Genetic analysis of nephrogenic diabetes insipidus patients: A study on the Iranian population.

INTRODUCTION: Nephrogenic diabetes insipidus (NDI) is a rare genetic disease that causes water imbalance. The kidneys play a crucial role in regulating body fluids by controlling water balance through urine excretion. This highlights their essential function in managing the body's water levels, but individuals with NDI may have excess urine production (polyuria), that leads to excessive thirst (polydipsia). Untreated affected individuals may exhibit poor feeding and failure to thrive. This disease is caused by mutations in the AVPR2 and the AQP2 genes which have the X-linked and autosomal recessive/dominant inheritance, respectively. Both of these genes are expressed in the kidney. METHODS: Twelve Iranian patients from 10 consanguineous families were studied in this project. DNA was extracted from the whole blood samples of the patients and their parents. All coding exons and exon-intron boundaries of the AVPR2 and AQP2 genes were sequenced in the affected individuals, and the identified variants were investigated in the parents. All variants were analyzed according to the ACMG (American College of Medical Genetics and Genomics) guidelines. RESULTS: In this study, 6 different mutations were identified in the patients, including 5 in the AQP2 gene (c.439G>A, c.538G>A, c.140C>T, c.450T>A, and the novel c.668T>C) and 1 in the AVPR2 gene (c.337C>T) in the present study. DISCUSSION: As expected, all the detected mutations in this study were missense. According to the ACMG guideline, the identified mutations were categorized as pathogenic or likely pathogenic. Unlike previous studies which showed more than 90% of mutations were in the AVPR2 gene, and only less than 10% of the mutations were in the AQP2 gene, it was found that more than 90% of our identified mutations located in the AQP2 gene, and only one mutation was observed in the AVPR2 gene, which seems it may be a result of the high rate of consanguineous marriages in the Iranian population. We observed genotype-phenotype correlation in some of our affected individuals, and some of the mutations were observed in unrelated families from same ethnicity which could be suggestive of a founder mutation.

The ß3-AR agonist BRL37344 ameliorates the main symptoms of X-linked nephrogenic diabetes insipidus in the mouse model of the disease.

X-linked nephrogenic diabetes insipidus (X-NDI) is a rare congenital disease caused by inactivating mutations of the vasopressin type-2 receptor (AVPR2), characterized by impaired renal concentrating ability, dramatic polyuria, polydipsia and risk of dehydration. The disease, which still lacks a cure, could benefit from the pharmacologic stimulation of other GPCRs, activating the cAMP-intracellular pathway in the kidney cells expressing the AVPR2. On the basis of our previous studies, we here hypothesized that the ß3-adrenergic receptor could be such an ideal candidate. We evaluated the effect of continuous 24 h stimulation of the ß3-AR with the agonist BRL37344 and assessed the effects on urine output, urine osmolarity, water intake and the abundance and activation of the key renal water and electrolyte transporters, in the mouse model of X-NDI. Here we demonstrate that the ß3-AR agonism exhibits a potent antidiuretic effect. The strong improvement in symptoms of X-NDI produced by a single i.p. injection of BRL37344 (1 mg/kg) was limited to 3 h but repeated administrations in the 24 h, mimicking the effect of a slow-release preparation, promoted a sustained antidiuretic effect, reducing the 24 h urine output by 27%, increasing urine osmolarity by 25% and reducing the water intake by 20%. At the molecular level, we show that BRL37344 acted by increasing the phosphorylation of NKCC2, NCC and AQP2 in the renal cell membrane, thereby increasing electrolytes and water reabsorption in the kidney tubule of X-NDI mice. Taken together, these data suggest that human ß3-AR agonists might represent an effective possible treatment strategy for X-NDI.

Ontogenetic Expression of Aquaporins in the Kidney and Urinary Bladder of the Japanese Tree Frog, Dryophytes japonicus.

