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Calcium oxalate (CaOx) kidney stones accumulate within the renal tubule due to high concentrations of insoluble deposits in the urine. Pb 2+ -induced Ca 2+ mobilization along with Pb 2+ -induced nephrotoxic effects within the proximal tubule have been well established; however, Pb 2+ mediated effects within the collecting duct remains insufficiently studied. Thus in vitro and ex vivo model systems were treated with increasing concentrations of lead (II) acetate (PbAc) ± sodium oxalate (Na 2 C 2 O 4 ) for 1 h, both individually and in combination. Pb 2+ -mediated solution turbidity increased 2 to 5 times greater post-exposure to 75, 100 and 200 μM Pb 2+ with the additional co-treatment of 10 mM oxalate in mouse inner medullary collecting duct (mIMCD-3) cells. Additionally, 100 μM and 200 μM Pb 2+ alone induced significant levels of intracellular Ca 2+ release. To validate Pb 2+ -mediated effects on the formation of CaOx crystals, alizarin red staining confirmed the presence of CaOx crystallization. Pb 2+ -induced intracellular Ca 2+ was also observed ex vivo in fly Malpighian tubules with significant increases in CaOx crystal formation via Pb 2+ -induced intracellular Ca 2+ release significantly increasing the average crystal number, size, and total area of crystal formation, which was ameliorated by tissue-specific SPoCk C transporter and Capa receptor knockdown. These studies demonstrate Pb 2+ -induced Ca 2+ release likely increases the formation of CaOx crystals, which is modulated by a G q -linked mechanism with concurrent Ca 2+ extracellular mobilization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Greg M. Landry reports financial support was provided by American Association of Colleges of Pharmacy. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Although the concept of the intrarenal renin-angiotensin system (RAS) in renal disease is well-described in the literature, the precise pathogenic role and mechanism of this local system have not been directly assessed in the absence of confounding influence from the systemic RAS. The present study used novel mouse models of collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) or renin together with pharmacological inhibition of soluble PRR production to unravel the precise contribution of the intrarenal RAS to renal injury induced by unilateral ureteral obstruction., Methods: We examined the impact of CD-specific deletion of PRR, CD-specific deletion of renin, and S1P (site-1 protease) inhibitor PF429242 treatment on renal fibrosis and inflammation and the indices of the intrarenal RAS in a mouse model of unilateral ureteral obstruction., Results: After 3 days of unilateral ureteral obstruction, the indices of the intrarenal RAS including the renal medullary renin content, activity and mRNA expression, and Ang (angiotensin) II content in obstructed kidneys of floxed mice were all increased. That effect was reversed with CD-specific deletion of PRR, CD-specific deletion of renin, and PF429242 treatment, accompanied by consistent improvement in renal fibrosis and inflammation. On the other hand, renal cortical renin levels were unaffected by unilateral ureteral obstruction, irrespective of the genotype. Similar results were obtained via pharmacological inhibition of S1P, the key protease for the generation of soluble PRR., Conclusions: Our results reveal that PRR-dependent/soluble PRR-dependent activation of CD renin represents a key determinant of the intrarenal RAS and, thus, obstruction-induced renal inflammation and fibrosis., Competing Interests: None.

Endoscopic and biopsy findings have identified two distinct phenotypes among individuals with calcium oxalate (CaOx) kidney stones. The first type has normal renal papillae but shows interstitial mineral deposition, known as Randall's plaque. The other phenotype presents with collecting duct plugging and a higher incidence of loss of papilla tissue mass. With Randall's plaque, renal papilla injury involves the loss of small patches of calcified tissue (Randall's plaque detaching with the stone), which likely results in damage to only a few nephrons. In contrast, collecting duct mineral plugs are very large, causing obstruction to tubular flow. Since each terminal collecting duct drains thousands of nephrons, ductal plugs could lead to the degeneration of many nephrons and a significant loss of renal glomeruli. New visualization techniques for immune cells in papillary biopsies have revealed that the Randall's plaque phenotype is marked by the accumulation of macrophages around the plaque regions. In contrast, preliminary data on the plugging phenotype shows collecting duct damage with mineral plugs and increased T-lymphocytes throughout the papilla. These regions also show tubulitis, i.e., T-cell infiltration into nearby collecting duct epithelium. This suggests that while some CaOx stone formers may have some papillary inflammation but with minimal damage to nephrons, others suffer from obstruction to flow for many nephrons that may also include destructive inflammation in the renal tissue. We propose that CaOx stone formers with the plugging phenotype will have a higher long-term risk for loss of renal function., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lithium induces nephrogenic diabetes insipidus (NDI) and microcystic chronic kidney disease (CKD). As previous clinical studies suggest that NDI is dose-dependent and CKD is time-dependent, we investigated the effect of low exposition to lithium in a long-term experimental rat model. Rats were fed with a normal diet (control group), with the addition of lithium (Li + group), or with lithium and amiloride (Li + /Ami group) for 6 months, allowing obtaining low plasma lithium concentrations (0.25 ± 0.06 and 0.43 ± 0.16 mmol/L, respectively). Exposition to low concentrations of plasma lithium levels prevented NDI but not microcystic dilations of kidney tubules, which were identified as collecting ducts (CDs) on immunofluorescent staining. Both hypertrophy, characterized by an increase in the ratio of nuclei per tubular area, and microcystic dilations were observed. The ratio between principal cells and intercalated cells was higher in microcystic than in hypertrophied tubules. There was no correlation between AQP2 messenger RNA levels and cellular remodeling of the CD. Additional amiloride treatment in the Li + /Ami group did not allow consistent morphometric and cellular composition changes compared to the Li + group. Low exposition to lithium prevented overt NDI but not microcystic dilations of the CD, with differential cellular composition in hypertrophied and microcystic CDs, suggesting different underlying cellular mechanisms., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Background: The literature on nephron-sparing surgery (NSS) in children with bilateral Wilms' tumors (BWT) involving the collection system is mostly comprised of case reports. The present study aimed to summarize the clinical characteristics, treatments, and prognosis of children with BWT involving the collecting system admitted to our pediatric surgery center compared with those whose tumors did not involve the collecting system. A secondary aim was to discuss how to preserve more kidney parenchyma and prevent long-term renal failure under the premise of preventing tumor recurrence., Methods: Patients with BWT admitted to our pediatric surgery center between January 2008 and June 2022 were reviewed. All included patients were grouped according to the relationship between the tumor and collecting system according to the intraoperative findings. Group I included children with tumor infiltrating the collecting system, group II included children with tumor growing into the collecting system, and group III included children whose tumor did not involve the collecting system. The clinical features, treatments and prognosis of the patients were analyzed., Results: Seventy patients were enrolled, including 20 patients with 25 sides of tumors infiltrating the collecting system in group I,10 patients with 13 sides of tumors growing into the collecting system in group II, and 40 patients in group III. There was no significant difference in patients age and gender between group I and group II. In total, 20 patients in group I and 9 patients in group II had partial response (PR) after neoadjuvant chemotherapy. In group I, 22 of 25 sides of tumors underwent NSS; in group II, 11 of 13 sides of tumors underwent NSS. During an average follow-up of 47 months, in group I, 6/20 patients relapsed and 2/20 patients died; in group II, 3/10 patients relapsed and 1/10 patient died. There was no significant difference in 4-year overall survival (OS) rate among groups I, II and III (86.36% vs. 85.71%vs. 91.40%, P = 0.902)., Conclusions: To preserve renal parenchyma, NSS is feasible for children with BWT involving the collecting system. There was no significant difference in postoperative long-term OS between patients with BWT involving the collecting system and not involving the collecting system., (© 2024. The Author(s).)

