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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]

Technical advancements in research techniques in science are made in slow increments. Even so, large advances from insight and hard work of an individual with a single technique can have astonishing ramifications. Here, we examine the impact of Dr. Maurice B. Burg and the isolated perfused renal tubule technique and celebrate the 50th anniversary of the publication by Dr. Burg and his colleagues of their landmark paper in the American Journal of Physiology in 1966. In this study, we have taken a scientific visualization approach to study the scientific contributions of Dr. Burg and the isolated perfused tubule preparation as determining research impact by the number of research students, postdoctoral fellows, visiting scientists, and national and international collaborators. Additionally, we have examined the research collaborations (first and second generation scientists), established the migrational visualization of the first generation scientists who worked directly with Dr. Burg, quantified the metrics indices, identified and quantified the network of coauthorship of the first generation scientists with their second generation links, and determined the citations analyses of outputs of Dr. Burg and/or his first generation collaborators as coauthors. We also review the major advances in kidney physiology that have been made with the isolated perfused tubule technique. Finally, we are all waiting for the discoveries that the isolated perfused preparation technique will bring during the next 50 years. [ABSTRACT FROM AUTHOR]

The textbook account of whole body acid-base balance in terms of endogenous acid production, renal net acid excretion, and gastrointestinal alkali absorption, which is the only comprehensive model around, has never been applied in clinical practice or been formally validated. To improve understanding of acid-base modeling, we managed to write up this conventional model as an expression solely on urine chemistry. Renal net acid excretion and endogenous acid production were already formulated in terms of urine chemistry, and we could from the literature also see gastrointestinal alkali absorption in terms of urine excretions. With a few assumptions it was possible to see that this expression of net acid balance was arithmetically identical to minus urine charge, whereby under the development of acidosis, urine was predicted to acquire a net negative charge. The literature already mentions unexplained negative urine charges so we scrutinized a series of seminal papers and confirmed empirically the theoretical prediction that observed urine charge did acquire negative charge as acidosis developed. Hence, we can conclude that the conventional model is problematic since it predicts what is physiologically impossible. Therefore, we need a new model for whole body acid-base balance, which does not have impossible implications. Furthermore, new experimental studies are needed to account for charge imbalance in urine under development of acidosis. [ABSTRACT FROM AUTHOR]

It has been long known that the kidney plays an essential role in the control of body fluids and blood pressure and that impairment of renal function may lead to the development of diseases such as hypertension (Guyton AC, Coleman TG, Granger Annu Rev Physiol 34: 13-46, 1972). In this review, we highlight recent advances in our understanding of renal hemodynamics, obtained from experimental and theoretical studies. Some of these studies were published in response to a recent Call for Papers of this journal: Renal Hemodynamics: Integrating with the Nephron and Beyond. [ABSTRACT FROM AUTHOR]

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]

Editorial. Calls for an increase in manuscript submissions to the 'American Journal of Physiology--Renal Physiology.' Request to reduce submissions in translational research; Goals of the journal.

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]

Due to recent advances in high-throughput techniques, we and others have generated multiple proteomic and transcriptomic databases to describe and quantify gene expression, protein abundance, or cellular signaling on the scale of the whole genome/proteome in kidney cells. The existence of so much data from diverse sources raises the following question: “How can researchers find information efficiently for a given gene product over all of these data sets without searching each data set individually?” This is the type of problem that has motivated the “Big-Data” revolution in Data Science, which has driven progress in fields such as marketing. Here we present an online Big-Data tool called BIG (Biological Information Gatherer) that allows users to submit a single online query to obtain all relevant information from all indexed databases. BIG is accessible at http://big.nhlbi.nih.gov/. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

