Search

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.)

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting β-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kuramoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lithium, mainstay treatment for bipolar disorder, frequently causes nephrogenic diabetes insipidus (NDI) and renal injury. However, the detailed mechanism remains unclear. Here we used the analysis of metabolomics and transcriptomics and metabolic intervention in a lithium-induced NDI model. Mice were treated with lithium chloride (40 mmol/kg chow) and rotenone (ROT, 100 ppm) in diet for 28 days. Transmission electron microscopy showed extensive mitochondrial structural abnormalities in whole nephron. ROT treatment markedly ameliorated lithium-induced NDI and mitochondrial structural abnormalities. Moreover, ROT attenuated the decrease of mitochondrial membrane potential in line with the upregulation of mitochondrial genes in kidney. Metabolomics and transcriptomics data demonstrated that lithium activated galactose metabolism, glycolysis, and amino sugar and nucleotide sugar metabolism. All these events were indicative of metabolic reprogramming in kidney cells. Importantly, ROT ameliorated metabolic reprogramming in NDI model. Based on transcriptomics analysis, we also found the activation of MAPK, mTOR and PI3K-Akt signaling pathways and impaired focal adhesion, ECM-receptor interaction and actin cytoskeleton in Li-NDI model were inhibited or attenuated by ROT treatment. Meanwhile, ROT administration inhibited the increase of Reactive Oxygen Species (ROS) in NDI kidneys along with enhanced SOD2 expression. Finally, we observed that ROT partially restored the reduced AQP2 and enhanced urinary sodium excretion along with the blockade of increased PGE2 output. Taken together, the current study demonstrates that mitochondrial abnormalities and metabolic reprogramming play a key role in lithium-induced NDI, as well as the dysregulated signaling pathways, thereby serving as a novel therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Nephrogenic diabetes insipidus is defined as an inability to concentrate urine due to a complete or partial alteration of the renal tubular response to arginine vasopressin hormone, resulting in excessive diluted urine excretion. Hereditary forms are caused by molecular defects in the genes encoding either of the two main renal effectors of the arginine vasopressin pathway: the AVPR2 gene, which encodes for the type 2 vasopressin receptor, or the AQP2 gene, which encodes for the water channel aquaporin-2. About 90% of cases of nephrogenic diabetes insipidus result from loss-of-function variants in the AVPR2 gene, which are inherited in a X-linked recessive manner. The remaining 10% of cases result from loss-of-function variants in the AQP2 gene, which can be inherited in either a recessive or a dominant manner. The main symptoms of the disease are polyuria, chronic dehydration and hypernatremia. These symptoms usually occur in the first year of life, although some patients present later. Diagnosis is based on abnormal response in urinary osmolality after water restriction and/or administration of exogenous vasopressin. Treatment involves ensuring adequate water intake on demand, possibly combined with thiazide diuretics, non-steroidal anti-inflammatory drugs, and a low-salt and protein diet. In this review, we provide an update on current understanding of the molecular basis of inherited nephrogenic insipidus diabetes., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We previously reported the novel finding that β3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of β3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the β3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

Disruption of water and electrolyte balance is frequently encountered in clinical medicine. Regulating water metabolism is critically important. Diabetes insipidus (DI) presented with excessive water loss from the kidney is a major disorder of water metabolism. To understanding the molecular and cellular mechanisms and pathophysiology of DI and rationales of clinical management of DI is important for both research and clinical practice. This chapter will first review various forms of DI focusing on central diabetes insipidus (CDI) and nephrogenic diabetes insipidus (NDI). This is followed by a discussion of regulatory mechanisms underlying CDI and NDI, with a focus on the regulatory axis of vasopressin, vasopressin receptor 2 (V2R) and the water channel molecule, aquaporin 2 (AQP2). The clinical manifestation, diagnosis, and management of various forms of DI will also be discussed with highlights of some of the latest therapeutic strategies that are developed from in vitro experiments and animal studies., (© 2023. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