For adult anuran amphibians, the kidney and urinary bladder play important osmoregulatory roles through water reabsorption. In the present study, we have examined ontogenetic expression of aquaporins, i.e., AQP2, AQPamU (AQP6ub, AQPa2U), and AQP3, in these organs using the Japanese tree frog, Dryophytes japonicus. Immunohistochemistry using the metamorphosing larvae at stages 40-43 localized AQP2 protein to the collecting ducts in the dorsal zone of the mesonephric kidney. At prometamorphic stages 40 and 41, labelling of AQP2 protein was observed in the apical/ subapical regions of the collecting duct cells. At climax stages 42 and 43, labels for AQP2 and AQP3 became observed in the apical/subapical regions and basolateral membrane of the collecting duct cells, respectively, as seen in the adults. As for the urinary bladder, immuno-positive labels for AQPamU were localized to the apical/subapical regions of granular cells in the mucosal epithelium at stages 40-43. On the other hand, AQP3 immunoreactivity was hardly observed in the urinary bladder at stage 40, and weakly appeared in many granular cells at stage 41. Thereafter, labels for AQP3 became evident along the basolateral membrane of granular cells at stages 42 and 43, together with AQPamU in the apical/subapical regions. These results suggest that the kidney and urinary bladder might be capable of water reabsorption, via AQP2, AQPamU, and AQP3, at stage 42, contributing to the acclimation of the tree frogs to terrestrial environments.

Recovery of Water Homeostasis in Adenine-Induced Kidney Disease Is Mediated by Increased AQP2 Membrane Targeting.

Chronic kidney disease (CKD) represents a major public health burden with increasing prevalence. Current therapies focus on delaying CKD progression, underscoring the need for innovative treatments. This necessitates animal models that accurately reflect human kidney pathologies, particularly for studying potential reversibility and regenerative mechanisms, which are often hindered by the progressive and irreversible nature of most CKD models. In this study, CKD was induced in mice using a 0.2% adenine-enriched diet for 4 weeks, followed by a recovery period of 1 or 2 weeks. The aim was to characterize the impact of adenine feeding on kidney function and injury as well as water and salt homeostasis throughout disease progression and recovery. The adenine diet induced CKD is characterized by impaired renal function, tubular injury, inflammation, and fibrosis. A significant decrease in urine osmolality, coupled with diminished aquaporin-2 (AQP2) expression and membrane targeting, was observed after adenine treatment. Intriguingly, these parameters exhibited a substantial increase after a two-week recovery period. Despite these functional improvements, only partial reversal of inflammation, tubular damage, and fibrosis were observed after the recovery period, indicating that the inclusion of the molecular and structural parameters is needed for a more complete monitoring of kidney status.

Effect of heat stress on the hypothalamic expression profile of water homeostasis-associated genes in low- and high-water efficient chicken lines.

With climate change, selection for water efficiency and heat resilience are vitally important. We undertook this study to determine the effect of chronic cyclic heat stress (HS) on the hypothalamic expression profile of water homeostasis-associated markers in high (HWE)- and low (LWE)-water efficient chicken lines. HS significantly elevated core body temperatures of both lines. However, the amplitude was higher by 0.5-1°C in HWE compared to their LWE counterparts. HWE line drank significantly less water than LWE during both thermoneutral (TN) and HS conditions, and HS increased water intake in both lines with pronounced magnitude in LWE birds. HWE had better feed conversion ratio (FCR), water conversion ratio (WCR), and water to feed intake ratio. At the molecular level, the overall hypothalamic expression of aquaporins (AQP8 and AQP12), arginine vasopressin (AVP) and its related receptor AVP2R, angiotensinogen (AGT), angiotensin II receptor type 1 (AT1), and calbindin 2 (CALB2) were significantly lower; however, CALB1 mRNA and AQP2 protein levels were higher in HWE compared to LWE line. Compared to TN conditions, HS exposure significantly increased mRNA abundances of AQPs (8, 12), AVPR1a, natriuretic peptide A (NPPA), angiotensin I-converting enzyme (ACE), CALB1 and 2, and transient receptor potential cation channel subfamily V member 1 and 4 (TRPV1 and TRPV4) as well as the protein levels of AQP2, however it decreased that of AQP4 gene expression. A significant line by environment interaction was observed in several hypothalamic genes. Heat stress significantly upregulated AQP2 and SCT at mRNA levels and AQP1 and AQP3 at both mRNA and protein levels, but it downregulated that of AQP4 protein only in LWE birds. In HWE broilers, however, HS upregulated the hypothalamic expression of renin (REN) and AVPR1b genes and AQP5 proteins, but it downregulated that of AQP3 protein. The hypothalamic expression of AQP (5, 7, 10, and 11) genes was increased by HS in both chicken lines. In summary, this is the first report showing improvement of growth performances in HWE birds. The hypothalamic expression of several genes was affected in a line- and/or environment-dependent manner, revealing potential molecular signatures for water efficiency and/or heat tolerance in chickens.

