine was significantly reduced in AKI-VC rats relative to sham rats (22.6 ± 2.2% vs. 13.8 ± 1.8%, P < 0.05). RBF responses were partially protected in AKI-HIFD rats relative to AKI-VC rats (17.0 ± 2.2%) and were not significantly different from sham rats. Capillary rarefaction observed in AKI-VC rats was significantly protected in AKI-HIFD rats. There was also a significant increase in T helper 17 cell infiltration and interstitial fibrosis in AKI-VC rats versus sham rats, which was not present in AKI-HIFD rats. These data suggest that recovery from AKI results in impaired hemodynamic reserve and that associated CKD progression may be mitigated by HIFD in the early post-AKI period. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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21. Tubular dysfunction impairs renal excretion of pseudouridine in diabetic kidney disease.
- Author
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Mathew, Anna V., Kayampilly, Pradeep, Byun, Jaeman, Nair, Viji, Afshinnia, Farsad, Biaoxin Chai, Brosius 3rd, Frank C., Kretzler, Matthias, and Pennathur, Subramaniam
- Subjects
DIABETIC nephropathies ,PSEUDOURIDINE ,METABOLIC flux analysis ,KIDNEY diseases ,LIQUID chromatography-mass spectrometry - Abstract
Plasma nucleosides-pseudouridine (PU) and N²N²-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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22. Vasopressin V2 receptor, tolvaptan, and ERK1/2 phosphorylation in the renal collecting duct.
- Author
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Khan, Shaza, Raghuram, Viswanathan, Lihe Chen, Chung-Lin Chou, Chin-Rang Yang, Khundmiri, Syed J., and Knepper, Mark A.
- Subjects
VASOPRESSIN ,POLYCYSTIC kidney disease ,PHOSPHORYLATION ,GENE expression ,PROTEIN kinases - Abstract
Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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23. Antinociceptive effect of the calcitonin gene-related peptide receptor antagonist BIBN4096BS in mice with bacterial cystitis.
- Author
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Montalbetti, Nicolas, Dalghi, Marianela G., Parakala-Jain, Tanmay, Clayton, Dennis, Apodaca, Gerard, and Carattino, Marcelo D.
- Subjects
CALCITONIN gene-related peptide ,URINARY tract infections ,PEPTIDE receptors ,FLUORESCENCE in situ hybridization ,CYSTITIS ,ESCHERICHIA coli - Abstract
Patients with urinary tract infections (UTIs) suffer from urinary frequency, urgency, dysuria, and suprapubic pain, but the mechanisms by which bladder afferents sense the presence of uropathogens and encode this information is not well understood. Calcitonin gene-related peptide (CGRP) is a 37-mer neuropeptide found in a subset of bladder afferents that terminate primarily in the lamina propria. Here, we report that the CGRP receptor antagonist BIBN4096BS lessens lower urinary tract symptoms and prevents the development of pelvic allodynia in mice inoculated with uropathogenic Escherichia coli (UPEC) without altering urine bacterial loads or the host immune response to the infection. These findings indicate that CGRP facilitates the processing of noxious/inflammatory stimuli during UPEC infection. Using fluorescent in situ hybridization, we identified a population of suburothelial fibroblasts in the lamina propria, a region where afferent fibers containing CGRP terminate, that expresses the canonical CGRP receptor components Calcrl and Ramp1. We propose that these fibroblasts, in conjunction with CGRP
+ afferents, form a circuit that senses substances released during the infection and transmit this noxious information to the central nervous system. NEW & NOTEWORTHY Afferent C fibers release neuropeptides including calcitonin gene-related peptide (CGRP). Here, we show that the specific CGRP receptor antagonist, BIBN409BS, ameliorates lower urinary tract symptoms and pelvic allodynia in mice inoculated with uropathogenic E. coli. Using fluorescent in situ hybridization, we identified a population of suburothelial fibroblasts in the lamina propria that expresses the canonical CGRP receptor. Our findings indicate that CGRP contributes to the transmission of nociceptive information arising from the bladder. [ABSTRACT FROM AUTHOR]- Published
- 2023
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24. Persistence and expansion of hypoxia detected by pimonidazole adduct immunostaining during progression of diabetic nephropathy in diabetic mice.
- Author
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Akari Inada and Atsushi Fukatsu
- Abstract
Hypoxia and oxidative stress are considered to be underlying factors in the deterioration of renal function and pathogenesis in acute kidney injury (AKI) and chronic kidney disease, including diabetic nephropathy (DN). However, the long-term role of hypoxia in DN is unknown. Here, we investigated the distribution, severity, and time course of hypoxia during DN development in our well-established severely diabetic transgenic (Tg) DN mouse model that mimics human DN up to 80 wk of age, using pimonidazole adduct immunohistochemistry. The relationship between pimonidazole adduct distribution and hypoxia-inducible factor (HIF) expression was also examined. We found 1) persistent pimonidazole immunostaining mainly in the outer zone of the outer medulla, extending into the inner zone, 2) significant expansion of area and intensity up to 40 wk of age, and 3) characteristic subcellular localization mainly at apical sites in vesicular form by laser scanning microscopy of thin slices. The distribution of pimonidazole adducts was different from that of HIF reported previously, indicating that hypoxia does not directly contribute to persistent abnormal HIF expression. These results suggest that pimonidazole adducts produced under low PO
2 conditions are sustained by a mechanism distinct from direct ischemia. We propose that in the long course of DN development, persistent hyperfiltration and hyperexcretion of glucose, albumin, and water increase metabolism and energy expenditure in the tubules, and such chronic stimulation leads to relative ischemia and local hypoxia, which may contribute in part to the loss of nephrons. [ABSTRACT FROM AUTHOR]- Published
- 2023
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25. Why is chronic kidney disease progressive? Evolutionary adaptations and maladaptations.
- Author
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Chevalier, Robert L.