The Indiana O'Brien Center for Advanced Microscopic Analysis is a National Institutes of Health (NIH) P30-funded research center dedicated to the development and dissemination of advanced methods of optical microscopy to support renal researchers throughout the world. The Indiana O'Brien Center was founded in 2002 as an NIH P-50 project with the original goal of helping researchers realize the potential of intravital multiphoton microscopy as a tool for understanding renal physiology and pathophysiology. The center has since expanded into the development and implementation of large-scale, high-content tissue cytometry. The advanced imaging capabilities of the center are made available to renal researchers worldwide via collaborations and a unique fellowship program. Center outreach is accomplished through an enrichment core that oversees a seminar series, an informational website, and a biennial workshop featuring hands-on training from members of the Indiana O'Brien Center and imaging experts from around the world. [ABSTRACT FROM AUTHOR]

Injured tubule epithelium stimulates a profibrotic milieu that accelerates loss of function in chronic kidney disease (CKD). This study tested the role of signal transducer and activator of transcription 1 (STAT1) in the progressive loss of kidney function in aristolochic acid (AA) nephropathy, a model of CKD. Mean serum creatinine concentration increased in wild-type (WT) littermates treated with AA, whereas Stat1-/- mice were protected. Focal increases in the apical expression of kidney injury molecule (KIM)- 1 were observed in the proximal tubules of WT mice with AA treatment but were absent in Stat1-/- mice in the treatment group as well as in both control groups. A composite injury score, an indicator of proximal tubule injury, was reduced in Stat1-/- mice treated with AA. Increased expression of integrin-b6 and phosphorylated Smad2/3 in proximal tubules as well as interstitial collagen and fibronectin were observed in WT mice following AA treatment but were all decreased in AA-treated Stat1-/- mice. The data indicated that STAT1 activation facilitated the development of progressive kidney injury and interstitial fibrosis in AA nephropathy. [ABSTRACT FROM AUTHOR]

Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na+ reabsorption in the renal medulla. A link between the renal ET-1 and purinergic systems was demonstrated in vitro, however, the in vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na+ excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague-Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mosmol/kgH2O) was infused into the medullary interstitium (500 µl/h) during a 30-min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH2O) for two further 30-min urine collection periods. Na+ excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the P2 receptor antagonist suramin inhibited the increase in Na+ excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary P2Y2/4 receptors by infusion of UTP increased urinary Na+ excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary P2 receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load. [ABSTRACT FROM AUTHOR]

The thiazide-sensitive NaCl cotransporter (NCC) is an important pharmacological target in the treatment of hypertension. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms. Only the third isoform has been extensively investigated. The aim of the present study was, therefore, to establish the abundance and localization of the almost identical isoforms 1 and 2 (NCC1/2) in the human kidney and to determine their functional properties and regulation in physiological conditions. Immunohistochemical analysis of NCC1/2 in the human kidney revealed that NCC1/2 localizes to the apical plasma membrane of the distal convoluted tubule. Importantly, NCC1/2 mRNA constitutes ~44% of all NCC isoforms in the human kidney. Functional analysis performed in the Xenopus laevis oocyte revealed that thiazide-sensitive 22Na+ transport of NCC1 was significantly increased compared with NCC3. Mimicking a constitutively active phosphorylation site at residue 811 (S811D) in NCC1 further augmented Na+ transport, while a nonphosphorylatable variant (S811A) of NCC1 prevented this enhanced response. Analysis of human urinary exosomes demonstrated that water loading in human subjects significantly reduces the abundance of NCC1/2 in urinary exosomes. The present study highlights that previously underrepresented NCC1/2 is a fully functional thiazide-sensitive NaCl-transporting protein. Being significantly expressed in the kidney, it may constitute a unique route of renal NaCl reabsorption and could, therefore, play an important role in blood pressure regulation. [ABSTRACT FROM AUTHOR]