Deciphering Molecular Mechanisms Involved in the Modulation of Human Aquaporins' Water Permeability by Zinc Cations: A Molecular Dynamics Approach.

Aquaporins (AQPs) constitute a wide family of water channels implicated in all kind of physiological processes. Zinc is the second most abundant trace element in the human body and a few studies have highlighted regulation of AQP0 and AQP4 by zinc. In the present work, we addressed the putative regulation of AQPs by zinc cations in silico through molecular dynamics simulations of human AQP0, AQP2, AQP4, and AQP5. Our results align with other scales of study and several in vitro techniques, hence strengthening the reliability of this regulation by zinc. We also described two distinct putative molecular mechanisms associated with the increase or decrease in AQPs' water permeability after zinc binding. In association with other studies, our work will help deciphering the interaction networks existing between zinc and channel proteins.

Effect of heat stress on the hypothalamic expression of water channel- and noncoding RNA biogenesis-related genes in modern broilers and their ancestor red jungle fowl.

Genetic selection for high growth rate has resulted in spectacular progress in feed efficiency in chickens. As feed intake and water consumption (WC) are associated and both are affected by environmental conditions, we evaluated WC and its hypothalamic regulation in three broiler-based research lines and their ancestor jungle fowl (JF) under heat stress (HS) conditions. Slow growing ACRB, moderate growing 95RB, fast growing MRB, and JF were exposed to daily chronic cyclic HS (36 °C, 9 h/d) or thermoneutral temperature (24 °C). HS increased WC in the MRB only. Arginine vasopressin (AVP) mRNA levels were decreased by HS in the MRB. Within the renin-angiotensin-aldosterone system (RAAS) system, renin expression was increased by HS in the JF, ACRB, and 95RB, while angiotensin I-converting enzyme (ACE), angiotensin II receptors (type 1, AT1, and type 2, AT2) were affected by line. The expression of aquaporin (AQP2, 7, 9, 10, 11, and 12) genes was upregulated by HS, whereas AQP4 and AQP5 expressions were influenced by line. miRNA processing components (Dicer1, Ago2, Drosha) were significantly different among the lines, but were unaffected by HS. In summary, this is the first report showing the effect of HS on hypothalamic water channel- and noncoding RNA biogenesis-related genes in modern chicken populations and their ancestor JF. These results provide a novel framework for future research to identify new molecular mechanisms and signatures involved in water homeostasis and adaptation to HS.

Pkd2 Deficiency in Embryonic Aqp2 + Progenitor Cells Is Sufficient to Cause Severe Polycystic Kidney Disease.

