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Animal models of a variety of acquired nephrogenic diabetes insipidus (NDI) disorders have identified a common feature: all such models are associated with the loss of aquaporin-2 (AQP2) from collecting duct principal cells, explaining the associated polyuria. To discover mechanisms of AQP2 loss, previous investigators have carried out either transcriptomics (lithium-induced NDI, unilateral ureteral obstruction, endotoxin-induced NDI) or proteomics (hypokalaemia-associated NDI, hypercalcaemia-associated NDI, bilateral ureteral obstruction), yielding contrasting views. Here, to address whether there may be common mechanisms underlying loss of AQP2 in acquired NDI disorders, we have used bioinformatic data integration techniques to combine information from all transcriptomic and proteomic data sets. The analysis reveals roles for autophagy/apoptosis, oxidative stress and inflammatory signalling as key elements of the mechanism that results in loss of AQP2. These processes can cause AQP2 loss through the combined effects of repression of Aqp2 gene transcription, generalized translational repression, and increased autophagic degradation of proteins including AQP2. Two possible types of stress-sensor proteins, namely death receptors and stress-sensitive protein kinases of the EIF2AK family, are discussed as potential triggers for signalling processes that result in loss of AQP2. KEY POINTS: Prior studies have shown in a variety of animal models of acquired nephrogenic diabetes insipidus (NDI) that loss of the aquaporin-2 (AQP2) protein is a common feature. Investigations of acquired NDI using transcriptomics (RNA-seq) and proteomics (protein mass spectrometry) have led to differing conclusions regarding mechanisms of AQP2 loss. Bioinformatic integration of transcriptomic and proteomic data from these prior studies now reveals that acquired NDI models map to three core processes: oxidative stress, apoptosis/autophagy and inflammatory signalling. These processes cause loss of AQP2 through translational repression, accelerated degradation of proteins, and transcriptional repression., (© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The symptoms of diabetes insipidus may be masked by the concurrence of adrenal insufficiency and emerge after the administration of hydrocortisone, occasionally at high doses. To elucidate the mechanism underlying polyuria induced by the administration of high-dose corticosteroids in the deficiency of arginine vasopressin (AVP), we first examined the secretion of AVP in three patients in whom polyuria was observed only after the administration of high-dose corticosteroids. Next, we examined the effects of dexamethasone or aldosterone on water balance in wild-type and familial neurohypophyseal diabetes insipidus (FNDI) model mice. A hypertonic saline test showed that AVP secretion was partially impaired in all patients. In one patient, there were no apparent changes in AVP secretion before and after the administration of high-dose corticosteroids. In FNDI mice, unlike dexamethasone, the administration of aldosterone increased urine volumes and decreased urine osmolality. Immunohistochemical analyses showed that, after the administration of aldosterone in FNDI mice, aquaporin-2 expression was decreased in the apical membrane and increased in the basolateral membrane in the collecting duct. These changes were not observed in wild-type mice. The present data suggest that treatment with mineralocorticoids induces polyuria by reducing aquaporin-2 expression in the apical membrane of the kidney in partial AVP deficiency.

Objective: Mutations in AQP2 (aquaporin-2) lead to rare congenital nephrogenic diabetes insipidus (NDI), which has been limitedly studied in Chinese population., Methods: Twenty-five subjects from seven families with NDI in a department (Beijing, PUMCH) were screened for AQP2 mutations. Clinical characteristics were described and genotype-phenotype correlation analysis was performed., Results: We identified 9 AQP2 mutations in 13 patients with NDI, including 3 novel AQP2 mutations (p.G165D, p.Q255RfsTer72 and IVS3-3delC). Missense mutations were the most common mutation type, followed by splicing mutations, and frameshift mutations caused by small deletion or insertion. The onset-age in our patients was younger than 1 year old. Common manifestations included polydipsia, polyuria (7/7) and intermittent fever (6/7). Less common presentations included short stature (3/7) and mental impairment (1/7). High osmotic hypernatremia and low osmotic urine were the main biochemical features. Dilation of the urinary tract was a common complication of NDI (3/6). Level of serum sodium in NDI patients with compound het AQP2 mutations was higher than non-compound het mutations., Conclusion: In the first and largest case series of NDI caused by AQP2 mutation in Chinese population, we identified 9 AQP2 mutations, including 3 novel mutations. Phenotype was found to correlate with genotypes, revealed by higher level of serum sodium in patients with compound het AQP2 mutations than non-compound het mutations. This knowledge broadens genotypic and phenotypic spectrum for rare congenital NDI and provided basis for studying molecular biology of AQP2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Tian, Cen, Duan and Xia.)

Background: MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression., Methods: Selective ablation of Dicer in AQP2-expressing cells (Dicer AQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice., Results: The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in Dicer AQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in Dicer AQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409-3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in Dicer AQP2Cre+ mice, resulting in decreased RNA Pol II association., Conclusions: Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression., (Copyright © 2021 by the American Society of Nephrology.)

As a rare hereditary disease, congenital nephrogenic diabetes insipidus (NDI) is clinically characterized by polyuria with hyposthenuria and polydipsia. NDI results from collecting duct principal cell hyporesponsiveness or insensitivity to the antidiuretic action of arginine vasopressin (AVP). The principal cell-specific water channel aquaporin-2 (AQP2) plays an essential role in water reabsorption along osmotic gradients. The capacity to accumulate AQP2 in the apical plasma membrane in response to decreased fluid volume or increased plasma osmolality is critically regulated by the antidiuretic hormone AVP and its receptor 2 (AVPR2). Mutations in AVPR2 result in X-linked recessive NDI, the most common form of inherited NDI. Genetic defects in AQP2 cause autosomal recessive or dominant NDI. In this review, we provide an updated overview of the genetic and molecular mechanisms of congenital NDI, with a focus on the potential disease-causing mutations in AVPR2 and AQP2 , the molecular defects in the AVPR2 and AQP2 mutants, post-translational modifications (i.e., phosphorylation, ubiquitination, and glycosylation) and various protein-protein interactions that regulate phosphorylation, ubiquitination, tetramerization, trafficking, stability, and degradation of AQP2.