This study aims to understand the extent to which modulation of the Na+-K+-2Cl- cotransporter NKCC2 differential splicing affects NaCl delivery to the macula densa. NaCl absorption by the thick ascending limb and macula densa cells is mediated by apical NKCC2. A recent study has indicated that differential splicing of NKCC2 is modulated by dietary salt (Schiel IM, Rosenauer A, Kattler V, Minuth WW, Oppermann M, Castrop H. Am J Physiol Renal Physiol 305: F1139-F1148, 2013). Given the markedly different ion affinities of its splice variants, modulation of NKCC2 differential splicing is believed to impact NaCl reabsorption. To assess the validity of that hypothesis, we have developed a mathematical model of macula densa cell transport and incorporated that cell model into a previously applied model of the thick ascending limb (Weinstein AM, Krahn TA. Am J Physiol Renal Physiol 298: F525-F542, 2010). The macula densa model predicts a 27.4- and 13.1-mV depolarization of the basolateral membrane [as a surrogate for activation of tubuloglomerular feedback (TGF)] when luminal NaCl concentration is increased from 25 to 145 mM or luminal K concentration is increased from 1.5 to 3.5 mM, respectively, consistent with experimental measurements. Simulations indicate that with luminal solute concentrations consistent with in vivo conditions near the macula densa, NKCC2 operates near its equilibrium state. Results also suggest that modulation of NKCC2 differential splicing by low salt, which induces a shift from NKCC2-A to NKCC2-B primarily in the cortical thick ascending limb and macula densa cells, significantly enhances salt reabsorption in the thick limb and reduces Na+ and Cl- delivery to the macula densa by 3.7 and 12.5%, respectively. Simulation results also predict that the NKCC2 isoform shift hyperpolarizes the macula densa basolateral cell membrane, which, taken in isolation, may inhibit the release of the TGF signal. However, excessive early distal salt delivery and renal salt loss during a low-salt diet may be prevented by an asymmetric TGF response, which may be more sensitive to flow increases. [ABSTRACT FROM AUTHOR]

We have developed a whole kidney model of the urine concentrating mechanism and renal autoregulation. The model represents the tubuloglomerular feedback (TGF) and myogenic mechanisms, which together affect the resistance of the afferent arteriole and thus glomerular filtration rate. TGF is activated by fluctuations in macula densa [Cl-] and the myogefnic mechanism by changes in hydrostatic pressure. The model was used to investigate the relative contributions of medullary blood flow autoregulation and inhibition of transport in the proximal convoluted tubule to pressure natriuresis in both diuresis and antidiuresis. The model predicts that medullary blood flow autoregulation, which only affects the interstitial solute composition in the model, has negligible influence on the rate of NaCl excretion. However, it exerts a significant effect on urine flow, particularly in the antidiuretic kidney. This suggests that interstitial washout has significant implications for the maintenance of hydration status but little direct bearing on salt excretion, and that medullary blood flow may only play a signaling role for stimulating a pressure-natriuresis response. Inhibited reabsorption in the model proximal convoluted tubule is capable of driving pressure natriuresis when the known actions of vasopressin on the collecting duct epithelium are taken into account. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is characterized by loss of renal function. The pathological processes involved in the progression of this condition are already known, but the molecular mechanisms have not been completely explained. Recent reports have shown the intrinsic capacity of the kidney to undergo repair after acute injury through the reexpression of repairing proteins (Villanueva S, Cespedes C, Vio CP. Am J Physiol Regul Integr Comp Physiol 290: R861-R870, 2006). Stimulation with basic fibroblast growth factor (bFGF) could accelerate this process. However, it is not known whether bFGF can induce this phenomenon in kidney cells affected by CKD. Our aim was to study the evolution of renal damage in animals with CKD treated with bFGF and to relate the amount of repairing proteins with renal damage progression. Male Sprague-Dawley rats were subjected to 5/6 nephrectomy (NPX) and treated with bFGF (30 βg/kg, NPXbFGF); a control NPX group was treated with saline (NPX+S). Animals were euthanized 35 days after bFGF administration. Functional effects were assessed based on serum creatinine levels; morphological damage was assessed by the presence of macrophages (ED-1), interstitial α-smooth muscle actin (α-SMA), and interstitial collagen through Sirius red staining. The angiogenic factors VEGF and Tie-2 and the epithelial/tubular factors Ncam, bFGF, Pax-2, bone morphogenic protein-7, Noggin, Lim-1, Wnt-4, and Smads were analyzed. Renal stem cells were evaluated by Oct-4. We observed a significant reduction in serum creatinine levels, ED-1, α-SMA, and Sirius red as well as an important induction of Oct-4, angiogenic factors, and repairing proteins in NPX+bFGF animals compared with NPX+S animals. These results open new perspectives toward reducing damage progression in CKD. [ABSTRACT FROM AUTHOR]