SIGNIFICANCE STATEMENT: Autosomal dominant polycystic kidney disease (ADPKD) is a devastating disorder caused by mutations in polycystin 1 ( PKD1 ) and polycystin 2 ( PKD2 ). Currently, the mechanism for renal cyst formation remains unclear. Here, we provide convincing and conclusive data in mice demonstrating that Pkd2 deletion in embryonic Aqp2 + progenitor cells (AP), but not in neonate or adult Aqp2 + cells, is sufficient to cause severe polycystic kidney disease (PKD) with progressive loss of intercalated cells and complete elimination of α -intercalated cells, accurately recapitulating a newly identified cellular phenotype of patients with ADPKD. Hence, Pkd2 is a new potential regulator critical for balanced AP differentiation into, proliferation, and/or maintenance of various cell types, particularly α -intercalated cells. The Pkd2 conditional knockout mice developed in this study are valuable tools for further studies on collecting duct development and early steps in cyst formation. The finding that Pkd2 loss triggers the loss of intercalated cells is a suitable topic for further mechanistic studies. BACKGROUND: Most cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by mutations in PKD1 or PKD2. Currently, the mechanism for renal cyst formation remains unclear. Aqp2 + progenitor cells (AP) (re)generate ≥5 cell types, including principal cells and intercalated cells in the late distal convoluted tubules (DCT2), connecting tubules, and collecting ducts. METHODS: Here, we tested whether Pkd2 deletion in AP and their derivatives at different developmental stages is sufficient to induce PKD. Aqp2Cre Pkd2f/f ( Pkd2AC ) mice were generated to disrupt Pkd2 in embryonic AP. Aqp2ECE/+Pkd2f/f ( Pkd2ECE ) mice were tamoxifen-inducted at P1 or P60 to inactivate Pkd2 in neonate or adult AP and their derivatives, respectively. All induced mice were sacrificed at P300. Immunofluorescence staining was performed to categorize and quantify cyst-lining cell types. Four other PKD mouse models and patients with ADPKD were similarly analyzed. RESULTS: Pkd2 was highly expressed in all connecting tubules/collecting duct cell types and weakly in all other tubular segments. Pkd2AC mice had obvious cysts by P6 and developed severe PKD and died by P17. The kidneys had reduced intercalated cells and increased transitional cells. Transitional cells were negative for principal cell and intercalated cell markers examined. A complete loss of α -intercalated cells occurred by P12. Cysts extended from the distal renal segments to DCT1 and possibly to the loop of Henle, but not to the proximal tubules. The induced Pkd2ECE mice developed mild PKD. Cystic α -intercalated cells were found in the other PKD models. AQP2 + cells were found in cysts of only 13/27 ADPKD samples, which had the same cellular phenotype as Pkd2AC mice. CONCLUSIONS: Hence, Pkd2 deletion in embryonic AP, but unlikely in neonate or adult Aqp2 + cells (principal cells and AP), was sufficient to cause severe PKD with progressive elimination of α -intercalated cells, recapitulating a newly identified cellular phenotype of patients with ADPKD. We proposed that Pkd2 is critical for balanced AP differentiation into, proliferation, and/or maintenance of cystic intercalated cells, particularly α -intercalated cells.

Urinary podocyte stress marker as a prognostic indicator for diabetic kidney disease.

BACKGROUND: Diabetic kidney diseases (DKD) is a the most common cause of end-stage kidney disease (ESKD) around the world. Previous studies suggest that urinary podocyte stress biomarker, e.g. podocin:nephrin mRNA ratio, is a surrogate marker of podocyte injury in non-diabetic kidney diseases. METHOD: We studied 118 patients with biopsy-proved DKD and 13 non-diabetic controls. Their urinary mRNA levels of nephrin, podocin, and aquaporin-2 (AQP2) were quantified. Renal events, defined as death, dialysis, or 40% reduction in glomerular filtration rate, were determined at 12 months. RESULTS: Urinary podocin:nephrin mRNA ratio of DKD was significantly higher than the control group (p = 0.0019), while urinary nephrin:AQP2 or podocin:AQP2 ratios were not different between groups. In DKD, urinary podocin:nephrin mRNA ratio correlated with the severity of tubulointerstitial fibrosis (r = 0.254, p = 0.006). and was associated with the renal event-free survival in 12 months (unadjusted hazard ratio [HR], 1.523; 95% confidence interval [CI] 1.157-2.006; p = 0.003). After adjusting for clinical and pathological factors, urinary podocin:nephrin mRNA ratio have a trend to predict renal event-free survival (adjusted HR, 1.327; 95%CI 0.980-1.797; p = 0.067), but the result did not reach statistical significance. CONCLUSION: Urinary podocin:nephrin mRNA ratio has a marginal prognostic value in biopsy-proven DKD. Further validation is required for DKD patients without kidney biopsy.

KDM6A Demethylase Regulates Renal Sodium Excretion and Blood Pressure.