The histomorphological features of normal kidneys in cats and dogs have been revealed despite the high susceptibility of cats to tubulointerstitial damage. Herein, the histological characteristics of the two species were compared. Cytoplasmic lipid droplets (LDs) were abundant in the proximal convoluted tubules (PCTs) of cats aged 23-27 months but scarce in dogs aged 24-27 months. LDs were rarely observed in the distal tubules (DTs) and collecting ducts (CDs) of either species, as visualized by the expression of Tamm-Horsfall protein 1, calbindin-D28K, and aquaporin 2. The occupational area ratio of proximal tubules (PTs) in the renal cortex was higher, but that of DTs or CDs was significantly lower in adult cats than in dogs. Single PT epithelial cells were larger, but PCT, DT, and CD lumens were significantly narrower in adult cats than in dogs. Unlike adults, young cats at 6 months exhibited significantly abundant cytoplasmic LDs in proximal straight tubules, indicating lipid metabolism-related development. Histochemistry of the 21 lectins also revealed variations in glycosylation across different renal tubules and CDs in both species. Sodium-glucose cotransporter 2 was expressed only in PTs, excluding the proximal straight tubules with few LDs in adult cats or the PCTs of young cats and adult dogs. These findings are crucial for understanding species-specific characteristics of renal histomorphology and pathogenesis., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright: © 2024 Kira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-β (TGF-β) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-β, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague-Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-β, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-β, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

Bisphenol A (BisPH-A) is a latent danger that threatens our health, which we frequently exposure in our modern life (e.g. the widespread use of drinking water in plastic pet bottles). But the BisPH-A induced transient receptor potential melastatin 2 (TRPM2)-mediated oxidative stress and apoptosis in these cells has not been studied yet. Calcium (Ca 2+ ) plays an important role in a versatile intracellular signal transduction that works over a wide range to regulate oxidative stress processes. TRPM2 is activated by oxidative stress and it has emerged as an important Ca 2+ signaling mechanism in a variety of cells, contributing many cellular functions including cell death. Resveratrol (RESV), which belongs to the polyphenol group, acts as an antioxidant, eliminating cellular oxidative stress and increasing the body's resistance to diseases. The current study aimed to elucidate the effect of antioxidant resveratrol on TRPM2-mediated oxidative stress induced by BisPH-A exposure in the mouse kidney cortical collecting duct cells (mpkCCD cl4 ). The cells were divided into four groups as control, resveratrol (50 µM for 24 h), BisPH-A (100 µM for 24 h) and BisPH-A + RESV. Intracellular free Ca 2+ concentrations and TRPM2 channel currents were high in BisPH-A treated cells, but decreased with resveratrol treatment. In addition, BisPH-A induced mitochondrial membrane depolarization, reactive oxygen species (ROS), caspase 3, caspase 9 and apoptosis values were decreased by the resveratrol treatment. In conclusion, resveratrol protected cells from BisPH-A induced oxidative damage. In this study, we showed that TRPM2 channel mediates this protective effect of resveratrol.

Background: It is believed that tubulo-interstitial fibrosis and atrophy in diabetic patients are directly associated with the progression of chronic kidney disease, CKD. AIF is one of the crucial factors responsible for mitochondrial apoptosis, however, it can also promote cell survival independently from its role in apoptosis, and therefore can be potentially used as a tool in prevention of the onset of CKD in diabetic patients. Our aim was to investigate the significance of AIF expression in the development of CKD by observing the expression of AIF in 2 weeks' and 2 months' kidneys of diabetic rats compared to their controls., Methods: Male Sprague-Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). After 2 weeks and 2 months kidney samples were collected and analysed in different renal areas., Results: Characteristic morphologic changes were found between the 2 months' control and 2 months' diabetic groups. Those changes, including fibrosis and possible replacement of podocytes with connective tissue were mainly present in the glomeruli. AIF expression was seen in the both cortex, and in the collecting ducts of the medulla. Strong intensity of AIF expression was seen in proximal and distal convoluted tubules in both diabetic groups. In the control groups the glomeruli showed no AIF staining but moderate staining was seen in both diabetic groups. Overall, the percentage of AIF positive cells in the glomeruli was the lowest. The greatest rise in cell positivity was displayed from the 2 weeks' control group to 2 weeks' diabetes group (38 %) in glomeruli. The cell positivity of the 2 weeks' diabetic group is significantly reduced to 18 % in the 2 months' diabetic group in glomeruli. A similar pattern was seen in the proximal tubular cells (92 % positivity 2 weeks diabetic groups; 89 % positivity 2 months diabetic groups), as well as in the distal tubules. The highest percentage of AIF positive cells was seen in the collecting ducts, more than 80 % in all groups., Conclusions: Our study provides insight into AIF expression pattern during short term diabetes model, confirming possible dual role of AIF, not only in apoptosis but also in cell function and homeostasis, and proving AIF as potential therapeutic target and marker of advancement of CKD., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals., Methods: Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined., Results: Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3 -/- mice significantly attenuated gentamicin-induced AKI., Conclusions: A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis., (Copyright © 2020 by the American Society of Nephrology.)

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2 -/- and FFA3 -/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lithium is widely used in psychiatry as the golden standard for more than 60 yr due to its effectiveness. However, its adverse effect has been limiting its long-term use in clinic. About 40% of patients taking lithium develop nephrogenic diabetes insipidus (NDI). Lithium can also induce proliferation of collecting duct cells, leading to microcyst formation in the kidney. Lithium was considered an autophagy inducer that might contribute to the therapeutic benefit of neuropsychiatric disorders. Thus, we hypothesized that autophagy may play a role in lithium-induced kidney nephrotoxicity. To address our hypothesis, we fed mice with a lithium-containing diet with chloroquine (CQ), an autophagy inhibitor, concurrently. Lithium-treated mice presented enhanced autophagy activity in the kidney cortex and medulla. CQ treatment significantly ameliorated lithium-induced polyuria, polydipsia, natriuresis, and kaliuresis accompanied with attenuated downregulation of aquaporin-2 and Na + -K + -2Cl - cotransporter protein. The protective effect of CQ on aquaporin-2 protein abundance was confirmed in cultured cortical collecting duct cells. In addition, we found that lithium-induced proliferation of collecting duct cells was also suppressed by CQ as detected by proliferating cell nuclear antigen staining. Moreover, both phosphorylated mammalian target of rapamycin and β-catenin expression, which have been reported to be increased by lithium and associated with cell proliferation, were reduced by CQ. Taken together, our study demonstrated that CQ protected against lithium-induced NDI and collecting duct cell proliferation possibly through inhibiting autophagy.