For decades, the Brattleboro rat has been a useful model in kidney physiology. These animals manifest central diabetes insipidus (lack of circulating vasopressin) due to a mutation in the vasopressin-neurophysin gene. V2 receptor-mediated vasopressin actions in the kidney can be assessed in these animals by infusing the V2-selective vasopressin analog 1-desamino-8-D-arginine vasopressin (dDAVP). However, the major commercial supplier in the United States has ceased production, creating the need for another reliable experimental model of V2 receptor-mediated vasopressin action in rodents. We designed an in vivo protocol to investigate vasopressin responses in the rat kidney using osmotic minipumps loaded with tolvaptan, a nonpeptide competitive inhibitor of the vasopressin V2 receptor. Tolvaptan-infused rats had a mean urinary osmolality of 300 vs. 2,000 mosmol/ kgH2O in vehicle-infused rats. The tolvaptan infusion produced large decreases in the renal abundance of aquaporin-2 (AQP2), aquaporin-3 (AQP3), the subunit of the epithelial sodium channel (ENaC), and ENaC that were comparable to the differences seen in vehicleinfused vs. vasopressin-infused Brattleboro rats. Thus we conclude that tolvaptan infusion in rats provides an additional model (besides dDAVP-infusion in the Brattleboro rat) for the assessment of V2 receptor-mediated vasopressin actions in the kidney. We also provide ancillary in vitro data in rat inner-medullary-collecting-duct suspensions showing that tolvaptan can block vasopressin's effects on phosphorylation of the water channel AQP2 in vitro. Specifically, tolvaptan almost completely inhibited the ability of vasopressin to increase AQP2 phosphorylation at Ser256, Ser264, and Ser269, while strongly inhibiting a vasopressin-induced decrease in AQP2 phosphorylation at Ser261. [ABSTRACT FROM AUTHOR]

A recent study demonstrated that the dopamine D1 receptor (D1R) is nonfunctional in human kidney cells, HK2 cells, in terms of their inability to couple to Gs protein in response to the D1R agonist fenoldopam. Since D1R also couples to Gq protein, we tested whether D1R is functional in HK2 cells in terms of their ability to couple to Gq and produce downstream signaling. For comparison, we also studied another receptor, angiotensin II type 1 receptor (AT1R) known to couple to Gq. Protein kinase C (PKC) and 86rubidium transport activities were determined as surrogate downstream signaling markers. Fenoldopam and angiotensin II increased PKC activity, which decreased in the presence of respective receptor antagonists (SCH23390 for D1R; candesartan for AT1R), PKC (chelerythrine chloride) and Gi protein (pertussis toxin) inhibitors and Gq/11α siRNA. Furthermore, fenoldopam and angiotensin II increased 35S-GTPαS binding, an index of receptor-G protein coupling, which decreased with pertussis toxin and in Gq/11α-depleted cells. Also, fenoldopam-mediated inhibition of 86rubidium transport (an index of Na-K-ATPase activity) was attenuated with SCH23390, chelerythrine chloride, pertussis toxin, and Gq/11α siRNA. Moreover, fenoldopam caused a decrease in cytosolic and increase in membranous abundance of Gq/11α. The immunoprecipitated levels of Gq/11α in the membranes were greater in fenoldopam-treated cells, and Giα coimmunoprecipitated with Gq/11α. Our results suggest that both D1R and AT1R are functional in HK2 cells, enabling Gq-mediated downstream signaling in a Gi dependent manner. [ABSTRACT FROM AUTHOR]