BACKGROUND: KDM6A (Lysine-Specific Demethylase 6A) is a specific demethylase for histone 3 lysine (K) 27 trimethylation (H3K27me3). The purpose of this study is to investigate whether KDM6A in renal tubule cells plays a role in the regulation of kidney function and blood pressure. METHODS: We first crossed Ksp-Cre+/- and KDM6Aflox/flox mice for generating inducible kidney-specific deletion of KDM6A gene. RESULTS: Notably, conditional knockout of KDM6A gene in renal tubule cells (KDM6A-cKO) increased H3K27me3 levels which leads to a decrease in Na excretion and elevation of blood pressure. Further analysis showed that the expression of NKCC2 (Na-K-2Cl cotransporter 2) and NCC (Na-Cl cotransporters) was upregulated which contributes to impaired Na excretion in KDM6A-cKO mice. The expression of AQP2 (aquaporin 2) was also increased in KDM6A-cKO mice, which may facilitate water reabsorption in KDM6A-cKO mice. The expression of Klotho was downregulated while expression of aging markers including p53, p21, and p16 was upregulated in kidneys of KDM6A-cKO mice, indicating that deletion of KDM6A in the renal tubule cells promotes kidney aging. Interestingly, KDM6A-cKO mice developed salt-sensitive hypertension which can be rescued by treatment with Klotho. KDM6A deficiency induced salt-sensitive hypertension likely through downregulation of the Klotho/ERK (extracellular signal-regulated kinase) signaling and upregulation of the WNK (with-no-lysine kinase) signaling. CONCLUSIONS: This study provides the first evidence that KDM6A plays an essential role in maintaining normal tubular function and blood pressure. Renal tubule cell specific KDM6A deficiency causes hypertension due to increased H3K27me3 levels and the resultant downregulation of Klotho gene expression which disrupts the Klotho/ERK/NCC/NKCC2 signaling.

Collecting duct water permeability inhibition by EGF is associated with decreased cAMP, PKA activity, and AQP2 phosphorylation at Ser269.

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-d-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis.NEW & NOTEWORTHY The authors used native rat collecting ducts to show that inhibition of vasopressin-stimulated water permeability by epidermal growth factor involves a reduction of aquaporin 2 phosphorylation at Ser269, a consequence of reduced cAMP production and PKA activity.

Lactobacillus plantarum supernatant inhibits growth of Riemerella anatipestifer and mediates intestinal antimicrobial defense in Muscovy ducks.

Riemerella anatipestifer (RA) is an important pathogen of waterfowl, with multiple serotypes and a lack of cross-protection between each serotype, which leads to the continued widespread in the world and causing significant economic losses to the duck industry. Thus, prevention and inhibition of RA infection are of great concern. Previous research has established that Lactobacillus plantarum supernatant (LPS) can prevents the pathogenic bacteria infection. However, LPS whether inhibits RA and underlying mechanisms have not yet been clarified. In this study, we investigated the direct and indirect effects of LPS-ZG7 against RA infection in Muscovy ducks. The results demonstrated that LPS-ZG7 prevented RA growth in the presence of pH-neutralized, and the inhibition was relatively stable and unaffected by heat, acid-base and ultraviolet light (UV). Following flow cytometry data found that LPS-ZG7 increased RA membrane permeability and leakage of intracellular molecules. And scanning electron microscopy revealed LPS-ZG7 damaged the RA membrane integrity and leading to RA death. Furthermore, quantitative real time polymerase chain reaction (qPCR) analysis represented that LPS-ZG7 upregulated mucosal tight junction proteins occludin, claudin-1, and Zo-1 in Muscovy ducks, and increasing mucosal transport channels SGLT-1, PepT1, AQP2, AQP3, and AQP10 in duodenum, jejunum, and colon, then decreased the intestinal permeability and intestinal barrier disruption which were caused from RA. From the data, it is apparent that LPS-ZG7 enhanced intestinal mucosal integrity by rising villus height, villus height-to-crypt depth ratio and lower crypt depth. LPS-ZG7 significantly decreased intestinal epithelia cells apoptosis caused by RA invasion, and enhanced intestinal permeability and contribute to barrier dysfunction, ultimately improving intestinal health of host, indirectly leading to reduce diarrhea rate and mortality caused by RA. Overall, this study strengthens the idea that LPS-ZG7 directly inhibited the RA growth by increased RA membrane permeability and damaged the RA membrane integrity, and then indirectly enhanced intestinal mucosal integrity, improved intestinal health of host and mediated intestinal antimicrobial defense.

TNF inhibits AQP2 expression via a miR137-dependent pathway.

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.

The B1 H + -ATPase ( Atp6v1b1 ) Subunit in Non-Type A Intercalated Cells is Required for Driving Pendrin Activity and the Renal Defense Against Alkalosis.