Aim: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1 -/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium., Methods: In Epac1 -/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [ 14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin., Results: Epac1 -/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1 -/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1 -/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide., Conclusion: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs., (© 2020 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Potassium depletion affects AQP2 expression and the cellular composition of the kidney collecting duct. This, in turn, contributes to the development of a secondary form of nephrogenic diabetes insipidus and hypokalemic nephropathy. Here we show that after 14 days of potassium depletion, the cellular fraction of A-type intercalated cells increases while the fraction of principal cells decreases along the outer medullary collecting duct in rats. The intercalated cells acquired a novel distribution pattern forming rows of cells attached to each other. These morphological changes occur progressively and reverse after 7 days of recovery on normal rat chow diet. The cellular remodeling mainly occurred in the inner stripe of outer medulla similar to the previously seen effect of lithium on the collecting duct cellular profile. The cellular remodeling is associated with the appearance of cells double labelled with both specific markers of principal and type-A intercalated cells. The appearance of this cell type was associated with the downregulation of the Notch signaling via the Hes1 pathways. These results show that the epithelium of the collecting duct has a high degree of plasticity and that Notch signaling likely plays a key role during hypokalemia.

The water channel aquaporin 2 (AQP2) is responsible for water reabsorption by kidney collecting duct cells. A substitution of amino acid leucine 137 to proline in AQP2 (AQP2-L137P) causes Nephrogenic Diabetes Insipidus (NDI). This study aimed to determine the cell biological consequences of this mutation on AQP2 function. Studies were performed in HEK293 and MDCK type I cells, transfected with wildtype (WT) AQP2 or an AQP2-L137P mutant. AQP2-L137P was predominantly detected as a high-mannose form of AQP2, whereas AQP2-WT was observed in both non-glycosylated and complex glycosylated forms. In contrast to AQP2-WT, the AQP2-L137P mutant did not accumulate on the apical plasma membrane following stimulation with forskolin. Ubiquitylation of AQP2-L137P was different from AQP2-WT, with predominance of non-distinct protein bands at various molecular weights. The AQP2-L137P mutant displayed reduced half-life compared to AQP2-WT. Treatment of cells with chloroquine increased abundance of AQP2-WT, but not AQP2-L137P. In contrast, treatment with MG132 increased abundance of AQP2-L137P but not AQP2-WT. Xenopus oocytes injected with AQP2-WT had increased osmotic water permeability when compared to AQP2-L137P, which correlated with lack of the mutant form in the plasma membrane. From the localization of the mutation and nature of the substitution it is likely that AQP2-L137P causes protein misfolding, which may be responsible for the observed functional defects. The data suggest that the L137P mutation results in altered AQP2 protein maturation, increased AQP2 degradation via the proteasomal pathway and limited plasma membrane expression. These combined mechanisms are likely responsible for the phenotype observed in this class of NDI patients.

Although anomalies of renal vessels and collecting system are relatively frequent, their concomitant occurrence is a rare event. During dissection of a 75-year-old male formalin-embalmed cadaver, we found multiple variations in the renal vessels and renal collecting system. Both kidneys were normal in size and anteriorly malrotated, with duplex collecting system and duplex ureter. One ureter drained the upper part of the kidney and the second ureter drained the lower part of the kidney. Superior and inferior collecting systems were separated by renal parenchyma. The right kidney had two renal arteries, the first renal artery (main renal artery) originating from the abdominal aorta, passing behind the inferior vena cava (IVC) and entering the kidney through the superior and inferior renal hilum. The second artery was the inferior polar artery. In addition, the right kidney had two renal veins as well. Three renal tributaries emerged from the upper and lower portion of the right renal hilum, and they joined to form the main renal vein which drained into the IVC. The lower renal vein was the inferior polar vein. The left kidney had four renal arteries (two hilar arteries and two polar arteries). The main left renal vein emerged from both superior and inferior left renal hilum, passed in front of the abdominal aorta and drained into the IVC. The left kidney also had the inferior polar vein which was divided behind the aorta (retro aortic vein) into two venous trunks. These venous trunks drained separately into posteromedial aspect of the IVC. Finally, the right testicular vein was formed by two tributaries and drained into the IVC, whereas the two left testicular veins drained separately into the left main renal vein.

Diabetes mellitus and its complications have become a major health concern in Western countries. Increased activity of the intrarenal renin-angiotensin system (RAS) contributes to diabetic nephropathy (DN). We previously reported that in mesangial cells, the high glucose concentration (HG) leads to upregulation of angiotensin-converting enzyme (ACE) messenger RNA, suggesting that ACE was modulated by angiotensin II (Ang II) release. However, this relation in the collecting duct has not yet been studied. We, therefore, aimed to evaluate RAS modulation in inner medullary collecting duct cells (IMCD) exposed to HG. The IMCD were divided into normal glucose (5 mM D-glucose, NG), high glucose (30 mM, HG), and mannitol (30 mM, M) groups. The cells were cultured 48 hr in their respective media. The intracellular and extracellular ACE activity was measured using hippuryl-His-Leu as substrate via a fluorimetric assay and expression was analyzed using western blot analysis. ACE activity, intracellular (27%) and extracellular (22%), was significantly lower in the HG group than in NG and M. ACE2 activity and Ang 1-7 levels were higher in the intracellular compartment. Our data suggest that the HG cannot modify ACE synthesis in IMCD cells but can modulate its activity. The decrease in ACE activity may result in decreased levels of Ang II to protect the IMCD against proliferative and inflammatory deleterious effects of this peptide. Conversely, the increase of ACE2 generating high levels of Ang 1-7, a vasodilator peptide, suggesting that this peptide can induce glucose uptake and protect cells against oxidative stress, which can elicit insulin resistance., (© 2019 Wiley Periodicals, Inc.)

Background: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied., Methods: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown., Results: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF- β signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice., Conclusions: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney., (Copyright © 2019 by the American Society of Nephrology.)