- Abstract
Despite significant advances in renal physiology, the global prevalence of chronic kidney disease (CKD) continues to increase. The emergence of multicellular organisms gave rise to increasing complexity of life resulting in trade-offs reflecting ancestral adaptations to changing environments. Three evolutionary traits shape CKD over the lifespan: 1) variation in nephron number at birth, 2) progressive nephron loss with aging, and 3) adaptive kidney growth in response to decreased nephron number. Although providing plasticity in adaptation to changing environments, the cell cycle must function within constraints dictated by available energy. Prioritized allocation of energy available through the placenta can restrict fetal nephrogenesis, a risk factor for CKD. Moreover, nephron loss with aging is a consequence of cell senescence, a pathway accelerated by adaptive nephron hypertrophy that maintains metabolic homeostasis at the expense of increased vulnerability to stressors. Driven by reproductive fitness, natural selection operates in early life but diminishes thereafter, leading to an exponential increase in CKD with aging, a product of antagonistic pleiotropy. A deeper understanding of the evolutionary constraints on the cell cycle may lead to manipulation of the balance between progenitor cell renewal and differentiation, regulation of cell senescence, and modulation of the balance between cell proliferation and hypertrophy. Application of an evolutionary perspective may enhance understanding of adaptation and maladaptation by nephrons in the progression of CKD, leading to new therapeutic advances. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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26. “Hi, how can i help you?”: embracing artificial intelligence in kidney research.
- Author
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Layton, Anita T.
- Subjects
ARTIFICIAL intelligence ,KIDNEY diseases ,MACHINE learning ,KIDNEY physiology ,KIDNEYS ,ELECTRONIC health records - Abstract
In recent years, biology and precision medicine have benefited from major advancements in generating large-scale molecular and biomedical datasets and in analyzing those data using advanced machine learning algorithms. Machine learning applications in kidney physiology and pathophysiology include segmenting kidney structures from imaging data and predicting conditions like acute kidney injury or chronic kidney disease using electronic health records. Despite the potential of machine learning to revolutionize nephrology by providing innovative diagnostic and therapeutic tools, its adoption in kidney research has been slower than in other organ systems. Several factors contribute to this underutilization. The complexity of the kidney as an organ, with intricate physiology and specialized cell populations, makes it challenging to extrapolate bulk omics data to specific processes. In addition, kidney diseases often present with overlapping manifestations and morphological changes, making diagnosis and treatment complex. Moreover, kidney diseases receive less funding compared with other pathologies, leading to lower awareness and limited public-private partnerships. To promote the use of machine learning in kidney research, this review provides an introduction to machine learning and reviews its notable applications in renal research, such as morphological analysis, omics data examination, and disease diagnosis and prognosis. Challenges and limitations associated with data-driven predictive techniques are also discussed. The goal of this review is to raise awareness and encourage the kidney research community to embrace machine learning as a powerful tool that can drive advancements in understanding kidney diseases and improving patient care. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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27. IL-6 mediates the hepatic acute phase response after prerenal azotemia in a clinically defined murine model.
- Author
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Kayo Okamura, Sizhao Lu, Zhibin He, Altmann, Chris, Montford, John R., Li, Amy S., Lucia, M. Scott, Orlicky, David J., Weiser-Evans, Mary, and Faubel, Sarah
- Subjects
ACUTE phase reaction ,GLOMERULAR filtration rate ,ACUTE kidney failure ,BLOOD proteins ,INTERLEUKIN-6 - Abstract
Prerenal azotemia (PRA) is a major cause of acute kidney injury and uncommonly studied in preclinical models. We sought to develop and characterize a novel model of PRA that meets the clinical definition: acute loss of glomerular filtration rate (GFR) that returns to baseline with resuscitation. Adult male C57BL/6J wild-type (WT) and IL-6
−/− mice were studied. Intraperitoneal furosemide (4 mg) or vehicle was administered at time = 0 and 3 h to induce PRA from volume loss. Resuscitation began at 6 h with 1 mL intraperitoneal saline for four times for 36 h. Six hours after furosemide administration, measured glomerular filtration rate was 25% of baseline and returned to baseline after saline resuscitation at 48 h. After 6 h of PRA, plasma interleukin (IL)-6 was significantly increased, kidney and liver histology were normal, kidney and liver lactate were normal, and kidney injury molecule-1 immunofluorescence was negative. There were 327 differentially regulated genes upregulated in the liver, and the acute phase response was the most significantly upregulated pathway; 84 of the upregulated genes (25%) were suppressed in IL-6−/− mice, and the acute phase response was the most significantly suppressed pathway. Significantly upregulated genes and their proteins were also investigated and included serum amyloid A2, serum amyloid A1, lipocalin 2, chemokine (C-X-C motif) ligand 1, and haptoglobin; hepatic gene expression and plasma protein levels were all increased in wild-type PRA and were all reduced in IL-6−/− PRA. This work demonstrates previously unknown systemic effects of PRA that includes IL-6-mediated upregulation of the hepatic acute phase response. NEW & NOTEWORTHY Prerenal azotemia (PRA) accounts for a third of acute kidney injury (AKI) cases yet is rarely studied in preclinical models. We developed a clinically defined murine model of prerenal azotemia characterized by a 75% decrease in measured glomerular filtration rate (GFR), return of measured glomerular filtration rate to baseline with resuscitation, and absent tubular injury. Numerous systemic effects were observed, such as increased plasma interleukin-6 (IL-6) and upregulation of the hepatic acute phase response. [ABSTRACT FROM AUTHOR]- Published
- 2023
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- View/download PDF
28. Optogenetic urothelial cell stimulation induces bladder contractions and pelvic nerve afferent firing.
- Author
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Robilotto, Gabriella L., Yang, Olivia J., Alom, Firoj, Johnson, Richard D., and Mickle, Aaron D.