Loss-of-function mutations of the arginine vasopressin receptor 2 gene (AVPR2) cause Nephrogenic diabetes insipidus (NDI). AVPR2 is a kind of G protein coupled receptor (GPCR) and mainly couples with Gαs protein leading to cAMP accumulation in the cell as a secondary messenger. Recent studies showed that some AVPR2 mutations could cause biased Gαq/11 protein coupling rather than Gαs. Investigation into the characterization of biased receptors may give insights into the relationship between the conformational change of the receptor because of the mutation and related downstream signaling. In this study, R68W and V162A were analyzed to whether they show a bias to Gαs or Gαq/11 proteins. Their functionality in terms of cAMP production via Gαs protein coupling was decreased compared to the wild-type receptor. On the other hand, they showed the ability to couple with Gαq/11 protein and make Ca 2+ mobilization at different levels in the cell. R68W showed bias to coupling with Gαq/11 protein rather than V162A and wild-type receptor. Studies about the Gα protein coupling bias of mutant AVPR2s may broaden our understanding of the relationship between the changed conformation of the receptor and consequently activated signaling pathways, and also may shed light on the development of more effective new therapeutics.

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5 PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5 PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8 h water restriction, NFAT5 PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5 PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Saving body water by optimal reabsorption of water filtered by the kidney leading to excretion of urine with concentrations of solutes largely above that of plasma allowed vertebrate species to leave the aquatic environment to live on solid ground. Filtered water is reabsorbed for 70% and 20% by proximal tubules and thin descending limbs of Henle, respectively. These two nephron segments express the water channel aquaporin-1 located along both apical and basolateral membranes. In the proximal tubule, the paracellular pathway accounts for at least 30% of water reabsorption, and the tight-junction core protein claudin-2 plays a key role in this permeability. The ascending limb of Henle and the distal convoluted tubule are impermeant to water and are responsible for urine dilution. The water balance is adjusted along the collecting system, i.e. connecting tubule and the collecting duct, under the control of arginine-vasopressin (AVP). AVP is synthesized by the hypothalamus and released in response to an increase in extracellular osmolality or stimulation of baroreceptors by decreased blood pressure. In response to AVP, aquaporin-2 water channels stored in subapical intracellular vesicles are translocated to the apical plasma membrane and raise the water permeability of the collecting system. The basolateral step of water reabsorption is mediated by aquaporin-3 and -4, which are constitutively expressed. Drugs targeting water transport include classical diuretics, which primarily inhibit sodium transport; the new class of SGLT2 inhibitors, which promotes osmotic diuresis and the non-peptidic antagonists of the V2 receptor, which are pure aquaretic drugs. Disturbed water balance includes diabetes insipidus and hyponatremias. Diabetes insipidus is characterized by polyuria and polydipsia. It is either related to a deficit in AVP secretion called central diabetes insipidus that can be treated by AVP analogs or to a peripheral defect in AVP response called nephrogenic diabetes insipidus. Diabetes insipidus can be either of genetic origin or acquired. Hyponatremia is a common disorder most often related to free water excess relying on overstimulated or inappropriate AVP secretion. The assessment of blood volume is key for the diagnosis and treatment of hyponatremia, which can be classified as hypo-, eu-, or hypervolemic., (© 2022. The Author(s).)

Ensuring the proper amount of water inside the body is essential for survival. One of the key factors in the maintenance of body water balance is water reabsorption in the collecting ducts of the kidney, a process that is regulated by aquaporin-2 (AQP2). AQP2 is a channel that is exclusively selective for water molecules and impermeable to ions or other small molecules. Impairments of AQP2 result in various water balance disorders, including nephrogenic diabetes insipidus (NDI), which is a disease characterized by a massive loss of water through the kidney and consequent severe dehydration. Dysregulation of AQP2 is also a cause of water retention with hyponatremia in heart failure, hepatic cirrhosis, and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Antidiuretic hormone vasopressin is an upstream regulator of AQP2. Its binding to the vasopressin V2 receptor promotes AQP2 targeting to the apical membrane and thus enables water reabsorption. Tolvaptan, a vasopressin V2 receptor antagonist, is effective and widely used for water retention with hyponatremia. However, there are no studies showing improvement in hard outcomes or long-term prognosis. A possible reason is that vasopressin receptors have many downstream effects other than AQP2 function. It is expected that the development of drugs that directly target AQP2 may result in increased treatment specificity and effectiveness for water balance disorders. This review summarizes recent progress in studies of AQP2 and drug development challenges for water balance disorders.