Renal ammonia excretion is a critical component of acid-base homeostasis, and changes in ammonia excretion are the predominant component of increased net acid excretion in response to metabolic acidosis. We recently reported substantial sex-dependent differences in basal ammonia metabolism that correlate with sex-dependent differences in renal structure and expression of key proteins involved in ammonia metabolism. The purpose of the present study was to investigate the effect of sex on the renal ammonia response to an exogenous acid load. We studied 4-mo-old C57BL/6 mice. Ammonia excretion, which was less in male mice under basal conditions, increased in response to acid loading to a greater extent in male mice, such that maximal ammonia excretion did not differ between the sexes. Fundamental structural sex differences in the nonacid-loaded kidney persisted after acid loading, with less cortical proximal tubule volume density in the female kidney than in the male kidney, whereas collecting duct volume density was greater in the female kidney. To further investigate sex-dependent differences in the response to acid loading, we examined the expression of proteins involved in ammonia metabolism. The change in expression of phosphoenolpyruvate carboxykinase and Rh family B glycoprotein with acid loading was greater in male mice than in female mice, whereas Na + -K + -2Cl - cotransporter and inner stripe of the outer medulla intercalated cell Rh family C glycoprotein expression were significantly greater in female mice than in male mice. There was no significant sex difference in glutamine synthetase, Na + /H + exchanger isoform 3, or electrogenic Na + -bicarbonate cotransporter 1 variant A protein expression in response to acid loading. We conclude that substantial sex-dependent differences in the renal ammonia response to acid loading enable a similar maximum ammonia excretion response.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment., Competing Interests: Competing interestsN.P. and A.D.K. are co-founders and shareholders of InVivo Analytics, Inc. The remaining authors declare no competing interests.

Flow-related bending of cilia results in Ca 2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca 2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with ( Ift88 flox/flox ) or without ( Ift88 -/- ) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na + channel subunits. The predominant channel activity from Ift88 flox/flox mice was from epithelial Na + channel [5-pS Na + -selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca 2+ -permeable pathway at the apical membrane of collecting duct cells that lack cilia.

Claudin-7 knockout (CLDN7 -/- ) mice display renal salt wasting and dehydration phenotypes. To address the role of CLDN7 in kidneys, we established collecting duct (CD) cell lines from CLDN7 +/+ and CLDN7 -/- mouse kidneys. We found that deletion of CLDN7 increased the transepithelial resistance (TER) and decreased the paracellular permeability for Cl - and Na + in CLDN7 -/- CD cells. Inhibition of transcellular Cl - and Na + channels has no significant effect on TER or dilution potentials. Current-voltage curves were linear in both CLDN7 +/+ and CLDN7 -/- CD cells, indicating that the ion flux was through the paracellular pathway. The impairment of Cl - and Na + permeability phenotype can be rescued by CLDN7 re-expression. We also found that WNK4 (its mutations lead to hypertension) expression, but not WNK1, was significantly increased in CLDN7 -/- CD cell lines as well as in primary CLDN7 -/- CD cells, suggesting that the expression of WNK4 was modulated by CLDN7. In addition, deletion of CLDN7 upregulated the expression level of the apical epithelial sodium channel (ENaC), indicating a potential cross-talk between paracellular and transcellular transport systems. This study demonstrates that CLDN7 plays an important role in salt balance in renal CD cells and modulating WNK4 and ENaC expression levels that are vital in controlling salt-sensitive hypertension.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Human autosomal dominant polycystic kidney disease (ADPKD) is characterized by bilateral renal cysts that lead to a decline in kidney function. Previous studies reported aquaporin (AQP)-3 expression in cysts derived from collecting ducts in ADPKD. To study the role of AQP3 in cyst development, we generated 2 polycystic kidney disease (PKD) mouse models: kidney-specific Pkd1 knockout mice and inducible Pkd1 knockout mice, each without and with AQP3 deletion. In both models, kidney sizes and cyst indexes were significantly reduced in AQP3-null PKD mice compared with AQP3-expressing PKD mice, with the difference seen mainly in collecting duct cysts. AQP3-deficient kidneys showed significantly reduced ATP content, increased phosphorylated (p)-AMPK, and decreased p-ERK and p-mammalian target of rapamycin (mTOR). In a matrix-grown Madin-Darby canine kidney cyst model, AQP3 expression promoted cyst enlargement and was associated with increased expression of hypoxia-inducible factor 1-α and glucose transporter 1 and increased glucose uptake. Our data suggest that the slowed renal cyst enlargement in AQP3 deficiency involves impaired energy metabolism in the kidney through AMPK and mTOR signaling and impaired cellular glucose uptake. These findings implicate AQP3 as a novel determinant of renal cyst enlargement and hence a potential drug target in ADPKD.-Wang, W., Geng, X., Lei, L., Jia, Y., Li, Y., Zhou, H., Verkman, A. S., Yang, B. Aquaporin-3 deficiency slows cyst enlargement in experimental mouse models of autosomal dominant polycystic kidney disease.

People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney's innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney's collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell-specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor-mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.

Klotho is a protein primarily expressed in renal tubular epithelial cells. Studies have suggested that Klotho is an antiaging protein that reduces renal fibrosis after acute kidney injury (AKI) and inhibits stem cell senescence. Bone marrow mesenchymal stem cells (BMSCs) have consistent proliferation ability and multidirectional differentiation ability and have been used to treat tissue injury. Thus, we hypothesized that Klotho expressed in BMSCs could increase the renal protective effects of BMSCs. To verify the hypothesis, we isolated BMSCs from C57BL/6 mice, transfected them with Klotho-GFP-adenovirus and investigated the change in BMSC proliferation. We then transplanted Klotho-GFP-BMSCs into mice with AKI and investigated the therapeutic effect compared with that of sham-treated mice and GFP-BMSC-transplanted mice. Kidney fibrosis after ischemia/reperfusion injury (IRI) was relieved by BMSC transplantation, and the antifibrotic effect of BMSCs was significantly enhanced by overexpressing the Klotho gene. Mechanistic studies showed that Klotho increased pluripotency gene expression in BMSCs. Klotho produced by Klotho-GFP-BMSCs inhibited the Wnt/β-catenin pathway in renal tubular epithelial cells (TECs). Klotho-GFP-BMSCs showed increased proliferative ability and more potent immuno-regulation ability than did GFP-BMSCs. Our findings suggested that Klotho gene-modified BMSCs may be a better choice for cell therapy after AKI., (© 2018 International Federation for Cell Biology.)

In polycystic kidney disease (PKD), persistent activation of cell proliferation and matrix production contributes to cyst growth and fibrosis, leading to progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is overexpressed by cystic epithelial cells of PKD kidneys. Periostin binds α V β 3 -integrins and activates integrin-linked kinase (ILK), leading to Akt/mammalian target of rapamycin (mTOR)-mediated proliferation of human PKD cells. By contrast, periostin does not stimulate the proliferation of normal human kidney cells. This difference in the response to periostin is due to elevated expression of α V β 3 -integrins by cystic cells. To determine whether periostin accelerates cyst growth and fibrosis, we generated mice with conditional overexpression of periostin in the collecting ducts (CDs). Ectopic CD expression of periostin was not sufficient to induce cyst formation or fibrosis in wild-type mice. However, periostin overexpression in pcy/pcy ( pcy) kidneys significantly increased mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis; and accelerated the decline in renal function. Moreover, CD-specific overexpression of periostin caused a decrease in the survival of pcy mice. These pathological changes were accompanied by increased renal expression of vimentin, α-smooth muscle actin, and type I collagen. We also found that periostin increased gene expression of pathways involved in repair, including integrin and growth factor signaling and ECM production, and it stimulated focal adhesion kinase, Rho GTPase, cytoskeletal reorganization, and migration of PKD cells. These results suggest that periostin stimulates signaling pathways involved in an abnormal tissue repair process that contributes to cyst growth and fibrosis in PKD.