SIGNIFICANCE STATEMENT: In the kidney, the B1 H + -ATPase subunit is mostly expressed in intercalated cells (IC). Its importance in acid-secreting type A ICs is evident in patients with inborn distal renal tubular acidosis and ATP6V1B1 mutations. However, the protein is also highly expressed in alkali-secreting non-type A ICs where its function is incompletely understood. We demonstrate in Atp6v1b1 knock out mice that the B1 subunit is critical for the renal response to defend against alkalosis during an alkali load or chronic furosemide treatment. These findings highlight the importance of non-type A ICs in maintaining acid-base balance in response to metabolic challenges or commonly used diuretics. BACKGROUND: Non-type A ICs in the collecting duct system express the luminal Cl - /HCO 3- exchanger pendrin and apical and/or basolateral H + -ATPases containing the B1 subunit isoform. Non-type A ICs excrete bicarbonate during metabolic alkalosis. Mutations in the B1 subunit (ATP6V1B1) cause distal renal tubular acidosis due to its role in acid secretory type A ICs. The function of B1 in non-type A ICs has remained elusive. METHODS: We examined the responses of Atp6v1b1-/- and Atp6v1b1+/+ mice to an alkali load and to chronic treatment with furosemide. RESULTS: An alkali load or 1 week of furosemide resulted in a more pronounced hypokalemic alkalosis in male ATP6v1b1-/- versus Atp6v1b1+/+ mice that could not be compensated by respiration. Total pendrin expression and activity in non-type A ICs of ex vivo microperfused cortical collecting ducts were reduced, and ß2 -adrenergic stimulation of pendrin activity was blunted in ATP6v1b1-/- mice. Basolateral H + -ATPase activity was strongly reduced, although the basolateral expression of the B2 isoform was increased. Ligation assays for H + -ATPase subunits indicated impaired assembly of V 0 and V 1 H + -ATPase domains. During chronic furosemide treatment, ATP6v1b1-/- mice also showed polyuria and hyperchloremia versus Atp6v1b1+/+ . The expression of pendrin, the water channel AQP2, and subunits of the epithelial sodium channel ENaC were reduced. CONCLUSIONS: Our data demonstrate a critical role of H + -ATPases in non-type A ICs function protecting against alkalosis and reveal a hitherto unrecognized need of basolateral B1 isoform for a proper H + -ATPase complexes assembly and ability to be stimulated.

Poly(ADP-ribose) polymerase-1 affects vasopressin-mediated AQP2 expression in collecting duct cells of the kidney.

Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.

Following the cellular itinerary of renal aquaporin-2 shuttling with 4.5x expansion microscopy.

The shuttling of renal collecting duct aquaporin-2 (AQP2) between intracellular vesicles and the apical plasma membrane is paramount for regulation of renal water reabsorption. The binding of the circulating antidiuretic hormone arginine vasopressin (AVP) to the basolateral AVP receptor increases intracellular cAMP, which ultimately leads to AQP2 plasma membrane accumulation via a dual effect on AQP2 vesicle fusion with the apical plasma membrane and reduced AQP2 endocytosis. This AQP2 plasma membrane accumulation increases water reabsorption and consequently urine concentration. Conventional fluorescent microscopy provides a lateral resolution of ∼250 nm, which is insufficient to resolve the AQP2-containing endosomes/vesicles. Therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x ExM, we detected AQP2 vesicles/endosomes as small as 79 nm considering an average expansion factor of 4.3 for endosomes. Using different markers of the endosomal system provided detailed information of the cellular AQP2 itinerary upon changes in endogenous cAMP levels. Before cAMP elevation, AQP2 colocalized with early and recycling, but not late endosomes. Forskolin-induced cAMP increase was characterized by AQP2 insertion into the plasma membrane and AQP2 withdrawal from large perinuclear endosomes as well as some localization to lysosomal compartments. Forskolin washout promoted AQP2 endocytosis where AQP2 localized to not only early and recycling endosomes but also late endosomes and lysosomes indicating increased AQP2 degradation. Thus, our results show that 4.5 ExM is an attractive approach to obtain detailed information regarding AQP2 shuttling.NEW & NOTEWORTHY Renal aquaporin-2 (AQP2) imaged by expansion microscopy provides unprecedented 3-D information regarding the AQP2 itinerary in response to changes in cellular cAMP.