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca 2+ ] i . While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1 -/- and Epac2 -/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca 2+ ] i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1 -/- and Epac2 -/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na + and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2.

Previous reports identify a voltage dependent distal renal tubular acidosis (dRTA) secondary to lithium (Li + ) salt administration. This was based on the inability of Li + -treated patients to increase the urine-blood (U-B) pCO 2 when challenged with NaHCO 3 and, the ability of sodium neutral phosphate or Na 2 SO 4 administration to restore U-B pCO 2 in experimental animal models. The underlying mechanisms for the Li + -induced dRTA are still unknown. To address this point, a 7 days time course of the urinary acid-base parameters was investigated in rats challenged with LiCl, LiCitrate, NaCl, or NaCitrate. LiCl induced the largest polyuria and a mild metabolic acidosis. Li + -treatment induced a biphasic response. In the first 2 days, proper urine volume and acidification occurred, while from the 3rd day of treatment, polyuria developed progressively. In this latter phase, the LiCl-treated group progressively excreted more NH 4 + and less pCO 2 , suggesting that NH 3 /NH 4 + became the main urinary buffer. This physiological parameter was corroborated by the upregulation of NBCn1 (a marker of increased ammonium recycling) in the inner stripe of outer medulla of LiCl treated rats. Finally, by investigating NH 4 + excretion in ENaC-cKO mice, a model resistant to Li + -induced polyuria, a primary role of the CD was confirmed. By definition, dRTA is characterized by deficient urinary ammonium excretion. Our data question the presence of a voltage-dependent Li + -induced dRTA in rats treated with LiCl for 7 days and the data suggest that the alkaline urine pH induced by NH 3 /NH 4 + as the main buffer has lead to the interpretation dRTA in previous studies.

Background and Objectives: Genetic heterogeneity and phenotypic variability are major challenges in familial nephronophthisis and related ciliopathies. To date, mutations in 20 different genes ( NPHP1 to -20 ) have been identified causing either isolated kidney disease or complex multiorgan disorders. In this study, we provide a comprehensive and detailed characterization of 152 children with a special focus on extrarenal organ involvement and the long-term development of ESRD., Design, Setting, Participants, & Measurements: We established an online-based registry (www.nephreg.de) to assess the clinical course of patients with nephronophthisis and related ciliopathies on a yearly base. Cross-sectional and longitudinal data were collected. Mean observation time was 7.5±6.1 years., Results: In total, 51% of the children presented with isolated nephronophthisis, whereas the other 49% exhibited related ciliopathies. Monogenetic defects were identified in 97 of 152 patients, 89 affecting NPHP genes. Eight patients carried mutations in other genes related to cystic kidney diseases. A homozygous NPHP1 deletion was, by far, the most frequent genetic defect ( n =60). We observed a high prevalence of extrarenal manifestations (23% [14 of 60] for the NPHP1 group and 66% [61 of 92] for children without NPHP1 ). A homozygous NPHP1 deletion not only led to juvenile nephronophthisis but also was able to present as a predominantly neurologic phenotype. However, irrespective of the initial clinical presentation, the kidney function of all patients carrying NPHP1 mutations declined rapidly between the ages of 8 and 16 years, with ESRD at a mean age of 11.4±2.4 years. In contrast within the non- NPHP1 group, there was no uniform pattern regarding the development of ESRD comprising patients with early onset and others preserving normal kidney function until adulthood., Conclusions: Mutations in NPHP genes cause a wide range of ciliopathies with multiorgan involvement and different clinical outcomes., (Copyright © 2017 by the American Society of Nephrology.)

The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of β1-integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of β1-integrin specifically in ICs and PCs, respectively, did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of β1-integrin in CD cells, specifically in the PCs. We conditionally deleted β1-integrin in mouse CD PCs using a specific aquaporin-2 (AQP2) promoter Cre-LoxP system. The resulting mutant mice, β-1 f/f AQP2-Cre+, had lower body weight, failed to thrive, and died around 8-12 wk. Their CD tubules were dilated, and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of transforming growth factor-β (TGF-β)-induced protein, fibronectin, and TGF-β receptor-1 mRNAs and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-β signaling pathway. Therefore, our data reveal that normal expression of β1-integrin in PCs is a critical determinant of CD structural and functional integrity and further support the previously reported critical role of β1-integrin in the development and/or maintenance of the CD structure and function., (Copyright © 2017 the American Physiological Society.)

The transcription factor hepatocyte nuclear factor-1 β (HNF-1 β ) is essential for normal kidney development and function. Inactivation of HNF-1 β in mouse kidney tubules leads to early-onset cyst formation and postnatal lethality. Here, we used Pkhd1/Cre mice to delete HNF-1 β specifically in renal collecting ducts (CDs). CD-specific HNF-1 β mutant mice survived long term and developed slowly progressive cystic kidney disease, renal fibrosis, and hydronephrosis. Compared with wild-type littermates, HNF-1 β mutant mice exhibited polyuria and polydipsia. Before the development of significant renal structural abnormalities, mutant mice exhibited low urine osmolality at baseline and after water restriction and administration of desmopressin. However, mutant and wild-type mice had similar plasma vasopressin and solute excretion levels. HNF-1 β mutant kidneys showed increased expression of aquaporin-2 mRNA but mislocalized expression of aquaporin-2 protein in the cytoplasm of CD cells. Mutant kidneys also had decreased expression of the UT-A urea transporter and collectrin, which is involved in apical membrane vesicle trafficking. Treatment of HNF-1 β mutant mIMCD3 cells with hypertonic NaCl inhibited the induction of osmoregulated genes, including Nr1h4 , which encodes the transcription factor FXR that is required for maximal urinary concentration. Chromatin immunoprecipitation and sequencing experiments revealed HNF-1 β binding to the Nr1h4 promoter in wild-type kidneys, and immunoblot analysis revealed downregulated expression of FXR in HNF-1 β mutant kidneys. These findings reveal a novel role of HNF-1 β in osmoregulation and identify multiple mechanisms, whereby mutations of HNF-1 β produce defects in urinary concentration., (Copyright © 2017 by the American Society of Nephrology.)