Background: Congenital nephrogenic diabetes insipidus (NDI) is primarily caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R). Renal unresponsiveness to the antidiuretic hormone vasopressin impairs aquaporin-2 (AQP2) water channel activity and water reabsorption from urine, resulting in polyuria. Currently available symptomatic treatments inadequately reduce patients' excessive amounts of urine excretion, threatening their quality of life. In the past 25 years, vasopressin/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) has been believed to be the most important signaling pathway for AQP2 activation. Although cAMP production without vasopressin is the reasonable therapeutic strategy for congenital NDI caused by V2R mutations, the efficacy of candidate drugs on AQP2 activation is far less than that of vasopressin., Results: Intracellular distribution and activity of PKA are largely controlled by its scaffold proteins, A-kinase anchoring proteins (AKAPs). Dissociating the binding of AKAPs and PKA significantly increased PKA activity in the renal collecting ducts and activated AQP2 phosphorylation and trafficking. Remarkably, the AKAPs-PKA disruptor FMP-API-1 increased transcellular water permeability in isolated renal collecting ducts to the same extent as vasopressin. Moreover, derivatives of FMP-API-1 possessed much more high potency. FMP-API-1/27 is the first low-molecular-weight compound to be discovered that can phosphorylate AQP2 more effectively than preexisting drug candidates., Conclusion: AKAP-PKA disruptors are a promising therapeutic target for congenital NDI. In this article, we shall discuss the pathophysiological roles of PKA and novel strategies to activate PKA in renal collecting ducts., (© 2021. The Author(s).)

The aquaporin 2 (AQP2) plays a critical role in water reabsorption to maintain water homeostasis. AQP2 mutation leads to nephrogenic diabetes insipidus (NDI), characterized by polyuria, polydipsia, and hypernatremia. We previously reported that a novel AQP2 mutation (G215S) caused NDI in a boy. In this study, we aimed to elucidate the cell biological consequences of this mutation on AQP2 function and clarify the molecular pathogenic mechanism for NDI in this patient. First, we analyzed AQP2 expression in Madin-Darby canine kidney (MDCK) cells by AQP2-G215S or AQP2-WT plasmid transfection and found significantly decreased AQP2-G215S expression in cytoplasmic membrane compared with AQP2-WT, independent of forskolin treatment. Further, we found co-localization of endoplasmic reticulum (ER) marker (Calnexin) with AQP2-G215S rather than AQP2-WT in MDCK cells by immunocytochemistry. The functional analysis showed that MDCK cells transfected with AQP2-G215S displayed reduced water permeability compared with AQP2-WT. Visualization of AQP2 structure implied that AQP2-G215S mutation might interrupt the folding of the sixth transmembrane α-helix and/or the packing of α-helices, resulting in the misfolding of monomer and further impaired formation of tetramer. Taken together, these findings suggested that AQP2-G215S was misfolded and retained in the ER and could not be translocated to the apical membrane to function as a water channel, which revealed the molecular pathogenic mechanism of AQP2-G215S mutation and explained for the phenotype of NDI in this patient., 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, Lu, Yang, Li, Li, Chen, Li, Duan, Gu, Zhang 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.)

X-linked nephrogenic diabetes insipidus (X-linked NDI) is a rare inherited disease mainly caused by lost-of-function mutations in human AVPR2 gene encoding arginine vasopressin receptor 2 (V2R). Our focus of the current study is on exploration of the functional and biochemical properties of Ile324Met (I324M) mutation identified in a pedigree showing as typical recessive X-linked NDI. We demonstrated that I324M mutation interfered with the conformation of complex glycosylation of V2R. Moreover, almost all of the I324M-V2R failed to express on the cell surface due to being captured by the endoplasmic reticulum control system. We further examined the signaling activity of DDAVP-medicated cAMP and ERK1/2 pathways and the results revealed that the mutant receptor lost the ability in response to DDAVP stimulation contributed to the failure of accumulation of cAMP and phosphorylated ERK1/2. Based on the characteristics of molecular defects of I324M mutant, we selected two reagents (SR49059 and alvespimycin) to determine whether the functions of I324M-V2R can be restored and we found that both compounds can significantly "rescue" I324M mutation. Our findings may provide further insights for understanding the pathogenic mechanism of AVPR2 gene mutations and may offer some implications on development of promising treatments for patients with X-linked NDI.