- Subjects
UROTHELIUM ,BLADDER ,SENSORY neurons ,NERVES ,CELL physiology ,AFFERENT pathways ,CELL communication ,LABORATORY mice - Abstract
Urothelial cells, which play an essential role in barrier function, are also thought to play a sensory role in bladder physiology by releasing signaling molecules in response to sensory stimuli that act upon adjacent sensory neurons. However, it is challenging to study this communication due to the overlap in receptor expression and proximity of urothelial cells to sensory neurons. To overcome this challenge, we developed a mouse model where we can directly stimulate urothelial cells using optogenetics. We crossed a uroplakin II (UPK2) cre mouse with a mouse that expresses the light-activated cation channel channelrhodopsin-2 (ChR2) in the presence of cre expression. Optogenetic stimulation of urothelial cells cultured from UPK2-ChR2 mice initiates cellular depolarization and release of ATP. Cystometry recordings demonstrated that optical stimulation of urothelial cells increases bladder pressure and pelvic nerve activity. Increases in bladder pressure persisted, albeit to a lesser extent, when the bladder was excised in an in vitro preparation. The P2X receptor antagonist PPADS significantly reduced optically evoked bladder contractions in vivo and ex vivo. Furthermore, corresponding nerve activity was also inhibited with PPADS. Our data suggest that urothelial cells can initiate robust bladder contractions via sensory nerve signaling or contractions through local signaling mechanisms. These data support a foundation of literature demonstrating communication between sensory neurons and urothelial cells. Importantly, with further use of these optogenetic tools, we hope to scrutinize this signaling mechanism, its importance for normal micturition and nociception, and how it may be altered in pathophysiological conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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29. Renal, but not platelet or skin, extracellular vesicles decrease oxidative stress, enhance nascent peptide synthesis, and protect from ischemic renal injury.
- Author
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Dominguez, Jesus H., Danhui Xie, and Kelly, K. J.
- Subjects
PEPTIDE synthesis ,EXTRACELLULAR vesicles ,OXIDATIVE stress ,ACUTE kidney failure ,BLOOD platelets ,THROMBIN receptors - Abstract
Acute kidney injury (AKI) is deadly and expensive, and specific, effective therapy remains a large unmet need. We have demonstrated the beneficial effects of transplanted adult tubular cells and extracellular vesicles (EVs; exosomes) derived from those renal cells on experimental ischemic AKI, even when administered after renal failure is established. To further examine the mechanisms of benefit with renal EVs, we tested the hypothesis that EVs from other epithelia or platelets (a rich source of EVs) might be protective, using a well-characterized ischemia-reperfusion model. When given after renal failure was present, renal EVs, but not those from skin or platelets, markedly improved renal function and histology. The differential effects allowed us to examine the mechanisms of benefit with renal EVs. We found significant decreases in oxidative stress postischemia in the renal EV-treated group with preservation of renal superoxide dismutase and catalase as well as increases in anti-inflammatory interleukin-10. In addition, we propose a novel mechanism of benefit: renal EVs enhanced nascent peptide synthesis following hypoxia in cells and in postischemic kidneys. Although EVs have been used therapeutically, these results serve as "proof of principle" to examine the mechanisms of injury and protection. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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30. PCK1 is a key regulator of metabolic and mitochondrial functions in renal tubular cells.
- Author
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Verissimo, Thomas, Dalga, Delal, Arnoux, Grégoire, Sakhi, Imene, Faivre, Anna, Auwerx, Hannah, Bourgeois, Soline, Paolucci, Deborah, Gex, Quentin, Rutkowski, Joseph M., Legouis, David, Wagner, Carsten A., Hall, Andrew M., and de Seigneux, Sophie
- Subjects
KIDNEY physiology ,PROXIMAL kidney tubules ,LACTATES ,CHRONIC kidney failure ,ORGANIC anion transporters ,ACIDOSIS ,KIDNEY diseases - Abstract
Phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C) is a cytosolic enzyme converting oxaloacetate to phosphoenolpyruvate, with a potential role in gluconeogenesis, ammoniagenesis, and cataplerosis in the liver. Kidney proximal tubule cells display high expression of this enzyme, whose importance is currently not well defined. We generated PCK1 kidney-specific knockout and knockin mice under the tubular cell-specific PAX8 promoter. We studied the effect of PCK1 deletion and overexpression at the renal level on tubular physiology under normal conditions and during metabolic acidosis and proteinuric renal disease. PCK1 deletion led to hyperchloremic metabolic acidosis characterized by reduced but not abolished ammoniagenesis. PCK1 deletion also resulted in glycosuria, lactaturia, and altered systemic glucose and lactate metabolism at baseline and during metabolic acidosis. Metabolic acidosis resulted in kidney injury in PCK1-deficient animals with decreased creatinine clearance and albuminuria. PCK1 further regulated energy production by the proximal tubule, and PCK1 deletion decreased ATP generation. In proteinuric chronic kidney disease, mitigation of PCK1 downregulation led to better renal function preservation. PCK1 is essential for kidney tubular cell acid-base control, mitochondrial function, and glucose/lactate homeostasis. Loss of PCK1 increases tubular injury during acidosis. Mitigating kidney tubular PCK1 downregulation during proteinuric renal disease improves renal function. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
31. Functional TFEB activation characterizes multiple models of renal cystic disease and loss of polycystin-1.
- Author
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Shillingford, Jonathan M. and Shayman, James A.