Tubuloid differentiation to model the human distal nephron and collecting duct in health and disease.

Organoid technology is rapidly gaining ground for studies on organ (patho)physiology. Tubuloids are long-term expanding organoids grown from adult kidney tissue or urine. The progenitor state of expanding tubuloids comes at the expense of differentiation. Here, we differentiate tubuloids to model the distal nephron and collecting ducts, essential functional parts of the kidney. Differentiation suppresses progenitor traits and upregulates genes required for function. A single-cell atlas reveals that differentiation predominantly generates thick ascending limb and principal cells. Differentiated human tubuloids express luminal NKCC2 and ENaC capable of diuretic-inhibitable electrolyte uptake and enable disease modeling as demonstrated by a lithium-induced tubulopathy model. Lithium causes hallmark AQP2 loss, induces proliferation, and upregulates inflammatory mediators, as seen in vivo. Lithium also suppresses electrolyte transport in multiple segments. In conclusion, this tubuloid model enables modeling of the human distal nephron and collecting duct in health and disease and provides opportunities to develop improved therapies.

Perfluorooctanoic acid (PFOA) exposure induces renal filtration and reabsorption disorders via down-regulation of aquaporins.

Perfluorooctanoic acid (PFOA) exposure is associated with kidney dysfunction, however the exact mechanisms by which PFOA induces nephrotoxicity and the specific involvement of aquaporins (AQPs) in kidney tissue remains unclear. In this study, adult male Sprague-Dawley (SD) rats were exposed to PFOA by oral gavage for 28 days and compared with controls. Body weight, water intake and urine volume were recorded daily. At the end of the experiment, blood and kidney samples were collected, and serum urea, creatine and uric acid levels were assessed. The renal expression levels of water channel proteins AQP1, AQP3, AQP2 and p-AQP2 (Ser256) were observed by immunohistochemical staining, and the corresponding transcription levels were detected by Western blot and qRT-PCR. The results showed that PFOA exposure inhibited weight gain and increased water intake, urine volume, kidney weight and renal visceral index. PASM staining and transmission electron microscopy revealed pathological thickening of the glomerular capsule and basement membrane. Serum urea levels were increased, while serum creatine levels were decreased compared to controls. Additionally, the expression levels of AQP1, AQP3, AQP2 and p-AQP2 in kidney tissues were decreased, and the phosphorylation of AQP2 at Ser256 was inhibited. In conclusion, we demonstrate that PFOA exposure can damage the renal filtration barrier and reduce the expression level of AQPs in renal tissues, leading to renal filtration and reabsorption disorders.

Impact of Ring Finger Protein 20 and Its Downstream Regulation on Renal Tubular Injury in a Unilateral Nephrectomy Mouse Model Fed a High-Fat Diet.

Abnormal lipid metabolism increases the relative risk of kidney disease in patients with a single kidney. Using transcriptome analysis, we investigated whether a high-fat diet leads to abnormalities in lipid metabolism and induces kidney cell-specific damage in unilateral nephrectomy mice. Mice with unilateral nephrectomy fed a high-fat diet for 12 weeks exhibited progressive renal dysfunction in proximal tubules, including lipid accumulation, vacuolization, and cell damage. Ring finger protein 20 (RNF20) is a ligase of nuclear receptor corepressor of peroxisome proliferator-activated receptors (PPARs). The transcriptome analysis revealed the involvement of RNF20-related transcriptome changes in PPAR signaling, lipid metabolism, and water transmembrane transporter under a high-fat diet and unilateral nephrectomy. In vitro treatment of proximal tubular cells with palmitic acid induced lipotoxicity by altering RNF20, PPARα, and ATP-binding cassette subfamily A member 1 (ABCA1) expression. PPARγ and aquaporin 2 (AQP2) expression decreased in collecting duct cells, regulating genetic changes in the water reabsorption process. In conclusion, a high-fat diet induces lipid accumulation under unilateral nephrectomy via altering RNF20-mediated regulation and causing functional damage to cells as a result of abnormal lipid metabolism, thereby leading to structural and functional kidney deterioration.