Two processes are associated with progressive loss of renal function: 1) decreased aquaporin-2 (AQP2) expression and urinary concentrating capacity (Nephrogenic Diabetes Insipidus, NDI); and 2) changes in extracellular matrix (ECM) composition, e.g. increased collagen I (Col I) deposition, characteristic of tubule-interstitial fibrosis. AQP2 expression is regulated by both the ECM-to-intracellular scaffold protein integrin-linked kinase (ILK) by NFATc/AP1 and other transcription factors. In the present work, we used in vivo and in vitro approaches to examine ILK participation in NFATc3/AP-1-mediated increases in AQP2 gene expression. Both NFATc3 knock-out mice and ILK conditional-knockdown mice (cKD-ILK) display symptoms of NDI (polyuria and reduced AQP2 expression). NFATc3 is upregulated in the renal medulla tubular cells of cKD-ILK mice but with reduced nuclear localization. Inner medullary collecting duct mIMCD3 cells were subjected to ILK depletion and transfected with reporter plasmids. Pharmacological activators or inhibitors determined the effect of ILK activity on NFATc/AP-1-dependent increases in transcription of AQP2. Finally, mIMCD3 cultured on Col I showed reduced activity of the ILK/GSK3β/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5 Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5 Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5 Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow-derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5 Y/- proximal tubule cells with bone marrow-derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow-derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow-derived cells and diseased tubular cells play a key role in the rescue mechanism., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Aims: The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (Clock Δ19/Δ19 ) mice in order to determine the effects of clock genes on NOC/NP., Methods: Male C57BL/6 mice aged 8-12 weeks (WT) and male C57BL/6 Clock Δ19/Δ19 mice aged 8 weeks were used. They were bred under 12 hr light/dark conditions for 2 weeks and voiding behavior was investigated by measuring water intake volume, urine volume, urine volume/void, and voiding frequency in metabolic cages in the dark and light periods., Results: No significant differences were observed in behavior patterns between Clock Δ19/Δ19 and WT mice. Clock Δ19/Δ19 mice showed greater voiding frequencies and urine volumes during the sleep phase than WT mice. The diurnal change in urine volume/void between the dark and light periods in WT mice was absent in Clock Δ19/Δ19 mice. Additionally, functional bladder capacity was significantly lower in Clock Δ19/Δ19 mice than in WT mice., Conclusions: We demonstrated that Clock Δ19/Δ19 mice showed the phenotype of NOC/NP. The Clock Δ19/Δ19 mouse may be used as an animal model of NOC and NP. Neurourol. Urodynam. 36:1034-1038, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2.

Background/aims: Glucose-galactose malabsorption (GGM) is a rare and potentially fatal disorder. The autosomal recessive mutation of the SGLT1 gene interferes with the active glucose transport in the gut resulting in osmotic diarrhea and failure to thrive (FTT). Two nonrelated infants with GGM are presented as well as a novel mutation in SGLT1., Case Presentation: The first case consulted for FTT and presented with hypercalcemia and hypercalciuria. His mother had self-medicated with high doses of vitamin D. The second case consulted for macroscopic hematuria, and presented with dehydration and secondary acute kidney injury. In both cases, the profuse diarrhea, initially mistaken for polyuria, promptly resolved after the introduction of glucose-galactose-free milk. Investigations showed bilateral nephrocalcinosis and high levels of 1,25(OH)2D3 in both patients. We hypothesize that the upregulation of epithelial calcium channels (TRPV6) and 1,25(OH)2D3 are possible factors involved in the pathophysiology of nephrocalcinosis sometimes seen in GGM. Furthermore, a novel intronic SGLT1 mutation (c.207+2dup) is described., Conclusion: These 2 cases demonstrate that a malabsorption disorder such as GGM can present with nephrocalcinosis and/or hypercalcemia, with increased 1,25(OH)2D3 levels in infants. Prompt recognition of GGM is sometimes difficult but crucial.
., (© 2017 S. Karger AG, Basel.)

Several aquaporin (AQP )-type water channels are expressed in kidney: AQP1 in the proximal tubule, thin descending limb of Henle, and vasa recta; AQP2 -6 in the collecting duct; AQP7 in the proximal tubule; AQP8 in the proximal tubule and collecting duct; and AQP11 in the endoplasmic reticulum of proximal tubule cells. AQP2 is the vasopressin-regulated water channel that is important in hereditary and acquired diseases affecting urine-concentrating ability. The roles of AQPs in renal physiology and transepithelial water transport have been determined using AQP knockout mouse models. This chapter describes renal physiologic insights revealed by phenotypic analysis of AQP knockout mice and the prospects for further basic and clinical studies.

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia, and alkalosis. In recent years, several Bartter syndrome-associated mutations have been described that result in truncations of the intracellular carboxyl terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation, and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at Trp-610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClC-Kb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyl terminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltage-dependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyl terminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxyl-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyl terminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mammalian class IX myosin Myo9a is a single-headed, actin-dependent motor protein with Rho GTPase-activating protein activity that negatively regulates Rho GTPase signaling. Myo9a is abundantly expressed in ciliated epithelial cells of several organs. In mice, genetic deletion of Myo9a leads to the formation of hydrocephalus. Whether Myo9a also has essential functions in the epithelia of other organs of the body has not been explored. In the present study, we report that Myo9a-deficient mice develop bilateral renal disease, characterized by dilation of proximal tubules, calyceal dilation, and thinning of the parenchyma and fibrosis. These structural changes are accompanied by polyuria (with normal vasopressin levels) and low-molecular-weight proteinuria. Immunohistochemistry revealed that Myo9a is localized to the circumferential F-actin belt of proximal tubule cells. In kidneys lacking Myo9a, the multiligand binding receptor megalin and its ligand albumin accumulated at the luminal surface of Myo9a-deficient proximal tubular cells, suggesting that endocytosis is dysregulated. In addition, we found, surprisingly, that levels of murine diaphanous-related formin-1, a Rho effector, were decreased in Myo9a-deficient kidneys as well as in Myo9a knockdown LLC-PK1 cells. In summary, deletion of the Rho GTPase-activating protein Myo9a in mice causes proximal tubular dilation and fibrosis, and we speculate that downregulation of murine diaphanous-related formin-1 and impaired protein reabsorption contribute to the pathophysiology., (Copyright © 2015 the American Physiological Society.)