- Subjects
CYSTIC kidney disease ,POLYCYSTIC kidney disease ,RENAL tubular transport disorders ,CATHEPSIN B ,TRANSCRIPTION factors - Abstract
Polycystic kidney disease is a disorder of renal epithelial growth and differentiation. Transcription factor EB (TFEB), a master regulator of lysosome biogenesis and function, was studied for a potential role in this disorder. Nuclear translocation and functional responses to TFEB activation were studied in three murine models of renal cystic disease, including knockouts of folliculin, folliculin interacting proteins 1 and 2, and polycystin-1 (Pkd1) as well as in mouse embryonic fibroblasts lacking Pkd1 and three-dimensional cultures of Madin-Darby canine kidney cells. Nuclear translocation of Tfeb characterized cystic but not noncystic renal tubular epithelia in all three murine models as both an early and sustained response to cyst formation. Epithelia expressed elevated levels of Tfeb-dependent gene products, including cathepsin B and glycoprotein nonmetastatic melanoma protein B. Nuclear Tfeb translocation was observed in mouse embryonic fibroblasts lacking Pkd1 but not wild-type fibroblasts. Pkd1 knockout fibroblasts were characterized by increased Tfeb-dependent transcripts, lysosomal biogenesis and repositioning, and increased autophagy. The growth of Madin-Darby canine kidney cell cysts was markedly increased following exposure to the TFEB agonist compound C1, and nuclear Tfeb translocation was observed in response to both forskolin and compound C1 treatment. Nuclear TFEB also characterized cystic epithelia but not noncystic tubular epithelia in human patients with autosomal dominant polycystic kidney disease. Noncanonical activation of TFEB is characteristic of cystic epithelia in multiple models of renal cystic disease including those associated with loss of Pkd1. Nuclear TFEB translocation is functionally active in these models and may be a component of a general pathway contributing to cystogenesis and growth. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
32. Circadian gene expression in mouse renal proximal tubule.
- Author
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Bingham, Molly A., Neijman, Kim, Chin-Rang Yang, Aponte, Angel, Mak, Angela, Hiroaki Kikuchi, Hyun Jun Jung, Poll, Brian G., Raghuram, Viswanathan, Euijung Park, Chung-Lin Chou, Lihe Chen, Leipziger, Jens, Knepper, Mark A., and Dona, Margo
- Subjects
PROXIMAL kidney tubules ,GENE expression ,RNA sequencing ,GLOMERULAR filtration rate ,KIDNEY physiology - Abstract
Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih. gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances. NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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- View/download PDF
33. Data resource: vasopressin-regulated protein phosphorylation sites in the collecting duct.
- Author
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Euijung Park, Chin-Rang Yang, Raghuram, Viswanathan, Deshpande, Venkatesh, Datta, Arnab, Poll, Brian G., Leo, Kirby T., Hiroaki Kikuchi, Lihe Chen, Chung-Lin Chou, and Knepper, Mark A.
- Subjects
CYTOSKELETAL proteins ,PHOSPHORYLATION ,PROTEIN kinases ,PHOSPHOPROTEINS ,WEBSITES ,DESMOPRESSIN ,AQUAPORINS - Abstract
Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressinregulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressininduced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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34. VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.
- Author
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Mingjun Shi, Maique, Jenny Omega, Cleaver, Ondine, Moe, Orson W., and Ming Chang Hu
- Subjects
RENAL fibrosis ,PROTEIN kinases ,GREEN fluorescent protein ,VASCULAR endothelial growth factors ,VASCULAR endothelial growth factor receptors ,HIGH cholesterol diet ,GENETIC overexpression ,HIGH-carbohydrate diet - Abstract
Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2þ/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2þ/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2þ/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wildtype mice but only modestly ameliorated these changes in the VEGFR2þ/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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35. 2024 Carl W. Gottschalk Distinguished Lectureship of the American Physiological Society Renal Section.
- Author
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Fenton, Robert A. and Ellison, David H.
- Subjects
WATER-electrolyte balance (Physiology) ,KIDNEY physiology ,MOLECULAR cloning ,MINERALOCORTICOID receptors ,CALCIUM-sensing receptors - Abstract
The American Physiological Society Renal Section has awarded Gerardo Gamba, MD, the 2024 Carl Gottschalk Distinguished Lectureship. Dr. Gamba is a Professor of Medicine and head of the Molecular Physiology Unit at the Biomedical Research Institute of the National Autonomous University of Mexico. He is a world leader in kidney physiology, with a focus on solute transport along the nephron. His research has led to significant advancements in understanding kidney function and the development of drugs used in clinical practice. Dr. Gamba is also known for his contributions to building research excellence in economically challenged areas and his passion for classical music and literature. [Extracted from the article]
- Published
- 2024
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36. Role of coagulation in persistent renal ischemia following reperfusion in an animal model.
- Author
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Dominguez, Jesus H., Danhui Xie, 2nd, James M. Dominguez, and Kelly, K. J.
- Subjects
BLOOD coagulation ,ACUTE kidney failure ,REPERFUSION ,CHRONIC kidney failure ,ISCHEMIA ,PLASMA flow - Abstract
Ischemic acute kidney injury is common, deadly, and accelerates the progression of chronic kidney disease, yet has no specific therapy. After ischemia, reperfusion is patchy with early and persistent impairment in regional renal blood flow and cellular injury. We tested the hypothesis that intrarenal coagulation results in sustained renal ischemia following reperfusion, using a well-characterized model. Markedly decreased, but heterogeneous, microvascular plasma flow with microthrombi was found postischemia by intravital microscopy. Widespread tissue factor expression and fibrin deposition were also apparent. Clotting was accompanied by complement activation and inflammation. Treatment with exosomes derived from renal tubular cells or with the fibrinolytic urokinase, given 24 h postischemia when renal failure was established, significantly improved microvascular flow, coagulation, serum creatinine, and histological evidence of injury. These data support the hypothesis that intrarenal clotting occurs early and the resultant sustained ischemia is a critical determinant of renal failure following ischemia; they demonstrate that the coagulation abnormalities are amenable to therapy and that therapy results in improvement in both function and postischemic inflammation. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
37. Urinary serpin-A3 is an early predictor of clinical response to therapy in patients with proliferative lupus nephritis.