Mitochondria are both a source and target of the actions of reactive oxygen species and possess a complex system of inter-related antioxidants that control redox signaling and protect against oxidative stress. Interestingly, the antioxidant enzyme heme oxygenase-1 (HO-1) is not present in the mitochondria despite the fact that the organelle is the site of heme synthesis and contains multiple heme proteins. Detoxification of heme is an important protective mechanism since the reaction of heme with hydrogen peroxide generates pro-oxidant ferryl species capable of propagating oxidative stress and ultimately cell death. We therefore hypothesized that a mitochondrially localized HO-1 would be cytoprotective. To test this, we generated a mitochondria-targeted HO-1 cell line by transfecting HEK293 cells with a plasmid construct containing the manganese superoxide dismutase mitochondria leader sequence fused to HO-1 cDNA (Mito-HO-1). Nontargeted HO-1- overexpressing cells were generated by transfecting HO-1 cDNA (HO-1) or empty vector (Vector). Mitochondrial localization of HO-1 with increased HO activity in the mitochondrial fraction of Mito- HO-1 cells was observed, but a significant decrease in the expression of heme-containing proteins occurred in these cells. Both cytosolic HO-1- and Mito-HO-1-expressing cells were protected against hypoxiadependent cell death and loss of mitochondrial membrane potential, but these effects were more pronounced with Mito-HO-1. Furthermore, decrement in production of tricarboxylic acid cycle intermediates following hypoxia was significantly mitigated in Mito-HO-1 cells. These data suggest that specific mitochondrially targeted HO-1 under acute pathological conditions may have beneficial effects, but the selective advantage of long-term expression is constrained by a negative impact on the synthesis of heme-containing mitochondrial proteins. [ABSTRACT FROM AUTHOR]

The thick ascending limb (TAL) is a major NaCl reabsorbing site in the nephron. Efficient reabsorption along that segment is thought to be a consequence of the establishment of a strong transepithelial potential that drives paracellular Na+ uptake. We used a multicell mathematical model of the TAL to estimate the efficiency of Na+ transport along the TAL and to examine factors that determine transport efficiency, given the condition that TAL outflow must be adequately dilute. The TAL model consists of a series of epithelial cell models that represent all major solutes and transport pathways. Model equations describe luminal flows, based on mass conservation and electroneutrality constraints. Empirical descriptions of cell volume regulation (CVR) and pH control were implemented, together with the tubuloglomerular feedback (TGF) system. Transport efficiency was calculated as the ratio of total net Na+ transport (i.e., paracellular and transcellular transport) to transcellular Na+ transport. Model predictions suggest that 1) the transepithelial Na+ concentration gradient is a major determinant of transport efficiency; 2) CVR in individual cells influences the distribution of net Na+ transport along the TAL; 3) CVR responses in conjunction with TGF maintain luminal Na+ concentration well above static head levels in the cortical TAL, thereby preventing large decreases in transport efficiency; and 4) under the condition that the distribution of Na+ transport along the TAL is quasi-uniform, the tubular fluid axial Cl- concentration gradient near the macula densa is sufficiently steep to yield a TGF gain consistent with experimental data. [ABSTRACT FROM AUTHOR]

Glutamatergic pathways mediate transmission of pain. Strategies to reduce glutamatergic neurotransmission may have beneficial effects to mitigate nociception. Recent work revealed that overexpression of the astrocytic glutamate transporter (GLT-1) by transgenic or pharmacologic approaches produced a diminished visceral nociceptive response to colonic distension. The purpose of this study was to determine the effect of GLT-1 overexpression on the visceromotor response to bladder distension. Increased glutamate uptake activity produced by 1-wk ceftriaxone (CTX) treatment attenuated 60-64% the visceromotor response to graded bladder distension compared with vehicle-treated mice. One-hour pretreatment with selective GLT-1 antagonist dihydrokainate reversed the blunted visceromotor response to bladder distension produced by 1-wk CTX, suggesting that GLT-1 overexpression mediated the analgesic effect of CTX. Moreover, sensitization of the visceromotor response to bladder distension produced by local bladder irritation (acrolein) was also attenuated by 1-wk CTX treatment. A model of cross-organ sensitization of bladder visceromotor response to distension was next studied to determine whether increased expression of GLT-1 can mitigate colon to bladder sensitization. Intracolonic trinitrobenzene sulfonic acid (TNBS) administered 1 h before eliciting the visceromotor response to graded bladder distension produced a 75- 138% increase in visceromotor response compared with animals receiving intracolonic vehicle. In marked contrast, animals treated with 1-wk CTX + intracolonic TNBS showed no enhanced viscero-motor response compared with the 1-wk vehicle + intracolonic vehicle group. The study suggests that GLT-1 overexpression attenuates the visceromotor response to bladder distension and both local irritant-induced and cross-organ-sensitized visceromotor response to bladder distension. [ABSTRACT FROM AUTHOR]