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive disorder caused by mutations in the CLDN16 or CLDN19 genes, encoding claudin-16 and claudin-19 in the thick ascending limb of Henle's loop. In patients with claudin-19 mutations, severe ocular involvement (macular coloboma, pigmentary retinitis, nystagmus, or visual loss) has been described. In this report, we presented a 12-year-old girl with rickets, polyuria, and polydipsia. She was the daughter of consanguineous parents, and she had a history of recurred hypocalcemic and hypomagnesemic tetany. On physical examination, bilateral horizontal nystagmus and severe myopia were detected. Laboratory examination revealed hypomagnesemia, hypocalcemia, hypercalciuria, nephrocalcinosis, and renal stone. A clinical diagnosis of FHHNC caused possibly by claudin-19 mutation was decided with the ocular findings. DNA analysis revealed a novel homozygous missense mutation c.241C>T in the CLDN19 gene. In conclusion, in a patient with hypomagnesemia, hypercalciuria, nephrocalcinosis, and ocular findings, a diagnosis of FHHNC caused by claudin-19 mutation should be considered. This is the first study of FHHNC in Chinese population. Our findings of the novel mutation c.241C>T in exon 2 add to the list of more than 16 mutations of CLDN19 gene reported.

In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To investigate the role of GSK3α in urine concentration, we compared GSK3α knockout (GSK3αKO) mice with wild-type (WT) littermates. Under normal conditions, GSK3αKO mice had higher water intake and urine output. GSK3αKO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3αKO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3α in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3αKO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3α could play a crucial role in renal urine concentration and suggest that GSK3α might be one of the initial targets of Li(+) in LiCl-induced nephrogenic diabetes insipidus., (Copyright © 2015 the American Physiological Society.)

A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane-anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2)(+) principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1., (Copyright © 2015 by the American Society of Nephrology.)

Context: Familial hyperaldosteronism type III (FH-III) is a rare and clinically heterogeneous condition, that can display mild as well as severe phenotypes. Point mutations in the KCNJ5 gene, affecting the ion selectivity of the inward rectifier K(+) channel 4 (Kir3.4), underlie the molecular basis of FH-III., Objective: The objective of the study was to investigate the effects of a de novo germline KCNJ5 mutation., Patients and Methods: We describe the case of a girl who came to medical attention at the age of 2 years because of polydipsia, polyuria, and failure to thrive. The patient, affected by hypertension and hypokalemia, was diagnosed with primary aldosteronism on the basis of extremely high aldosterone levels and suppressed plasma renin activity. Genomic DNA was isolated and KCNJ5 sequenced. Human adrenocortical cells were used as an in vitro model for the functional characterization of the mutant channel., Results: KCNJ5 sequencing in the index case and her parents revealed a de novo p.Glu145Gln germline mutation. The substitution resulted in Na(+)-dependent depolarization of adrenal cells and increased intracellular calcium concentration, which activated the transcription of NR4A2 and, in turn, CYP11B2. Pharmacological studies revealed that the mutant channel was insensitive to tertiapin-Q and calcium-channel blocker verapamil., Conclusions: Herein we report the identification of a novel KCNJ5 germline mutation responsible for severe hyperaldosteronism that presented in infancy with symptoms of diabetes insipidus. The findings of this study further elucidate the etiology of FH-III and expand our knowledge of this rare condition.

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular-to-intracellular scaffold protein integrin-linked kinase (ILK) regulates expression of tubular water channel aquaporin-2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional-knockdown (cKD-ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine-vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD-ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD-ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI., (© FASEB.)

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and autophagy. Despite widespread clinical use of mTORC1 inhibitors, the role of mTORC1 in renal tubular function and kidney homeostasis remains elusive. By using constitutive and inducible deletion of conditional Raptor alleles in renal tubular epithelial cells, we discovered that mTORC1 deficiency caused a marked concentrating defect, loss of tubular cells, and slowly progressive renal fibrosis. Transcriptional profiling revealed that mTORC1 maintains renal tubular homeostasis by controlling mitochondrial metabolism and biogenesis as well as transcellular transport processes involved in countercurrent multiplication and urine concentration. Although mTORC2 partially compensated for the loss of mTORC1, exposure to ischemia and reperfusion injury exaggerated the tubular damage in mTORC1-deficient mice and caused pronounced apoptosis, diminished proliferation rates, and delayed recovery. These findings identify mTORC1 as an important regulator of tubular energy metabolism and as a crucial component of ischemic stress responses.

Background: Familial hyperaldosteronism type III (FH-III) is a rare autosomal dominant disease for which five missense mutations in KCNJ5 have been identified. FH-III has a wide phenotypic variability from spironolactone-responsive hyperaldosteronism to massive adrenal hypertrophy with drug-resistant hypertension. This variation has mainly been attributed to genotype, because, in contrast to other genotypes (G151R, T158A, I157S, and Y152C), (1) FH-III patients with G151E have shown milder phenotype, and (2) G151E-harboring cells were found to have rapid lethality due to much larger sodium conductance of the encoded channel (Kir3.4), which prevents adrenal hypertrophy., Methods: Here we describe the clinical course of a sporadic case of FH-III, with de novo G151R mutation., Results: The patient developed polyuria at around 1.5 years of age and developed hypertension and hypokalemia by 4 years of age. Thereafter, spironolactone treatment successfully ameliorated hyperaldosteronism for 7 years with no discernible adrenal enlargement., Conclusion: Diverse clinical severity in FH-III cannot be defined solely by KCNJ5 genotype., (© 2014 S. Karger AG, Basel.)