- Author
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Martínez-Rojas, Miguel Ángel, Sánchez-Navarro, Andrea, Mejia-Vilet, Juan Manuel, Pérez-Villalva, Rosalba, Uribe, Norma, and Bobadilla, Norma A.
- Subjects
LUPUS nephritis ,TREATMENT effectiveness ,WESTERN immunoblotting ,MEXICANS - Abstract
We have previously reported that urinary excretion of serpin-A3 (uSerpA3) is significantly elevated in patients with active lupus nephritis (LN). Here, we evaluated the course of uSerpA3 during the first year of treatment and its association with response to therapy in patients with proliferative LN. The observational longitudinal study included 60 Mexican adults with proliferative LN followed during the first year after LN flare. uSerpA3 was detected by Western blot analysis at flare and after 3, 6, and 12 mo. The response to therapy was determined 1 yr after the LN flare. We evaluated the correlation between uSerpA3 and histological parameters at LN flare. The temporal association between uSerpA3 and response to therapy was analyzed with linear mixed models. uSerpA3 prognostic performance for response was evaluated with receiver-operating characteristic curves. Among the 60 patients studied, 21 patients (35%) were class III and 39 patients (65%) were class IV. uSerpA3 was higher in class IV than in class III LN (6.98 vs. 2.89 dots per in./mg creatinine, P = 0.01). Furthermore, uSerpA3 correlated with the histological activity index (r = 0.29, P = 0.02). There was a significant association between the temporal course of uSerpA3 and response to therapy. Responders showed a significant drop in uSerpA3 at 6 mo compared with LN flare (P < 0.001), whereas nonresponders persisted with elevated uSerpA3. Moreover, uSerpA3 was significantly lower at flare in responders compared with nonresponders (2.69 vs. 6.98 dots per in./mg creatinine, P < 0.05). Furthermore, uSerpA3 was able to identify nonresponders since 3 mo after LN flare (area under the curve: 0.77). In conclusion, uSerpA3 is an early indicator of kidney inflammation and predictor of the clinical response to therapy in patients with proliferative LN. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
38. Novel method for kinetic analysis applied to transport by the uniporter OCT2.
- Author
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Wright, Stephen H. and Secomb, Timothy W.
- Subjects
CHO cell ,ORGANIC cation transporters ,MICHAELIS-Menten equation ,ABSOLUTE value - Abstract
The kinetics of solute transport shed light on the roles these processes play in cellular physiology, and the absolute values of the kinetic parameters that quantitatively describe transport are increasingly used to model its impact on drug clearance. However, accurate assessment of transport kinetics is challenging. Although most carrier-mediated transport is adequately described by the Michaelis-Menten equation, its use presupposes that the rates of uptake used in the analysis of maximal rates of transport (J
max ) and half-saturation constants (Kt) reflect true unidirectional rates of influx from known concentrations of substrate. Most experimental protocols estimate the initial rate of transport from net substrate accumulation determined at a single time point (typically between 0.5 and 5 min) and assume it reflects unidirectional influx. However, this approach generally results in systematic underestimates of Jmax and overestimates of Kt; the former primarily due to the unaccounted impact of efflux of accumulated substrate, and the latter due to the influence of unstirred water layers. Here, we describe the bases of these timedependent effects and introduce a computational model that analyzes the time course of net substrate uptake at several concentrations to calculate Jmax and Kt for unidirectional influx, taking into account the influence of unstirred water layers and mediated efflux. This method was then applied to calculate the kinetics of transport of 1-methyl-4-phenylpryridinium and metformin by renal organic cation transporter 2 as expressed in cultured Chinese hamster ovary cells. NEW & NOTEWORTHY Here, we describe a mathematical model that uses the time course of net substrate uptake into cells from several increasing concentrations to calculate unique kinetic parameters [maximal rates of transport (Jmax) and half-saturation constants (Kt)] of the process. The method is the first to take into consideration the common complicating factors of unstirred layers and carrier-mediated efflux in the experimental determination of transport kinetics. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
39. Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA+ interstitial cells are fibroblasts.
- Author
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Clayton, Dennis R., Ruiz, Wily G., Dalghi, Marianela G., Montalbetti, Nicolas, Carattino, Marcelo D., and Apodaca, Gerard
- Subjects
INTERSTITIAL cells ,CONNECTIVE tissue cells ,FIBROBLASTS ,GENE expression ,BLADDER - Abstract
Fibroblasts are crucial to normal and abnormal organ and tissue biology, yet we lack basic insights into the fibroblasts that populate the bladder wall. Candidates may include bladder interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells), which express the fibroblast-associated marker PDGFRA along with VIM and CD34 but whose form and function remain enigmatic. By applying the latest insights in fibroblast transcriptomics, coupled with studies of gene expression, ultrastructure, and marker analysis, we observe the following: 1) that mouse bladder PDGFRA
+ cells exhibit all of the ultrastructural hallmarks of fibroblasts including spindle shape, lack of basement membrane, abundant endoplasmic reticulum and Golgi, and formation of homotypic cell-cell contacts (but not heterotypic ones); 2) that they express multiple canonical fibroblast markers (including Col1a2, CD34, LY6A, and PDGFRA) along with the universal fibroblast genes Col15a1 and Pi16 but they do not express Kit; and 3) that PDGFRA+ fibroblasts include suburothelial ones (which express ACTA2, CAR3, LY6A, MYH10, TNC, VIM, Col1a2, and Col15a1), outer lamina propria ones (which express CD34, LY6A, PI16, VIM, Col1a2, Col15a1, and Pi16), intermuscular ones (which express CD34, VIM, Col1a2, Col15a1, and Pi16), and serosal ones (which express CD34, PI16, VIM, Col1a2, Col15a1, and Pi16). Collectively, our study revealed that the ultrastructure of PDFRA+ interstitial cells combined with their expression of multiple canonical and universal fibroblast-associated gene products indicates that they are fibroblasts. We further propose that there are four regionally distinct populations of fibroblasts in the bladder wall, which likely contribute to bladder function and dysfunction. NEW & NOTEWORTHY We currently lack basic insights into the fibroblasts that populate the bladder wall. By exploring the ultrastructure of mouse bladder connective tissue cells, combined with analyses of their gene and protein expression, our study revealed that PDGRA+ interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells) are fibroblasts and that the bladder wall contains multiple, regionally distinct populations of these cells. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