The association between diabetes insipidus (DI) and chronic dietary K + deprivation is well known, but it remains uncertain how the disorder develops and whether it is influenced by the sexual dimorphism in K + handling. Here, we determined the plasma K + (P K ) threshold for DI in male and female mice and ascertained if DI is initiated by polydipsia or by a central or nephrogenic defect. C57BL6J mice were randomized to a control diet or to graded reductions in dietary K + for 8 days, and kidney function and transporters involved in water balance were characterized. We found that male and female mice develop polyuria and secondary polydipsia. Altered water balance coincided with a decrease in aquaporin-2 (AQP2) phosphorylation and apical localization despite increased levels of the vasopressin surrogate marker copeptin. No change in the protein abundance of urea transporter-A1 was observed. The Na + -K + -2Cl - cotransporter decreased only in males. Desmopressin treatment failed to reverse water diuresis in K + -restricted mice. These findings indicate that even a small fall in P K is associated with nephrogenic DI (NDI), coincident with the development of altered AQP2 regulation, implicating low P K as a causal trigger of NDI. We found that P K decreased more in females, and, consequently, females were more prone to develop NDI. Together, these data indicate that AQP2 regulation is disrupted by a small decrease in P K and that the response is influenced by sexual dimorphism in K + handling. These findings provide new insights into the mechanisms linking water and K + balances and support defining the disorder as "potassium-dependent NDI." NEW & NOTEWORTHY This study shows that aquaporin-2 regulation is disrupted by a small fall in plasma potassium levels and the response is influenced by sexual dimorphism in renal potassium handling. The findings provided new insights into the mechanisms by which water balance is altered in dietary potassium deficiency and support defining the disorder as "potassium-dependent nephrogenic diabetes insipidus."

Nephrogenic diabetes insipidus (NDI) patients produce large amounts of dilute urine. NDI can be congenital, resulting from mutations in the type-2 vasopressin receptor (V2R), or acquired, resulting from medications such as lithium. There are no effective treatment options for NDI. Activation of PKA is disrupted in both congenital and acquired NDI, resulting in decreased aquaporin-2 phosphorylation and water reabsorption. We show that adenosine monophosphate-activated protein kinase (AMPK) also phosphorylates aquaporin-2. We identified an activator of AMPK, NDI-5033, and we tested its ability to increase urine concentration in animal models of NDI. NDI-5033 increased AMPK phosphorylation by 2.5-fold, confirming activation. It increased urine osmolality in tolvaptan-treated NDI rats by 30%-50% and in V2R-KO mice by 50%. Metformin, another AMPK activator, can cause hypoglycemia, which makes it a risky option for treating NDI patients, especially children. Rats with NDI receiving NDI-5033 showed no hypoglycemia in a calorie-restricted, exercise protocol. Congenital NDI therapy needs to be effective long-term. We administered NDI-5033 for 3 weeks and saw no reduction in efficacy. We conclude that NDI-5033 can improve urine concentration in animals with NDI and holds promise as a potential therapy for patients with congenital NDI due to V2R mutations.