A pregnant woman presented at gestational week 28 with loss of consciousness and profound polyuria. Further characterization revealed osmotic diuresis due to massive glycosuria without hyperglycemia. Glycosuria reduced substantially postpartum, from approximately 100 to approximately 30 g/1.73 m2 per day. DNA sequencing analysis of the SLC5A2 gene encoding the renal glucose transporter SGLT2 showed a homozygous frame-shift mutation (occurring after the glutamine at amino acid 168 and leading to premature termination of the protein at amino acid 186) diagnostic of familial renal glycosuria. Pregnant women with familial renal glycosuria can be at risk of profound polyuria during pregnancy due to the associated increase in glycosuria. These findings also have implications for the use of SGLT2 inhibitors in clinical practice., (Published by Elsevier Inc.)

The mammalian collecting duct comprises principal and intercalated cells, which maintain sodium/water and acid/base balance, respectively, but the epigenetic contributors to the differentiation of these cell types remain unknown. Here, we investigated whether the histone H3 K79 methyltransferase Dot1l, which is highly expressed in principal cells, participates in this process. Taking advantage of the distribution of aquaporin 2 (Aqp2), which localizes to principal cells of the collecting duct, we developed mice lacking Dot1l in Aqp2-expressing cells (Dot1l(AC)) and found that these mice had approximately 20% fewer principal cells and 13%-16% more intercalated cells than control mice. This deletion of Dot1l in principal cells abolished histone H3 K79 methylation in these cells, but unexpectedly, most intercalated cells also had undetectable di-methyl K79, suggesting that Aqp2(+) cells give rise to intercalated cells. These Aqp2(+) cell-derived intercalated cells were present in both developing and mature kidneys. Furthermore, compared with control mice, Dot1l(AC) mice had 40% higher urine volume and 18% lower urine osmolarity with relatively normal electrolyte and acid-base homeostasis. In conclusion, these data suggest that Dot1l deletion facilitates the differentiation of some α- and β-intercalated cells from Aqp2-expressing progenitor cells or mature principal cells.

Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC)) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC) vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC) kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC) vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC) mice and in patients with diabetic nephropathy.

Background and Aims: The thiazide-sensitive Na(+)-Cl(-) cotransporter NCC and the Cl(-)/HCO3(-)exchanger pendrin are expressed on apical membranes of distal cortical nephron segments and mediate salt absorption, with pendrin working in tandem with the epithelial Na(+) channel (ENaC) and the Na(+)-dependent chloride/bicarbonate exchanger (NDCBE), whereas NCC is working by itself. A recent study showed that NCC and pendrin compensate for loss of each other under basal conditions, therefore masking the role that each plays in salt reabsorption. Carbonic anhydrase II (CAII, CA2 or CAR2) plays an important role in acid-base transport and salt reabsorption in the proximal convoluted tubule and acid-base transport in the collecting duct. Animals with CAII deletion show remodeling of intercalated cells along with the downregulation of pendrin. NCC KO mice on the other hand show significant upregulation of pendrin and ENaC. Neither model shows any significant salt wasting under baseline conditions. We hypothesized that the up-regulation of pendrin is essential for the prevention of salt wasting in NCC KO mice., Methods and Results: To test this hypothesis, we generated NCC/CAII double KO (dKO) mice by crossing mice with single deletion of NCC and CAII. The NCC/CAII dKO mice displayed significant downregulation of pendrin, along with polyuria and salt wasting. As a result, the dKO mice developed volume depletion, which was associated with the inability to concentrate urine., Conclusions: We conclude that the upregulation of pendrin is essential for the prevention of salt and water wasting in NCC deficient animals and its downregulation or inactivation will result in salt wasting, impaired water conservation and volume depletion in the setting of NCC inactivation or inhibition., (© 2014 S. Karger AG, Basel.)

A 3-year 5-month-old boy was seen for second opinion regarding polydipsia and polyuria. Previously, a diagnosis of primary polydipsia was made after normal urine concentration after overnight water deprivation testing. The boy's father, paternal grandfather, and paternal aunt had diabetes insipidus treated with desmopressin acetate. Based on this young boy's symptoms, ability to concentrate urine after informal overnight water deprivation, and family history of diabetes insipidus, we performed AVP gene mutation testing. Analysis of the AVP gene revealed a novel mutation G54E that changes a normal glycine to glutamic acid, caused by a guanine to adenine change at nucleotide g.1537 (exon 2) of the AVP gene. Commonly, patients with familial neurohypophyseal diabetes insipidus (FNHDI) present within the first 6 years of life with progressively worsening polyuria and compensatory polydipsia. Since these patients have progressive loss of arginine vasopressin (AVP), they may initially respond normally to water deprivation testing and have normal pituitary findings on brain MRI. Genetic testing may be helpful in these patients, as well as preemptively diagnosing those with a mutation, thereby avoiding unnecessary surveillance of those unaffected.

Recent discoveries have shed new light on the understanding of water metabolism: (1.) in addition to hypothalamic osmoreceptor cells expressing a TRPV1 variant, there are peripheral TRPV4 receptors sensing tonicity in the portal vein and changing central vasopressin secretion and peripheral autonomic activity; (2.) the central osmoregulatory gain of angiotensin action participates in the non-osmotic release of vasopressin induced by hypovolaemia; (3.) prostaglandins EP2 receptors on principal cells of the collecting ducts positively regulate urine concentration mechanisms. These new developments are important clinically for the understanding of hereditary polyuric states. We recommend sequencing of the nephrogenic diabetes insipidus genes in all affected patients. This genomic information is key to the routine care of patients with congenital polyuria and, as in other genetic diseases, reduces health costs and confers psychological benefits on patients and families.