40. Single cell versus single nucleus: transcriptome differences in the murine kidney after ischemia-reperfusion injury.
- Author
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Gaedcke, Svenja, Sinning, Julius, Dittrich-Breiholz, Oliver, Haller, Hermann, Soerensen-Zender, Inga, Chieh Ming Liao, Nordlohne, Alexandra, Sen, Payel, von Vietinghoff, Sibylle, DeLuca, David S., and Schmitt, Roland
- Subjects
KIDNEYS ,RNA sequencing ,TRANSCRIPTOMES ,RETINAL injuries ,REPERFUSION injury ,CELL populations - Abstract
The kidney is a complex organ, which consists of multiple components with highly diverse cell types. A detailed understanding of these cell types in health and disease is crucial for the future development of preventive and curative treatment strategies. In recent years, single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) technology has opened up completely new possibilities in investigating the variety of renal cell populations in physiological and pathological states. Here, we systematically assessed differences between scRNAseq and snRNAseq approaches in transcriptome analysis of murine kidneys after ischemia-reperfusion injury. We included tissues from control kidneys and from kidneys harvested 1 wk after mild (17-min clamping time) and severe (27-min clamping time) transient unilateral ischemia. Our findings revealed important methodological differences in the discovery of inflammatory cells, tubular cells, and other specialized cell types. Although the scRNAseq approach was advantageous for investigating immune cells, the snRNAseq approach allowed superior insights into healthy and damaged tubular cells. Apart from differences in the quantitative discovery rate, we found important qualitative discrepancies in the captured transcriptomes with crucial consequences for the interpretation of cell states and molecular functions. Together, we provide an overview of method-dependent differences between scRNAseq and snRNAseq results from identical postischemic kidney tissues. Our results highlight the importance of choosing the right approach for specific research questions. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
41. Inhibition of hypoxia-inducible factor-prolyl hydroxylation protects from cyclophosphamide-induced bladder injury and urinary dysfunction.
- Author
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Clayton, Douglass B., Ching Man Carmen Tong, Li, Belinda, Taylor, Abby S., Shuvro De, Mason, Matthew D., Dudley, Anne G., Davidoff, Olena, Hanako Kobayashi, and Haase, Volker H.
- Subjects
CYCLOPHOSPHAMIDE ,BLADDER ,CYSTITIS ,HYPOXIA-inducible factors ,CHRONIC kidney failure ,HYDROXYLATION - Abstract
Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Activation of the oxygen-regulated hypoxia-inducible factor (HIF) pathway has been shown to protect mucosal membranes by increasing the expression of cytoprotective genes and by suppressing inflammation. The activity of HIF is controlled by prolyl hydroxylase domain (PHD) dioxygenases, which have been exploited as therapeutic targets for the treatment of anemia of chronic kidney disease. Here, we established a mouse model of acute cyclophosphamide (CYP)-induced blood-urine barrier disruption associated with inflammation and severe urinary dysfunction to investigate the HIF-PHD axis in inflammatory bladder injury. We found that systemic administration of dimethyloxalylglycine or molidustat, two small-molecule inhibitors of HIF-prolyl hydroxylases, profoundly mitigated CYP-induced bladder injury and inflammation as assessed by morphological analysis of transmural edema and urothelial integrity and by measuring tissue cytokine expression. Void spot analysis to examine bladder function quantitatively demonstrated that HIF-prolyl hydroxylase inhibitor administration normalized micturition patterns and protected against CYP-induced alteration of urinary frequency and micturition patterns. Our study highlights the therapeutic potential of HIF-activating small-molecule compounds for the prevention or therapy of bladder injury and urinary dysfunction due to blood-urine barrier disruption. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
42. Physiology assays in human kidney organoids.
- Author
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Freedman, Benjamin S.
- Subjects
ORGANOIDS ,HUMAN physiology ,PLURIPOTENT stem cells ,KIDNEY physiology ,HUMAN stem cells ,KIDNEY diseases - Abstract
Kidney organoids derived from human pluripotent stem cells constitute a novel model of disease, development, and regenerative therapy. Organoids are human, experimentally accessible, high throughput, and enable reconstitution of tissue-scale biology in a petri dish. Although gene expression patterns in organoid cells have been analyzed extensively, less is known about the functionality of these structures. Here, we review assays of physiological function in human kidney organoids, including best practices for quality control, and future applications. Tubular structures in organoids accumulate specific molecules through active transport, including dextran and organic anions, and swell with fluid in response to cAMP stimulation. When engrafted into animal models in vivo, organoids form vascularized glomerulus-like structures capable of size-selective filtration. Organoids exhibit metabolic, endocrine, injury, and infection phenotypes, although their specificity is not yet fully clear. To properly interpret organoid physiology assays, it is important to incorporate appropriate negative and positive controls, statistical methods, data presentation, molecular mechanisms, and clinical data sets. Improvements in organoid perfusion, patterning, and maturation are needed to enable branching morphogenesis, urine production, and renal replacement. Reconstituting renal physiology with kidney organoids is a new field with potential to provide fresh insights into classical phenomena. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
43. High-calorie diet results in reversible obesity-related glomerulopathy in adult zebrafish regardless of dietary fat.
- Author
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Zeitler, Evan M., Jennette, J. Charles, Flythe, Jennifer E., Falk, Ronald J., and Poulton, John S.