Renal Na+ and K+ excretion was measured in rats with varying dietary K+ intake. The requirement for channel-mediated distal nephron Na+ reabsorption was assessed by infusing the animals with the K+-sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, the concentration of amiloride in the urine was 38 μM, corresponding to concentrations of 9-23 μM in the distal tubular fluid, sufficient to block >98% of Na+ transport through apical Na+ channels (ENaC). With a control K+ intake (0.6% KC1), amiloride reduced K+ excretion rates (UKV) from 0.85 ± 0.15 to 0.05 ± 0.01 μmol/min during the first 2 h of infusion, suggesting that distal nephron K+ secretion was completely dependent on the activity of Na+ channels. When K+ intake was increased by feeding overnight with a diet containing 10% KCI, amiloride reduced UKV from 7.5 ± 0.7 to 1.3 ± 0.1 μmol/min despite an increased plasma K+ of 9 mM, again suggesting a major but not exclusive role for the Na+ channel-dependent pathway of K+ secretion. The maximal measured rates of amiloride-sensitive K+ excretion correspond well with estimates based on apical K+ channel activity in distal nephron segments. However, when the animals were adapted to the high-K+ diet for 7-9 days, the diuretic decreased UKV less, from 6.1 ± 0.6 to 3.0 ± 0.8 μmol/min, indicating an increasing fraction of K+ excretion that was independent of Na+ channels. This indicates the upregulation of a Na+ channel-independent mechanism for secreting K+. [ABSTRACT FROM AUTHOR]

In the companion study (Am J Physiol Renal Physiol. First published April 29, 2009; doi: 10.11 52/ajprenal.90496.2008), we extended the regionbased mathematical model of the urine-concentrating mechanism in the rat outer medulla (OM) developed by Layton and Layton (Am J Physiol Renal Physiol 289: F1346-F1366, 2005) to examine the impact of the complex structural organization of the OM on O2 transport and distribution. In the present study, we investigated the sensitivity of predicted Po2 profiles to several parameters that characterize the degree of OM regionalization, boundary conditions, structural dimensions, transmural transport properties, and relative positions and distributions of tubules and vessels. Our results suggest that the fraction of O2 supplied to descending vasa recta (DVR) that reaches the inner medulla, i.e., a measure of the axial Po2 gradient in the OM, is insensitive to parameter variations as a result of the sequestration of long DVR in the vascular bundles. In contrast, O2 distribution among the regions surrounding the vascular core strongly depends on the radial positions of medullary thick ascending limbs (mTALs) relative to the vascular core, the degree of regionalization, and the distribution of short DVR along the corticomedullary axis. Moreover, if it is assumed that the mTAL active Na+ transport rate decreases when mTAL Po2 falls below a critical level, O2 availability to mTALs has a significant impact on the concentrating capability of the model OM. The model also predicts that when the OM undergoes hypertrophy, its concentrating capability increases significantly only when anaerobic metabolism supports a substantial fraction of the mTAL active Na+ transport and is otherwise critically reduced by low interstitial and mTAL luminal Po2 in a hypertrophied OM. [ABSTRACT FROM AUTHOR]