We report the molecular findings for the CTNS gene in 12 Turkish cystinosis patients aged 7-29 years. All presented initially with severe failure to thrive, polyuria, and polydipsia. Cystinosis was diagnosed at age 1 month to 9 years. Seven patients reached end-stage renal failure at ages ranging from 6.5 to 15 years. Whereas three of the remaining five have renal Fanconi syndrome with proteinuria, two have had kidney failure of varying degrees. Molecular analyses involved an initial multiplex polymerase chain reaction (PCR) to determine the presence or absence of the 57-kb northern European founder deletion in CTNS, followed by sequencing of the ten coding exons of CTNS. Comprehensive mutation analysis verified that none of the 12 patients carried the common 57-kb deletion. We identified four previously reported nucleotide variations associated with cystinosis and five new variants: a 10-kb deletion, three missense variants, and a nucleotide substitution in a potential branch point site of intron 4. This study is the first molecular analysis of Turkish cystinosis patients and provides guidance for the molecular diagnosis of cystinosis in this population.

Familial hyperaldosteronism (FH) encompasses 3 types of autosomal dominant hyperaldosteronisms leading to inheritable hypertension. FH type II (FH-II), undistinguishable from sporadic hyperaldosteronism, represents the most frequent cause of inheritable hypertension and is believed to only manifest in adults. FH-III is a severe variety of PA resistant to pharmacotherapy and recently demonstrated to be caused by mutations in the gene encoding the potassium channel KCNJ5. In this report, we describe a FH pediatric patient, remarkable both for age at onset and unusual presentation: a two-years old girl with polyuric-polydipsic syndrome and severe hypertension, successfully treated with canrenone and amiloride. The girl had severe hypertension, hypokalemia, hypercalciuria, suppressed renin activity, high aldosterone, and unremarkable adrenal imaging. FH type I was ruled out by glucocorticoid suppression test, PCR test for CYP11B1/CYP11B2 gene, and urinary 18-oxo-cortisol and 18-hydroxy-cortisol excretion, which was in FH-II range. In spite of a clear-cut FH-II phenotype, the girl and her mother were found to harbor a FH-III genotype with KCNJ5 mutation (c.452G>A). Treatment with canrenone was started, resulting in prompt normalization of electrolytes and remission of polyuric-polydypsic syndrome. The addition of amiloride led to a complete normalization of blood pressure. This report expands the phenotypic spectrum of FH-III to a milder end, mimiking FH-II phenotype demonstrating that pharmacotherapy may be effective. This also implies that FH-II/III should be considered in the differential diagnosis of hypertensive children and, perhaps, that the offspring of patients with hyperaldosteronism should be screened for hypertension.

Suboptimal kidney development resulting from a genetic deficit in nephron number can have lifelong consequences that may lead to cardiorenal complications upon exposure to secondary insults in later life. To determine whether the inherited reduced renal reserve compromises the ability to handle osmotic stress in the adult animal, we challenged the heterozygous 3H1 Brachyrrhine (Br/+) mouse, which displays heritable renal hypoplasia associated with reduced embryonic six2 expression, to a solution of 2% NaCl for 5 days or to fluid restriction for 48 h. Blood chemistry, fluid intake, and physiological parameters, including renal measurements, were determined. Systemic hypertonicity by prolonged salt loading led to significant increases in plasma osmolality and plasma Na(+), along with polydipsia and polyuria, with a significant urine-concentrating defect that was resistant to DDAVP treatment in the adult Br/+ mouse compared with wild-type littermates. The Br/+ mouse also developed a significant increase in blood urea nitrogen at baseline that was further elevated when 2% NaCl was given. Fluid restriction for 48 h further enhanced plasma osmolality and plasma Na(+) responses, although the Br/+ mouse was evidently able to produce a small amount of concentrated urine at this time. Hypothalamic c-Fos expression was appropriately activated in the Br/+ mouse in response to both osmotic challenges, indicating an intact central neuroendocrine pathway that was not affected by the lack of congenital six2 expression. Collectively, our results demonstrate impaired osmoregulatory mechanisms consistent with chronic renal failure in the Br/+ mouse and indicate that six2 haploinsufficiency has a direct effect on postnatal fluid and electrolyte handling associated with fluid imbalance.

Objective: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI), a disorder caused by mutations in the vasopressin (AVP)-neurophysin II (NPII) gene, manifests gradually during early childhood with progressive polyuria and polydipsia. Patients are usually treated with synthetic AVP analog. If unlimited access to water is provided, prognosis is usually good even in the absence of specific treatment. In this study, we describe three families with adFNDI, in which growth failure was a prominent complaint, on the clinical and molecular level., Design/methods: Histories from affected and unaffected family members were taken. Height and weight of index patients were recorded longitudinally. Patients underwent water deprivation tests, magnetic resonance imaging, and genetic analysis. One mutant was studied by heterologous expression in cell culture., Results: A total of ten affected individuals were studied. In two of the three pedigrees, a novel mutation in the AVP-NPII gene was found. The index children in each pedigree showed growth retardation, which was the reason for referral in two. In these cases, water intake was tightly restricted by the parents in an attempt to overcome suspected psychogenic polydipsia and to improve appetite. Once the children were treated by hormone replacement, they rapidly caught up to normal weight and height., Conclusions: Genetic testing and appropriate parent counseling should be enforced in adFNDI families to ensure adequate treatment and avoid chronic water deprivation, which causes failure to thrive.