- Abstract
Obesity is a risk factor for the development of kidney disease. The role of diet in this association remains undetermined, in part due to practical limitations in studying nutrition in humans. In particular, the relative importance of calorie excess versus dietary macronutrient content is poorly understood. For example, it is unknown if calorie restriction modulates obesity-related kidney pathology. To study the effects of diet-induced obesity in a novel animal model, we treated zebrafish for 8 wk with diets varied in both calorie and fat content. Kidneys were evaluated by light and electron microscopy. We evaluated glomerular filtration barrier function using a dextran permeability assay. We assessed the effect of diet on podocyte sensitivity to injury using an inducible podocyte injury model. We then tested the effect of calorie restriction on the defects caused by diet-induced obesity. Fish fed a high-calorie diet developed glomerulomegaly (mean: 1,211 vs. 1,010 µm
2 in controls, P = 0.007), lower podocyte density, foot process effacement, glomerular basement membrane thickening, tubular enlargement (mean: 1,038 vs. 717 µm2 in controls, P < 0.0001), and ectopic lipid deposition. Glomerular filtration barrier dysfunction and increased susceptibility to podocyte injury were observed with high-calorie feeding regardless of dietary fat content. These pathological changes resolved with 4 wk of calorie restriction. Our findings suggest that calorie excess rather than dietary fat drives obesity-related kidney dysfunction and that inadequate podocyte proliferation in response to glomerular enlargement may cause podocyte dysfunction. We also demonstrate the value of zebrafish as a novel model for studying diet in obesity-related kidney disease. NEW & NOTEWORTHY Obesity is a risk factor for kidney disease. The role of diet in this association is difficult to study in humans. In this study, zebrafish fed a high-calorie diet, regardless of fat macronutrient composition, developed glomerulomegaly, foot process effacement, and filtration barrier dysfunction, recapitulating the changes seen in humans with obesity. Calorie restriction reversed the changes. This work suggests that macronutrient composition may be less important than total calories in the development of obesity-related kidney disease. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
44. A mathematical model of the rat kidney. IV. Whole kidney response to hyperkalemia.
- Author
-
Weinstein, Alan M.
- Subjects
HYPERKALEMIA ,KIDNEYS ,MATHEMATICAL models ,KIDNEY cortex ,EXCRETION - Abstract
The renal response to acute hyperkalemia is mediated by increased K
+ secretion within the connecting tubule (CNT), flux that is modulated by tubular effects (e.g., aldosterone) in conjunction with increased luminal flow. There is ample evidence that peritubular K+ blunts Naþ reabsorption in the proximal tubule, thick ascending Henle limb, and distal convoluted tubule (DCT). Although any such reduction may augment CNT delivery, the relative contribution of each is uncertain. The kidney model of this laboratory was recently advanced with representation of the cortical labyrinth and medullary ray. Model tubules capture the impact of hyperkalemia to blunt Naþ reabsorption within each upstream segment. However, this forces the question of the extent to which increased Naþ delivery is transmitted past the macula densa and its tubuloglomerular feedback (TGF) signal. Beyond increasing macula densa Naþ delivery, peritubular K+ is predicted to raise cytosolic Cl- and depolarize macula densa cells, which may also activate TGF. Thus, although the upstream reduction in Naþ transport may be larger, it appears that the DCT effect is critical to increasing CNT delivery. Beyond the flow effect, hyperkalemia reduces ammoniagenesis and reduced ammoniagenesis enhances K+ excretion. What this model provides is a possible mechanism. When cortical NHþ 4 is taken up via peritubular Naþ-K+ (NH+ 4 )-ATPase, it acidifies principal cells. Consequently, reduced ammoniagenesis increases principal cell pH, thereby increasing conductance of both the epithelial Naþ channel and renal outer medullary K+ channel, enhancing K+ excretion. In this model, the effect of aldosterone on principal cells, diminished DCT Naþ reabsorption, and reduced ammoniagenesis all provide relatively equal and additive contributions to renal K+ excretion. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
45. Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients.
- Author
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Isaksson, Gustaf L., Nielsen, Marie B., Hinrichs, Gitte R., Krogstrup, Nicoline V., Zachar, Rikke, Stubmark, Heidi, Svenningsen, Per, Madsen, Kirsten, Bistrup, Claus, Jespersen, Bente, Palarasah, Yaseelan, and Jensen, Boye L.
- Subjects
COMPLEMENT activation ,KIDNEY transplantation ,UROMODULIN ,KIDNEY physiology ,AQUAPORINS - Abstract
Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of -300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Naþ-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
46. Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.
- Author
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Montalbetti, Nicolas, Dalghi, Marianela G., Bastacky, Sheldon I., Clayton, Dennis R., Ruiz, Wily G., Apodaca, Gerard, and Carattino, Marcelo D.
- Subjects
URINARY tract infections ,ESCHERICHIA coli diseases ,BLADDER ,SENSORY neurons ,BLADDER obstruction ,BACTERIAL cells ,AFFERENT pathways ,MEMBRANE potential - Abstract
Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
47. Acid-sensing ion channels modulate bladder nociception.
- Author
-
Montalbetti, Nicolas and Carattino, Marcelo D.
- Subjects
ACID-sensing ion channels ,BLADDER ,SENSORY neurons ,AFFERENT pathways ,CHEMICAL models - Abstract
Sensitization of neuronal pathways and persistent afferent drive are major contributors to somatic and visceral pain. However, the underlying mechanisms that govern whether afferent signaling will give rise to sensitization and pain are not fully understood. In the present report, we investigated the contribution of acid-sensing ion channels (ASICs) to bladder nociception in a model of chemical cystitis induced by cyclophosphamide (CYP). We found that the administration of CYP to mice lacking ASIC3, a subunit primarily expressed in sensory neurons, generates pelvic allodynia at a time point at which only modest changes in pelvic sensitivity are apparent in wild-type mice. The differences in mechanical pelvic sensitivity between wild-type and Asic3 knockout mice treated with CYP were ascribed to sensitized bladder C nociceptors. Deletion of Asic3 from bladder sensory neurons abolished their ability to discharge action potentials in response to extracellular acidification. Collectively, the results of our study support the notion that protons and their cognate ASIC receptors are part of a mechanism that operates at the nerve terminals to control nociceptor excitability and sensitization. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
48. Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes.