The mechanism of albuminuria is perhaps one of the most complex yet important questions in renal physiology today. Recent studies have directly demonstrated that the normal glomerulus filters substantial amounts of albumin and that charge selectivity plays little or no role in preventing this process. This filtered albumin is then processed by proximal tubular cells by two distinct pathways; dysfunction in either one of these pathways gives rise to discrete forms of albuminuria. Most of the filtered albumin is returned to the peritubular blood supply by a retrieval pathway. Albuminuria in the nephrotic range would arise from retrieval pathway dysfunction. The small quantities of filtered albumin that are not retrieved undergo obligatory lysosomal degradation before urinary excretion as small peptide fragments. This degradation pathway is sensitive to metabolic factors responsible for hypertrophy and fibrosis, particularly molecules such as angiotensin II and transforming growth factor-β1, whose production is stimulated by hyperglycemic and hypertensive environments. Dysfunction in this degradation pathway leads to albuminuria below the nephrotic range. These new insights into albumin filtration and processing argue for a reassessment of the role of podocytes and the slit diaphragm as major direct determinants governing albuminuria, provide information on how glomerular morphology and "tubular" albuminuria may be interrelated, and offer a new rationale for drug development. [ABSTRACT FROM AUTHOR]

Although the concentrating process in the renal outer medulla is well understood, the concentrating mechanism in the renal inner medulla remains an enigma. The purposes of this review are fourfold. 1) We summarize a theoretical basis for classifying all possible steady-state inner medullary countercurrent concentrating mechanisms based on mass balance principles. 2) We review the major hypotheses that have been proposed to explain the axial osmolality gradient in the interstitium of the renal inner medulla. 3) We summarize and expand on the SchmidtNielsen hypothesis that the contractions of the renal pelvocalyceal wall may provide an important energy source for concentration in the inner medulla. 4) We discuss the special properties of hyaluronan, a glycosaminoglycan that is the chief component of a gel-like renal inner medullary interstitial matrix, which may allow it to function as a mechanoosmotic transducer, converting energy from the contractions of the pelvic wall to an axial osmolality gradient in the medulla. These considerations set the stage for renewed experimental investigation of the urinary concentrating process and a new generation of mathematical models of the renal concentrating mechanism, which treat the inner medullary interstitium as a viscoelastic system rather than a purely hydraulic system. [ABSTRACT FROM AUTHOR]

We used a mathematical model to explore the possibility that metabolic production of net osmoles in the renal inner medulla (IM) may participate in the urine-concentrating mechanism. Anaerobic glycolysis (AG) is an important source of energy for cells of the IM, because this region of the kidney is hypoxic. AG is also a source of net osmoles, because it splits each glucose into two lactate molecules, which are not metabolized within the IM. Furthermore, these sugars exert their full osmotic effect across the epithelia of the thin descending limb of Henle's loop and the collecting duct, so they are apt to fulfill the external osmole role previously attributed to interstitial urea (whose role is compromised by the high urea permeability of long descending limbs). The present simulations show that physiological levels of IM glycolytic lactate production could suffice to significantly amplify the IM accumulation of NaCl. The model predicts that for this to be effective, IM lactate recycling must be efficient, which requires high lactate permeability of descending vasa recta and reduced IM blood flow during antidiuresis, two conditions that are probably fulfilled under normal circumstances. The simulations also suggest that the resulting IM osmotic gradient is virtually insensitive to the urea permeability of long descending limbs, thus lifting a longstanding paradox, and that this high urea permeability may serve for independent regulation of urea balance. [ABSTRACT FROM AUTHOR]

An ATP-regulated inwardly rectifying K[sup +] channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K[sup +] channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 µM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5523 (1 @M), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKAspecific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 µM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 riM) in cell-attached patches stimuluted channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K[sup +] channel and producing the stimulatory effect of ANP on channel activity. [ABSTRACT FROM AUTHOR]