Most of kidney stones are supposed to originate from Randall's plaque at the tip of the papilla or from papillary tubular plugs. Nevertheless, the frequency and the composition of crystalline plugs remain only partly described. The objective was to assess the frequency, the composition and the topography of papillary plugs in human kidneys. A total of 76 papillae from 25 kidneys removed for cancer and without stones were analysed by immunohistochemistry combined with Yasue staining, field emission-scanning electron microscopy and Fourier transformed infrared micro-spectroscopy. Papillary tubular plugs have been observed by Yasue staining in 23/25 patients (92%) and 52/76 papillae (68%). Most of these plugs were made of calcium phosphate, mainly carbonated apatite and amorphous calcium phosphate, and rarely octacalcium phosphate pentahydrate. Calcium and magnesium phosphate (whitlockite) have also been observed. Based upon immunostaining coupled to Yasue coloration, most of calcium phosphate plugs were located in the deepest part of the loop of Henle. Calcium oxalate monohydrate and dihydrate tubular plugs were less frequent and stood in collecting ducts. At last, we observed calcium phosphate plugs deforming and sometimes breaking adjacent collecting ducts. Papillary tubular plugging, which may be considered as a potential first step toward kidney stone formation, is a very frequent setting, even in kidneys of non-stone formers. The variety in their composition and the distal precipitation of calcium oxalate suggest that plugs may occur in various conditions of urine supersaturation. Plugs were sometimes associated with collecting duct deformation.

Prolonged lithium treatment is associated with various renal side effects and is known to induce inner medullary collecting duct (IMCD) remodeling. In animals treated with lithium, the fraction of intercalated cells (ICs), which are responsible for acid-base homeostasis, increases compared with renal principal cells (PCs). To investigate the intricacies of lithium-induced IMCD remodeling, male Sprague-Dawley rats were fed a lithium-enriched diet for 0,1, 2, 3, 6, 9, or 12 wk. Urine osmolality was decreased at 1 wk, and from 2 to 12 wk, animals were severely polyuric. After 6 wk of lithium treatment, approximately one-quarter of the cells in the initial IMCD expressed vacuolar H + -ATPase, an IC marker. These cells were localized in portions of the inner medulla, where ICs are not normally found. Pendrin, a Cl - /[Formula: see text] exchanger, is normally expressed only in two IC subtypes found in the convoluted tubule, the cortical collecting duct, and the connecting tubule. At 6 wk of lithium treatment, we observed various patterns of pendrin localization and expression in the rat IMCD, including a novel phenotype wherein pendrin was coexpressed with aquaporin-4. These observations collectively suggest that renal IMCD cell plasticity may play an important role in lithium-induced IMCD remodeling.

Acute nephron reduction such as after living kidney donation may increase the risk of hypertension. Uninephrectomy induces major hemodynamic changes in the remaining kidney, resulting in rapid increase of single-nephron glomerular filtration rate (GFR) and fluid delivery in the distal nephron. Decreased sodium (Na) fractional reabsorption after the distal tubule has been reported after uninephrectomy in animals preserving volume homeostasis. In the present study, we thought to specifically explore the effect of unilateral nephrectomy on epithelial Na channel (ENaC) subunit expression in mice. We show that γ-ENaC subunit surface expression was specifically downregulated after uninephrectomy, whereas the expression of the aldosterone-sensitive α-ENaC and α 1 -Na-K-ATPase subunits as well as of kidney-specific Na-K-Cl cotransporter isoform and Na-Cl cotransporter were not significantly altered. Because acute nephron reduction induces a rapid increase of single-nephron GFR, resulting in a higher tubular fluid flow, we speculated that local mechanical factors such as fluid shear stress (FSS) were involved in Na reabsorption regulation after uninephrectomy. We further explore such hypothesis in an in vitro model of FSS applied on highly differentiated collecting duct principal cells. We found that FSS specifically downregulates β-ENaC and γ-ENaC subunits at the transcriptional level through an unidentified heat-insensitive paracrine basolateral factor. The primary cilium as a potential mechanosensor was not required. In contrast, protein kinase A and calcium-sensitive cytosolic phospholipase A 2 were involved, but we could not demonstrate a role for cyclooxygenase or epoxygenase metabolites.

Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations.

Mineralocorticoids trigger a profibrotic process in the kidney. In mouse cortical collecting duct cells, the present study addressed two main questions: 1) what are microRNAs (miRNAs) and their target genes that are changed by aldosterone? and 2) what do miRNAs, in response to aldosterone, regulate regarding signaling pathways related to fibrosis? A microarray chip assay was done in cells in the absence or presence of aldosterone treatment (10 -6 M; 3 days). The candidate miRNAs were identified by the criteria of >30% of fold change among the significantly changed miRNAs ( P < 0.05). Twenty-nine miRNAs were upregulated (>1.3-fold), and 27 miRNAs were downregulated (<0.7-fold). Putative target genes of identified miRNAs were associated with 74 Kyoto Encyclopedia of Genes and Genomes pathways. Among them, the wingless-related integration site (Wnt) signaling pathway was highly ranked, where 15 mature miRNAs were observed. These miRNAs were further analyzed by real-time quantitative PCR, and among them, miR-130b-3p, miR-34c-5p, and miR-146a-5p were selected. Through the identification of putative target genes of these three miRNAs, mRNA and protein expression of the Ca 2+ /calmodulin-dependent protein kinase type II β-chain ( Camk2b) gene (a target gene of miR-34c-5p) were found to be increased significantly in aldosterone-treated cells, where fibronectin (FN) and α-smooth muscle actin were induced. When CaMKIIβ small interfering RNA or the miR-34c-5p mimic was transfected, aldosterone-induced FN expression was significantly attenuated, along with reduced CaMKIIβ protein expression. A luciferase reporter assay revealed a decrease of CaMKIIβ translation in cells transfected with miRNA mimics of miR-34c-5p. In conclusion, aldosterone-induced downregulation of miR-34c-5p in the Wnt signaling pathway and a consequent increase of CaMKIIβ expression are likely to be involved in aldosterone-induced fibrosis.