- Author
-
Eroglu, Fehime K., Yazar, Volkan, Guler, Ulku, Yıldırım, Muzaffer, Yildirim, Tugce, Gungor, Tulin, Celikkaya, Evra, Karakaya, Deniz, Turay, Nilsu, Dede, Eda Ciftci, Korkusuz, Petek, Salih, Bekir, Bulbul, Mehmet, and Gursel, Ihsan
- Subjects
EXTRACELLULAR vesicles ,NEPHROTIC syndrome ,PHENOTYPES ,WESTERN immunoblotting ,DISEASE remission - Abstract
Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with steroid-sensitive NS in relapse and remission, 10 healthy controls, and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry, and Western blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. Gene Ontology classification analysis and Ingenuity Pathway Analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho-p38 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
49. Bayesian identification of candidate transcription factors for the regulation of Aqp2 gene expression.
- Author
-
Hiroaki Kikuchi, Hyun Jun Jung, Raghuram, Viswanathan, Leo, Kirby T., Euijung Park, Chin-Rang Yang, Chun-Lin Chou, Lihe Chen, and Knepper, Mark A.
- Subjects
GENETIC regulation ,TRANSCRIPTION factors ,BAYES' theorem ,RNA sequencing ,STAT proteins - Abstract
Aquaporin-2 (Aqp2) gene transcription is strongly regulated by vasopressin in the renal collecting duct. However, the transcription factors (TFs) responsible for the regulation of expression of Aqp2 remain largely unknown. We used Bayes' theorem to integrate several -omics data sets to stratify the 1,344 TFs present in the mouse genome with regard to probabilities of regulating Aqp2 gene transcription. Also, we carried out new RNA sequencing experiments mapping the time course of vasopressin-induced changes in the transcriptome of mpkCCD cells to identify TFs that change in tandem with Aqp2. The analysis identified 17 of 1,344 TFs that are most likely to be involved in the regulation of Aqp2 gene transcription. These TFs included eight that have been proposed in prior studies to play a role in Aqp2 regulation, viz., Cebpb, Elf1, Elf3, Ets1, Jun, Junb, Nfkb1, and Sp1. The remaining nine represent new candidates for future studies (Atf1, Irf3, Klf5, Klf6, Mef2d, Nfyb, Nr2f6, Stat3, and Nr4a1). Conspicuously absent is CREB (Creb1), which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription (Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper MA. Physiol Rev 82: 205-244, 2002; Kortenoeven ML, Fenton RA. Biochim Biophys Acta 1840: 1533-1549, 2014; Bockenhauer D, Bichet DG. Nat Rev Nephrol 11: 576-588, 2015; Pearce D, Soundararajan R, Trimpert C, Kashlan OB, Deen PM, Kohan DE. Clin J Am Soc Nephrol 10: 135-146, 2015). Instead, another CREB-like TF, Atf1, ranked fourth among all TFs. RNA sequencing time-course experiments showed a rapid increase in Aqp2 mRNA, within 3 h of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, which we have shown by chromatin immunoprecipitation-sequencing studies to bind downstream from the Aqp2 gene. The identified TFs provide a roadmap for future studies to understand regulation of Aqp2 gene expression. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
50. Podocyte endowment and the impact of adult body size on kidney health.
- Author
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Cullen-McEwen, Luise A., van der Wolde, James, Kotaro Haruhara, Tribolet, Leon, Dowling, John P., Bertram, Michael G., de Matteo, Robert, Haas, Fabian, Czogalla, Jan, Yusuke Okabayashi, Armitage, James A., Black, M. Jane, Hoy, Wendy E., Puelles, Victor G., and Bertram, John F.
- Subjects
LOW birth weight ,BODY size ,ADIPOSE tissues ,ADULTS ,LOW-protein diet ,DIABETIC nephropathies - Abstract
Low birth weight is a risk factor for chronic kidney disease, whereas adult podocyte depletion is a key event in the pathogenesis of glomerulosclerosis. However, whether low birth weight due to poor maternal nutrition is associated with low podocyte endowment and glomerulosclerosis in later life is not known. Female Sprague-Dawley rats were fed a normal-protein diet (NPD; 20%) or low-protein diet (LPD; 8%), to induce low birth weight, from 3 wk before mating until postnatal day 21 (PN21), when kidneys from some male offspring were taken for quantitation of podocyte number and density in whole glomeruli using immunolabeling, tissue clearing, and confocal microscopy. The remaining offspring were fed a normal- or high-fat diet until 6 mo to induce catchup growth and excessive weight gain, respectively. At PN21, podocyte number per glomerulus was 15% lower in low birth weight (LPD) than normal birth weight (NPD) offspring, with this deficit greater in outer glomeruli. Surprisingly, podocyte number in LPD offspring increased in outer glomeruli between PN21 and 6 mo, although an overall 9% podocyte deficit persisted. Postnatal fat feeding to LPD offspring did not alter podometric indexes or result in glomerular pathology at 6mo, whereas fat feeding in NPD offspring was associated with far greater body and fat mass as well as podocyte loss, reduced podocyte density, albuminuria, and glomerulosclerosis. This is the first report that maternal diet can influence podocyte endowment. Our findings provide new insights into the impact of low birth weight, podocyte endowment, and postnatal weight on podometrics and kidney health in adulthood. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
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