Transcriptional repression is a key mechanism for the spatial specification of gene expression and cell fate determination. During kidney development, proliferating cell nuclear antigen (PCNA) is expressed in the nephrogenic zone and is downregulated rapidly as renal epithelial cells enter terminal differentiation and acquire functional characteristics. Our laboratory reported that the transcription factor p53 stimulates the terminal differentiation of renal epithelial cells by means of transcriptional activation of renal function genes (Saifudeen Z, Dipp S, and El-Dahr SS. J Clin Invest 109: 1021-1030, 2002). Because p53-induced growth arrest correlates with downregulation of PCNA gene expression, we examined the impact of p53 inactivation on PCNA expression in mice and evaluated the effect of p53 on PCNA transcription. Immunohistochemistry revealed that the transition from nephrogenesis to terminal epithelial cell differentiation correlates with accumulation of the transcription factor p53. Importantly, the spatially restricted pattern of PCNA expression is disrupted in kidneys of p53-deficient pups, in which there was a redistribution of PCNA expression into the differentiation zone (without a change in total kidney PCNA content) and distortion of the tubular architecture. Electrophoretic mobility shift assays revealed that the binding of kidney nuclear extracts to the p53 response elements in human and rat PCNA promoters is developmentally regulated. Transient transfection assays performed in p53-deficient HeLa cells revealed that exogenous p53 strongly represses transcription from human PCNA promoter-reporter constructs. Interestingly, deletion of the p53-binding site confers enhanced responsiveness to p53mediated repression, suggesting that transcriptional repression of PCNA by p53 is achieved by a mechanism other than direct DNA binding. On the basis of these results, we propose the hypothesis that p53-mediated transcriptional repression plays a role in... [ABSTRACT FROM AUTHOR]

Determines whether chronic metabolic acidosis (CMA) affects the abundance of Na-HCO cotransporters (NBCs) in kidneys using two conventional protocols. Upgregulation of NBCn1 and NBC3 with no change in the segmental distribution along the nephron; Association of CMA with a marked increase in the abundance of NBCn1 in the medullary thick ascending limb and NBC3 in intercalated cells.

Studies whether hypertension could be controlled in genetically hypertensive rats by the administration of the immunosuppressive drug mycophenolate mofetil (MMF). Reduction of systemic hypertension to normal levels during MMF treatment; Decrease in lymphocyte, macrophage, and angiotensin II-positive cells infiltrating the kidney; Decline in oxidative stress.

Examines the tetracycline-inducible gene expression in renal collecting duct (CD) cells from transgenic mice. Role of renal CD in the control of ion and fluid homeostasis; Description of genetic diseases involving gene mutations for ion transport or hormone receptors expressed in CD; Development of a CD cell line using a tetracycline-inducible system.

Determines the localization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR analyses. Use of peptide-derived antibodies to AQP8; Presence of AQP8 messenger RNA in all kidney zones; Exhibition of strong labeling of affinity-purified antibodies by AQP8.

Presents a study which examined the expression of gamma in rat kidney sections to sodium-potassium-ATPase alpha1-, beta1-, and gamma-subunits. Materials and methods; Results; Discussion.

Presents information on a study which investigated the pathogenesis of impaired urinary concentrating ability of chloride channel mice kidney. Methodology; Results; Discussion.

Presents information on a study which identified cellular and molecular mechanisms that underlie the effects of bone morphogenetic proteins in the embryonic kidney. Methodology; Results of the study; Discussion.

Presents information on a study which identified and characterized the podocyte-specific elements of the nephrin promoter. Discussion on podocytes; Methodology; Results.

Discusses a study which determined the cellular and subcellular localization of sodium bicarbonate in the rat kidney. Methods; Results; Discussion.

Focuses on a study which examined the peptide transport activity in renal basolateral membranes. Materials and methods; Results; Discussion.

Focuses on a study which examined the role of CLC chloride channels in the kidney. CLC chloride channels; Molecular identification and intrarenal and cellular localization; Mutations in human kidney diseases.

Focuses on a study which demonstrated the localization of organic cation transporters at the basolateral membrane of renal proximal tubules. Methods; Results; Discussion.

Discusses a study which investigated the effects of high glucose and glucosamine on the synthesis of the extracellular matrix protein laminin in the kidney. Materials and methods; Results; Discussion.

Presents information on a study which identified and localized an adenosine triphosphate diphosphohydrolase (ATPDase) in porcine kidney and established its identity to CD39. Materials and methods; Identification of the ATPDase with monoclonal antibodies; Characterization of the ATPDase; Immune and enzyme histochemical localization by light and electron microscopy.