Renal medullary carcinomas (RMCs) and collecting duct carcinomas (CDCs) are rare subsets of lethal high-stage, high-grade distal nephron-related adenocarcinomas with a predilection for the renal medullary region. Recent findings have established an emerging group of fumarate hydratase (FH)-deficient tumors related to hereditary leiomyomatosis and renal cell carcinoma (HLRCC-RCCs) syndrome within this morphologic spectrum. Recently developed, reliable ancillary testing has enabled consistent separation between these tumor types. Here, we present the clinicopathologic features and differences in the morphologic patterns between RMC, CDC, and FH-deficient RCC in consequence of these recent developments. This study included a total of 100 cases classified using contemporary criteria and ancillary tests. Thirty-three RMCs (SMARCB1/INI1-deficient, hemoglobinopathy), 38 CDCs (SMARCB1/INI1-retained), and 29 RCCs defined by the FH-deficient phenotype (FH/2SC or FH/2SC with FH mutation, regardless of HLRCC syndromic stigmata/history) were selected. The spectrum of morphologic patterns was critically evaluated, and the differences between the morphologic patterns present in the 3 groups were analyzed statistically. Twenty-five percent of cases initially diagnosed as CDC were reclassified as FH-deficient RCC on the basis of our contemporary diagnostic approach. Among the different overlapping morphologic patterns, sieve-like/cribriform and reticular/yolk sac tumor-like patterns favored RMCs, whereas intracystic papillary and tubulocystic patterns favored FH-deficient RCC. The tubulopapillary pattern favored both CDCs and FH-deficient RCCs, and the multinodular infiltrating papillary pattern favored CDCs. Infiltrating glandular and solid sheets/cords/nested patterns were not statistically different among the 3 groups. Viral inclusion-like macronucleoli, considered as a hallmark of HLRCC-RCCs, were observed significantly more frequently in FH-deficient RCCs. Despite the overlapping morphology found among these clinically aggressive infiltrating high-grade adenocarcinomas of the kidney, reproducible differences in morphology emerged between these categories after rigorous characterization. Finally, we recommend that definitive diagnosis of CDC should only be made if RMC and FH-deficient RCC are excluded.

Objective: To study the pathological features, immunophenotypes, differential diagnoses and prognostic parameters of collecting duct carcinoma of the kidney (CDC). Methods: Clinical imaging, histopathology, immunohistochemistry, and survival data of 10 patients at First Affiliated Hospital of Nanjing Medical University from January 2009 to August 2017 were retrospectively analyzed along with a review of literatures. Results: The clinical symptoms of CDC were not specific, and image examinations showed space-occupying mass lesions. Tumors were mainly located in renal medulla with grey and firm cut face and the presence of focal hemorrhage and necrosis. Microscopically, there were predominant tubular or tubular-papillary structures with associated focal sarcomatoid areas, desmoplastic stromal reaction and lymphoplasmacytic cells infiltration. Tumor cells had marked cytological atypia with high grade nuclei, conspicuous nucleolus and numerous mitoses. Immunohistochemically, tumor cells were strongly positive for CK19, E-cadherin, vimentin, HCK, CK7 and PAX8. The main treatment was radical nephrectomy in the patients. Seven cases died of CDC with median survival of 10 months. Conclusions: CDC is a rare, highly aggressive malignancy of kidney with poor prognosis. Definitive diagnosis should be made by histology and immunohistochemistry. Differential diagnoses include papillary renal cell carcinoma(type Ⅱ), renal medullary carcinoma, infiltrating high grade urothelial carcinoma, renal pelvis adenocarcinoma and metastatic adenocarcinomas.

Lithium, given to bipolar disorder patients, causes nephrogenic diabetes insipidus (Li-NDI), a urinary-concentrating defect. Li-NDI occurs due to downregulation of principal cell AQP2 expression, which coincides with principal cell proliferation. The metabolic effect of lithium on principal cells, however, is unknown and investigated here. In earlier studies, we showed that the carbonic anhydrase (CA) inhibitor acetazolamide attenuated Li-induced downregulation in mouse-collecting duct (mpkCCD) cells. Of the eight CAs present in mpkCCD cells, siRNA and drug treatments showed that downregulation of CA9 and to some extent CA12 attenuated Li-induced AQP2 downregulation. Moreover, lithium induced cell proliferation and increased the secretion of lactate. Lithium also increased urinary lactate levels in wild-type mice that developed Li-NDI but not in lithium-treated mice lacking ENaC, the principal cell entry site for lithium. Inhibition of aerobic glycolysis with 2-deoxyglucose (2DG) attenuated lithium-induced AQP2 downregulation in mpkCCD cells but did not attenuate Li-NDI in mice. Interestingly, NMR analysis demonstrated that lithium also increased the urinary succinate, fumarate, citrate, and NH 4 + levels, which were, in contrast to lactate, not decreased by 2DG. Together, our data reveal that lithium induces aerobic glycolysis and glutaminolysis in principal cells and that inhibition of aerobic glycolysis, but not the glutaminolysis, does not attenuate Li-NDI.

microRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. The physiologic function of these noncoding RNAs in postnatal renal tubules still remains unclear. Surprisingly, they appear to be dispensable for mammalian proximal tubule (PT) function. Here, we examined the effects of miRNA suppression in collecting ducts (CDs). To conclusively evaluate the role of miRNAs, we generated three mouse models with CD-specific inactivation of key miRNA pathway genes Dicer , Dgcr8 , and the entire Argonaute gene family ( Ago1 , 2 , 3 , and 4 ). Characterization of these three mouse models revealed that inhibition of miRNAs in CDs spontaneously evokes a renal tubule injury-like response, which culminates in progressive tubulointerstitial fibrosis (TIF) and renal failure. Global miRNA profiling of microdissected renal tubules showed that miRNAs exhibit segmental distribution along the nephron and CDs. In particular, the expression of miR-200c is nearly 70-fold higher in CDs compared with PTs. Accordingly, miR-200s are downregulated in Dicer- KO CDs, its direct target genes Zeb1 , Zeb2 , and Snail2 are upregulated, and miRNA-depleted CDs undergo partial epithelial-to-mesenchymal transition (EMT). Thus, miRNAs are essential for CD homeostasis. Downregulation of CD-enriched miRNAs and the subsequent induction of partial EMT may be a new mechanism for TIF progression., (Copyright © 2018 by the American Society of Nephrology.)