Background: A family was identified with autosomal dominant inheritance of anemia, polyuria, hyperuricemia, and chronic kidney disease. Mutational analysis revealed a novel heterozygous mutation c.58T > C resulting in the amino acid substitution of cysteine for arginine in the preprorenin signal sequence (p.cys20Arg) occurring in all affected members., Methods: Effects of the identified mutation were characterized using in vitro and in vivo studies. Affected individuals were clinically characterized before and after administration of fludrocortisone., Results: The mutation affects endoplasmic reticulum co-translational translocation and posttranslational processing, resulting in massive accumulation of non-glycosylated preprorenin in the cytoplasm. This affects expression of intra-renal RAS components and leads to ultrastructural damage of the kidney. Affected individuals suffered from anemia, hyperuricemia, decreased urinary concentrating ability, and progressive chronic kidney disease. Treatment with fludrocortisone in an affected 10-year-old child resulted in an increase in blood pressure and estimated glomerular filtration rate., Conclusions: A novel REN gene mutation resulted in an alteration in the amino acid sequence of the renin signal sequence and caused childhood anemia, polyuria, and kidney disease. Treatment with fludrocortisone improved renal function in an affected child. Nephrologists should consider REN mutational analysis in families with autosomal dominant inheritance of chronic kidney disease, especially if they suffer from anemia, hyperuricemia, and polyuria in childhood.

The renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role within individual tissues such as the brain. The brain RAS is thought to control blood pressure through effects on fluid intake, vasopressin release, and sympathetic nerve activity (SNA), and may regulate metabolism through mechanisms which remain undefined. We used a double-transgenic mouse model that exhibits brain-specific RAS activity to examine mechanisms contributing to fluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenal steroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptor blockade. They are also hyperphagic but lean because of a marked increase in body temperature and metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalized metabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluid intake and energy expenditure., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). The analyses of knock-in mice expressing a mutant NPII that causes FNDI in humans demonstrated that polyuria progressed substantially in the absence of loss of AVP neurones. Morphological analyses revealed that inclusion bodies were present in the AVP neurones in the supraoptic nucleus and that the size and numbers of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) of AVP neurones. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregate formation in the ER is likely to be related to the pathogenesis of the progressive polyuria.

The bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter NKCC2, located in the thick ascending limb of Henle's loop, plays a critical role in the kidney's ability to concentrate urine. In humans, loss-of-function mutations of the solute carrier family 12 member 1 gene (SLC12A1), coding for NKCC2, cause type I Bartter syndrome, which is characterized by prenatal onset of a severe polyuria, salt-wasting tubulopathy, and hyperreninemia. In this study, we describe a novel chemically induced, recessive mutant mouse line termed Slc12a1(I299F) exhibiting late-onset manifestation of type I Bartter syndrome. Homozygous mutant mice are viable and exhibit severe polyuria, metabolic alkalosis, marked increase in plasma urea but close to normal creatininemia, hypermagnesemia, hyperprostaglandinuria, hypotension,, and osteopenia. Fractional excretion of urea is markedly decreased. In addition, calcium and magnesium excretions are more than doubled compared with wild-type mice, while uric acid excretion is twofold lower. In contrast to hyperreninemia present in human disease, plasma renin concentration in homozygotes is not increased. The polyuria observed in homozygotes may be due to the combination of two additive factors, a decrease in activity of mutant NKCC2 and an increase in medullary blood flow, due to prostaglandin-induced vasodilation, that impairs countercurrent exchange of urea in the medulla. In conclusion, this novel viable mouse line with a missense Slc12a1 mutation exhibits most of the features of type I Bartter syndrome and may represent a new model for the study of this human disease.

Dent's disease is an X-linked renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis or nephrolithiasis. The disease is caused by mutations in a renal chloride channel gene, CLCN5. We report on three boys, of Indian origin, with Dent's disease that presented at an early age (1-4 years), with polyuria, polydipsia, salt craving, recurrent vitamin A-responsive night blindness, hypophosphataemic rickets, hypercalciuria and low molecular weight proteinuria. All these patients were found to have novel mutations in the CLCN5 gene.

Vitamin D receptor (VDR)-null mice develop polyuria, but the underlying mechanism remains unknown. In this study, we investigated the relationship between vitamin D and homeostasis of water and electrolytes. VDR-null mice had polyuria, but the urine osmolarity was normal as a result of high salt excretion. The urinary responses to water restriction and to vasopressin were similar between wild-type and VDR-null mice, suggesting intact fluid-handling capacity in VDR-null mice. Compared with wild-type mice, however, renin and angiotensin II were dramatically upregulated in the kidney and brain of VDR-null mice, leading to a marked increase in water intake and salt appetite. Angiotensin II-mediated upregulation of intestinal NHE3 expression partially explained the increased salt absorption and excretion in VDR-null mice. In the brain of VDR-null mice, expression of c-Fos, which is known to associate with increased water intake, was increased in the hypothalamic paraventricular nucleus and the subfornical organ. Treatment with an angiotensin II type 1 receptor antagonist normalized water intake, urinary volume, and c-Fos expression in VDR-null mice. Furthermore, despite a salt-deficient diet to reduce intestinal salt absorption, VDR-null mice still maintained the increased water intake and urinary output. Together, these data indicate that the polyuria observed in VDR-null mice is not caused by impaired renal fluid handling or increased intestinal salt absorption but rather is the result of increased water intake induced by the increase in systemic and brain angiotensin II.

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowman's capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.

Aquaporin 2 (AQP2) is responsible for regulating the concentration of urine in the collecting tubules of the kidney under the control of vasopressin (Vp). Studies using Vp-deficient Brattleboro rats, however, indicated the existence of substantial Vp-independent mechanisms for membrane insertion, as well as transcriptional regulation, of this water channel. The Vp-independent mechanism(s) is clinically relevant to patients with X-linked nephrogenic diabetes insipidus (NDI) by therapeutically bypassing the dysfunctional Vp receptor. On the basis of studies with secretin receptor-null (SCTR(-/-)) mice, we report here for the first time that mutation of the SCTR gene could lead to mild polydipsia and polyuria. Additionally, SCTR(-/-) mice were shown to have reduced renal expression of AQP2 and AQP4, as well as altered glomerular and tubular morphology, suggesting possible disturbances in the filtration and/or water reabsorption process in these animals. By using SCTR(-/-) mice as controls and comparing them with wild-type animals, we performed both in vivo and in vitro studies that demonstrated a role for secretin in stimulating (i) AQP2 translocation from intracellular vesicles to the plasma membrane in renal medullary tubules and (ii) expression of this water channel under hyperosmotic conditions. The present study therefore provides information for at least one of the Vp-independent mechanisms that modulate the process of renal water reabsorption. Future investigations in this direction should be important in developing therapeutic means for treating NDI patients.