Background: From a clinical point of view, uric acid has been dismissed as a cause of injury and renal progression, and the mechanisms by which uric acid directly causes renal injury have not been fully understood. Hyperuricemia is associated with metabolic syndrome, diabetes, hypertension, and kidney and cardiovascular diseases. Although it remains controversial whether hyperuricemia is a causal factor for kidney disease, kidneys play a major role in the regulation of serum uric acid levels., Summary: Similar to the management of other substances, renal uric acid management occurs mainly in the proximal tubule. The elevation of uric acid in blood is mainly due to an increase in its intake or a defect in its secretion. The mechanisms of renal damage go beyond the deposition of crystals at the tubular level; in this sense, renal damage also contributes to the production of chemotactic cytokines, cell proliferation, and inflammation, with the development of afferent arteriolopathy, glomerular hypertrophy and tubulointersticial fibrosis. Nevertheless, whether or not hyperuricemia plays a causal role or simply is a marker arising in the course of each related disorder remains unresolved. Although a strong relationship between hyperuricemia and metabolic syndrome has been established through animal and epidemiological studies, the potential pathophysiological mechanisms by which uric acid contributes to this disease state are just beginning to be explained and clarified. Key Messages: Experimental studies performed in animals have limitations due to the differences that exist between humans and other mammals in purine metabolism and in renal uric acid handling. Additional experimental studies aimed at evaluating the effects of lowering urate levels on the courses of these disorders are warranted in the future., (© 2018 S. Karger AG, Basel.)

Urinary tract infections (UTIs) mainly due to uropathogenic Escherichia coli (UPEC) are one of the most frequent complications in kidney-transplanted patients, causing significant morbidity. However, the mechanisms underlying UTI in renal grafts remain poorly understood. Here, we analysed the effects of the potent immunosuppressive agent cyclosporine A (CsA) on the activation of collecting duct cells that represent a preferential site of adhesion and translocation for UPEC. CsA induced the inhibition of lipopolysaccharide- induced activation of collecting duct cells due to the downregulation of the expression of TLR4 via the microRNA Let-7i. Using an experimental model of ascending UTI, we showed that the pretreatment of mice with CsA prior to infection induced a marked fall in cytokine production by collecting duct cells, neutrophil recruitment, and a dramatic rise of bacterial load, but not in infected TLR4-defective mice kidneys. This effect was also observed in CsA-treated infected kidneys, where the expression of Let-7i was increased. Treatment with a synthetic Let-7i mimic reproduced the effects of CsA. Conversely, pretreatment with an anti-Let-7i antagonised the effects of CsA and rescued the innate immune response of collecting duct cells against UPEC. Thus, the utilisation of an anti-Let-7i during kidney transplantation may protect CsA-treated patients from ascending bacterial infection., (© 2017 S. Karger AG, Basel.)

Intercalated cells of the collecting duct (CD) are critical for acid-base homeostasis and innate immune defense of the kidney. Little is known about the impact of acidosis on innate immune defense in the distal nephron. Urinary tract infections are mainly due to Escherichia coli and are an important risk factor for development of chronic kidney disease. While the effect of urinary pH on growth of E. coli is well established, in this study, we demonstrate that acidosis increases urine antimicrobial activity due, at least in part, to induction of cathelicidin expression within the CD. Acidosis was induced in rabbits by adding NH 4 Cl to the drinking water and reducing food intake over 3 days or by casein supplementation. Microdissected CDs were examined for cathelicidin mRNA expression and antimicrobial activity, and cathelicidin protein levels in rabbit urine were measured. Cathelicidin expression in CD cells was detected in kidney sections. CDs from acidotic rabbits expressed three times more cathelicidin mRNA than those isolated from normal rabbits. Urine from acidotic rabbits had significantly more antimicrobial activity (vs. E. coli ) than normal urine, and most of this increased activity was blocked by cathelicidin antibody. The antibody had little effect on antimicrobial activity of normal urine. Urine from acidotic rabbits had at least twice the amount of cathelicidin protein as did normal urine. We conclude that metabolic acidosis not only stimulates CD acid secretion but also induces expression of cathelicidin and, thereby, enhances innate immune defense against urinary tract infections via induction of antimicrobial peptide expression., (Copyright © 2017 the American Physiological Society.)

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic hereditary disorders in humans characterized by fluid-filled cysts, primarily in the kidneys. Cux1, a cell cycle regulatory gene highly expressed during kidney development, is elevated in the cyst-lining cells of Pkd1 mutant mice, and in human ADPKD cells. However, forced expression of Cux1 is insufficient to induce cystic disease in transgenic mice or to induce rapid cyst formation after cilia disruption in the kidneys of adult mice. Here we report a double mutant mouse model that has a conditional deletion of the Pkd1 gene in the renal collecting ducts together with a targeted mutation in the Cux1 gene (Pkd1 CD ;Cux1 tm2Ejn ). While kidneys isolated from newborn Pkd1 CD mice exhibit cortical and medullary cysts, kidneys isolated from newborn Pkd1 CD ;Cux1 tm2Ejn-/- mice did not show any cysts. Because Cux1 tm2Ejn-/- are perinatal lethal, we evaluated Pkd1 CD mice that were heterozygote for the Cux1 mutation. Similar to the newborn Pkd1 CD ;Cux1 tm2Ejn-/- mice, newborn Pkd1 CD ;Cux1 tm2Ejn+/- mice did not show any cysts. Comparison of Pkd1 CD and Pkd1 CD ;Cux1 tm2Ejn+/- mice at later stages of development showed a reduction in the severity of PKD in the Pkd1 CD ;Cux1 tm2Ejn+/- mice. Moreover, we observed an increase in expression of the cyclin kinase inhibitor p27, a target of Cux1 repression, in the rescued collecting ducts. Taken together, our results suggest that Cux1 expression in PKD is not directly involved in cystogenesis but promotes cell proliferation required for expansion of existing cysts, primarily by repression of p27., (Copyright © 2017 the American Physiological Society.)

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by innumerous fluid-filled cysts and progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is markedly overexpressed by cyst epithelial cells. Periostin promotes cell proliferation, cyst growth, interstitial fibrosis, and the decline in renal function in PKD mice. Here, we investigated the regulation of these processes by the integrin-linked kinase (ILK), a scaffold protein that links the extracellular matrix to the actin cytoskeleton and is stimulated by periostin. Pharmacologic inhibition or shRNA knockdown of ILK prevented periostin-induced Akt/mammalian target of rapamycin (mTOR) signaling and ADPKD cell proliferation in vitro Homozygous deletion of ILK in renal collecting ducts (CD) of Ilk fl/fl ; Pkhd1-Cre mice caused tubule dilations, apoptosis, fibrosis, and organ failure by 10 weeks of age. By contrast, Ilk fl/+ ; Pkhd1-Cre mice had normal renal morphology and function and survived >1 year. Reduced expression of ILK in Pkd1 fl/fl ; Pkhd1-Cre mice, a rapidly progressive model of ADPKD, decreased renal Akt/mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis, and significantly improved renal function and animal survival. Additionally, CD-specific knockdown of ILK strikingly reduced renal cystic disease and fibrosis and extended the life of pcy/pcy mice, a slowly progressive PKD model. We conclude that ILK is critical for maintaining the CD epithelium and renal function and is a key intermediate for periostin activation of signaling pathways involved in cyst growth and fibrosis in PKD., (Copyright © 2017 by the American Society of Nephrology.)

Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1 -/- kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD.

Carcinoma of the collecting ductsof Bellini isa rare histological subtype of renal cell carcinoma and mostly has unfavorable prognosis. Radical nephrectomy is generally chosen for the 1st line treatment but therapeutic approaches for the metastasis/recurrence have not been established. We report a case of carcinoma of collecting ducts of Bellini in a patient receiving hemodialysis treated with temsirolimus. A 62- year-old man receiving hemodialysis was admitted to our hospital with drug-resistant anemia and high-grade cyclic fever. Computed tomography revealed the right renal tumor and multiple metastatic lung tumors. Open radical nephrectomy wasperformed. Pathological findingswere compatible with carcinoma of the collecting ducts of Bellini. He was given weekly temsirolimus treatment. The disease progressed modestly but kept the stable disease (SD) status for six months. He died of the cancer 11 months after the initial diagnosis.

Aquaporin-3 (AQP3), a transporter of water, glycerol and H 2 O 2 , is expressed in basolateral membranes of principal cells in kidney collecting duct. Here, we report that AQP3 deletion in mice affects renal function and modulates renal injury. We found collecting duct hyperplasia and cell swelling in kidneys of adult AQP3 null mice. After mild renal ischemia-reperfusion (IR), AQP3 null mice had significantly greater blood urea nitrogen (57mg/dl) and creatinine (136μM) than wild-type mice (35mg/dl and 48μM, respectively), and showed renal morphological changes, including tubular dilatation, erythrocyte diapedesis and collecting duct incompletion. MPO, MDA and SOD following IR in AQP3 null mice were significantly different from that in wild-type mice (1.7U/g vs 0.8U/g, 3.9μM/g vs 2.4μM/g, 6.4U/mg vs 11U/mg, respectively). Following IR, AQP3 deletion inhibited activation of mitogen-activated protein kinase (MAPK) signaling and produced an increase in the ratios of Bax/Bcl-2, cleaved caspase-3/caspase-3 and p-p53/p53. Studies in transfected MDCK cells showed that AQP3 expression attenuated reduced cell viability following hypoxia-reoxygenation, with reduced apoptosis and increased MAPK signaling. Our results support a novel role for AQP3 in modulating renal injury and suggest the mechanisms involved in protection against hypoxic injury., (Copyright © 2017. Published by Elsevier B.V.)

Increased renal reabsorption of sodium is a significant risk factor in hypertension. An established clinical marker for essential hypertension is elevated sodium lithium countertransport (SLC) activity. NHA2 is a newly identified Na + (Li + )/H + antiporter with potential genetic links to hypertension, which has been shown to mediate SLC activity and H + -coupled Na + (Li + ) efflux in kidney-derived MDCK cells. To evaluate a putative role in sodium homeostasis, we determined the effect of dietary salt on NHA2. In murine kidney sections, NHA2 localized apically to distal convoluted (both DCT1 and 2) and connecting tubules, partially overlapping in distribution with V-ATPase, AQP2, and NCC1 transporters. Mice fed a diet high in sodium chloride showed elevated transcripts and expression of NHA2 protein. We propose a model in which NHA2 plays a dual role in salt reabsorption or secretion, depending on the coupling ion (sodium or protons). The identified novel regulation of Na + /H + antiporter in the kidney suggests new roles in salt homeostasis and disease.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced predominantly by gut microbiota fermentation of dietary fiber. SCFAs are newly identified as endogenous ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, which have the potential to modulate inflammation. Therefore, GPR41 and GPR43 may mediate the link between the gut microbiome status and various disease conditions including renal inflammation. This study aimed at investigating whether SCFAs activate GPR41 and GPR43, and thereby exert anti-inflammatory effects in human renal cortical epithelial cells (HRCEs) as a main component of kidney tissue. Immunohistochemical analyses of human renal biopsy specimens revealed the expression of GPR41 and GPR43 protein in the distal renal tubules and collecting tubules. TNF-α increased the expression of monocyte chemoattractant protein-1 (MCP-1), a potential fibrotic inducer, at least partly via enhancing phosphorylation of p38 and JNK in HRCEs. SCFAs, especially propionate, attenuated TNF-α- stimulated MCP-1 expression by inhibiting the phosphorylation of p38 and JNK. This inhibitory effect was considerably attenuated by an inactivator of the Gi/o-type G protein and a Gβγ (i/o) blocker, but not by a Gα (i/o) blocker. Furthermore, SCFA-mediated inhibition of MCP-1 expression was significantly blocked by siRNA-induced gene silencing of GPR41 and GPR43. In conclusion, SCFAs lowered TNF-α-induced MCP-1 expression by reducing phosphorylation of p38 and JNK in a GPR41/43-dependent manner in HRCEs, suggesting that SCFA modification may be a new therapeutic tool for preventing progression of renal inflammation and fibrosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An original model of nephrogenic hypertension in rats was used for histochemical mapping of NADPH diaphorase (NO synthase) in various renal segments to examine the effect of hypotensive drugs furosemide, bendazol, and clonidine on the time course of nitroxide production in the kidneys. In various nephron segments, these drugs modulated NO synthesis in different ways. Clonidine induced a stable up-regulation of NO synthesis, which can maintain active vasodilation and gradually diminish the rennin production. Bendazol also enhanced NO synthase activity in renal glomeruli and collecting tubules, but this effect was less pronounced and short lasting. During the first week after injection of bendazol, insignificant elevation of NO synthase activity was observed in the proximal nephron segments. Furosemide exerted the least effect on NO production in kidneys.

The collecting duct of the mammalian kidney is important for the regulation of extracellular volume, osmolarity, and pH. There are two major structurally and functionally distinct cell types: principal cells and intercalated cells. The former regulates Na + and water homeostasis, while the latter participates in acid-base homeostasis. In vivo lineage tracing using Cre recombinase or its derivatives such as CreGFP and CreER T2 is a powerful new technique to identify stem/progenitor cells in their native environment and to decipher the origins of the tissue that they give rise to. Recent studies using this technique in mice have revealed multiple renal progenitor cell populations that differentiate into various nephron segments and collecting duct. In particular, emerging evidence suggests that like principal cells, most of intercalated cells originate from the progenitor cells expressing water channel Aquaporin 2. Mutations or malfunctions of the channels, pumps, and transporters expressed in the collecting duct system cause various human diseases. For example, gain-of-function mutations in ENaC cause Liddle's syndrome, while loss-of-function mutations in ENaC lead to Pseudohypoaldosteronism type 1. Mutations in either AE1 or V-ATPase B1 result in distal renal tubular acidosis. Patients with disrupted AQP2 or AVPR2 develop nephrogenic diabetes insipidus. A better understanding of the function and development of the collecting duct system may facilitate the discovery of new therapeutic strategies for treating kidney disease.

Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and β subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3β1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6β1 and α6β4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or β4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa., (Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)