Most cases of hydronephrosis are caused by urinary tract obstruction. However, excessive polyuric syndrome rarely gives rise to non-obstructive hydronephrosis, megaureter, and a distended bladder. The authors report here on two cases of congenital nephrogenic diabetes insipidus (NDI) with severe bilateral hydronephrosis and megaureter. It is Interesting that the patients were symptomless except for their polyuria, and they both presented with bilateral hydronephrosis. Fluid deprivation testing revealed the presence of AVP resistant NDI. Gene analysis for these patients showed the AVP receptor 2 (V2R) missense mutations (Q225X and S126F), which have previously been reported on in other studies. We made the diagnosis of NDI by using a physiologic test, and we confirmed it by mutation analysis of the V2R gene.

Aquaporin-2 (AQP2) is responsible for the concentration of urine in the kidney collecting tubule under the regulation of vasopressin. The mRNA level of this water channel in polydipsic STR/N mice was extremely reduced compared with that in normal ICR mice. In male mice, reduction of the AQP2 mRNA level was not evident at 3 wk of age, at which time water intake was not increased. At 10 wk of age, however, the AQP2 mRNA level was reduced to 10% of that in control mice, whereas water intake was increased by 36%. At 44 wk, the water intake became five times that of the control ICR mice, and the AQP2 mRNA level in these polydipsic mice was only approximately 5% of control. Similar changes were observed in the AQP2 protein level, suggesting that the mRNA level of AQP2 reflects the protein level of AQP2. These inverse changes in the AQP2 mRNA level and water intake were also evident in female mice. The data imply that polydipsia in STR/N mice may have affected AQP2 mRNA transcription in the kidney, resulting in reduced AQP2 expression, which would contribute to a reduction in overretention of water.

Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c-/-) are born at the expected ratio, but approximately 80% die of dehydration within a few days. The surviving Ren1c-/- mice have no renin mRNA expression in the kidney, hydronephrosis, thickening of renal arterial walls, and fibrosis in the kidney. Plasma renin and angiotensins I and II are undetectable. Urinary aldosterone is 6% wild-type. They have low tail-cuff BP (84 +/- 4 versus 116 +/- 5 mmHg in +/+) and excrete large amounts of urine (5.2 +/- 0.8 ml/d, 725 +/- 34 mOsm versus 1.1 +/- 0.1 ml/d, 2460 +/- 170 mOsm in +/+). After 5 d of drinking 5% dextrose, desmopressin does not increase the osmolality of the urine in -/- mice (624 +/- 19 to 656 +/- 25 mOsm), whereas in +/+, it increases severalfold (583 +/- 44 to 2630 +/- 174 mOsm). Minipump infusion of angiotensin II to Ren1c-/- mice restores BP to wild-type level, but preexisting damage to the medulla prevents complete restoration of the ability of the kidney to concentrate urine. Heterozygous Ren1c+/- mice, in contrast, are indistinguishable from +/+ in BP, urine volume, and osmolality. Kidney renin mRNA, the number of kidney cells producing renin, and plasma renin concentration in the Ren1c+/- mice are also indistinguishable from +/+. These results demonstrate that renin is the only enzyme capable of maintaining plasma angiotensins and that renin expression in the kidney is very tightly regulated at the mRNA level.

We describe a novel missense mutant of arginine vasopressin (AVP)-dependent neurohypophyseal diabetes insipidus in an autosomal dominant family. A 54-year-old woman was admitted to our hospital because of thyroidectomy for thyroid cancer. After thyroidectomy she was found to have hypernatremia and polyuria and polydipsia both of which had been present from childhood. She had no obstructive hydronephrosis. Her father, father's younger sister and her third son also had polyuria and polydipsia. Basal plasma AVP concentration at normal plasma osmolality was normal but did not respond to increased plasma osmolality despite hyperosmolality during infusion of hypertonic saline infusion, indicating that plasma AVP secretion was impaired. Sodium concentration in urine and urine osmolality were low and increased after nasal administration of DDAVP. There was a diminished but bright signal of pituitary posterior gland on magnetic resonance T1 weighted image. Molecular genetic analysis demonstrated that the patient and her son had a single heterozygous missense mutation (G-->A) at nucleotide 1829 in 1 AVP allele, yielding an abnormal AVP precursor with lacking Glu-47 in its neurophysin II moiety. The abnormal AVP precursor may be related to the impaired AVP secretion.

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.

Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a defect in free water conservation caused by mutations in the single gene that encodes both vasopressin (VP) and its binding protein, neurophysin II (NP II). Most of the human mutations in this gene have been in the portion encoding the NP molecule; the resultant abnormal gene products are believed to cause cellular toxicity as improperly folded precursor molecules accumulate in the endoplasmic reticulum. We identified a new American kindred with ADNDI and found a novel mutation in the VP molecule. A 78-yr-old man was noted to have hypotonic polyuria and plasma hyperosmolarity; the urinary concentration defect was reversed by administration of VP. His symptomatology dated to childhood, and his family history was consistent with autosomal transmission of the polyuric syndrome, with affected members in three generations, including several females. Affected individuals were found to be heterozygous for a 3-bp deletion in exon 1 of arginine VP (AVP)-NP II, predicting a deletion of phenylalanine 3 (known to be critical for receptor binding) in the VP nonapeptide. Neuro 2A cells stably transfected with the mutant AVP-NP construct showed increased rates of apoptosis as assessed by flow cytometric methods. These observations support the concept that cellular toxicity of abnormal AVP-NP gene products underlies the development of ADNDI, and the data further demonstrate that mutations affecting the AVP moiety can result in initiation of these pathological processes.