Nephrogenic diabetes insipidus (NDI) is characterized by renal resistance to the antidiuretic hormone arginine vasopressin (AVP), which leads to polyuria, plasma hyperosmolarity, polydipsia, and impaired quality of living. Inherited forms are caused by X-linked loss-of-function mutations in the gene encoding the vasopressin 2 receptor (V2R) or autosomal recessive/dominant mutations in the gene encoding aquaporin 2 (AQP2). A common acquired form is lithium-induced NDI. AVP facilitates reabsorption of water through increased abundance and insertion of AQP2 in the apical membrane of principal cells in the collecting ducts. In X-linked NDI, V2R is dysfunctional, which leads to impaired water reabsorption. These patients have functional AQP2, and thus the challenge is to achieve AQP2 membrane insertion independently of V2R. The current treatment is symptomatic and is based on distally acting diuretics (thiazide or amiloride) and cyclooxygenase inhibitors (indomethacin). This mini-review covers published data from trials in preclinical in vivo models and a few human intervention studies to improve NDI by more causal approaches. Promising effects on NDI in preclinical studies have been demonstrated by the use of pharmacological approaches with secretin, Wnt5a, protein kinase A agonist, fluconazole, prostaglandin E 2 EP2 and EP4 agonists, statins, metformin, and soluble prorenin receptor agonists. In patients, only casuistic reports have evaluated the effect of statins, phosphodiesterase inhibitors (rolipram and sildenafil), and the guanylate cyclase stimulator riociguat without amelioration of symptoms. It is concluded that there is currently no established intervention that causally improves symptoms or quality of life in patients with NDI. There is a need to collaborate to improve study quality and conduct formal trials.

Most cases of acquired central diabetes insipidus are caused by destruction of the neurohypophysis by: 1) anatomic lesions that destroy the vasopressin neurons by pressure or infiltration, 2) damage to the vasopressin neurons by surgery or head trauma, and 3) autoimmune destruction of the vasopressin neurons. Because the vasopressin neurons are located in the hypothalamus, lesions confined to the sella turcica generally do not cause diabetes insipidus because the posterior pituitary is simply the site of the axon terminals that secrete vasopressin into the bloodstream. In addition, the capacity of the neurohypophysis to synthesize vasopressin is greatly in excess of the body's needs, and destruction of 80-90% of the hypothalamic vasopressin neurons is required to produce diabetes insipidus. As a result, even large lesions in the sellar and suprasellar area generally are not associated with impaired water homeostasis until they are surgically resected. Regardless of the etiology of central diabetes insipidus, deficient or absent vasopressin secretion causes impaired urine concentration with resultant polyuria. In most cases, secondary polydipsia is able to maintain water homeostasis at the expense of frequent thirst and drinking. However, destruction of the osmoreceptors in the anterior hypothalamus that regulate vasopressin neuronal activity causes a loss of thirst as well as vasopressin section, leading to severe chronic dehydration and hyperosmolality. Vasopressin deficiency also leads to down-regulation of the synthesis of aquaporin-2 water channels in the kidney collecting duct principal cells, causing a secondary nephrogenic diabetes insipidus. As a result, several days of vasopressin administration are required to achieve maximal urine concentration in patients with CDI. Consequently, the presentation of patients with central diabetes insipidus can vary greatly, depending on the size and location of the lesion, the magnitude of trauma to the neurohypophysis, the degree of destruction of the vasopressin neurons, and the presence of other hormonal deficits from damage to the anterior pituitary., (Copyright © 2020. Published by Elsevier Ltd.)

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

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

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

Diabetes insipidus (DI), be it from central or from nephrogenic origin, has to be differentiated from primary polydipsia. This differentiation is crucial since wrong treatment can have dangerous consequences. For decades, the "gold standard" for differential diagnosis has been the standard water deprivation test. However, this test has several limitations leading to an overall limited diagnostic accuracy. In addition, the test has a long duration of 17 h and is cumbersome for patients. Also clinical signs and symptoms and MRI characteristics overlap between patients with DI and primary polydipsia. Direct measurement of arginine vasopressin (AVP) upon osmotic stimulation was first shown to overcome these limitations, but failed to enter clinical practice mainly due to technical limitations of the AVP assay. Copeptin is secreted in equimolar ratio to AVP, mirroring AVP concentrations in the circulation. We have shown that copeptin, without prior fluid deprivation, identifies patients with nephrogenic DI. For the more difficult differentiation between central DI and primary polydipsia, a copeptin level of 4.9 pmol/L stimulated with hypertonic saline infusion differentiates between these 2 entities with a high diagnostic accuracy and is superior to the water deprivation test. However, it is important to note that close and regular sodium monitoring every 30 min during the hypertonic saline test is a prerequisite, which is not possible in all hospitals. Furthermore, side effects are common. Therefore, a nonosmotic stimulation test would be advantageous. Arginine significantly stimulates copeptin and therefore is a novel, so far unknown stimulus of this peptide. Consequently, infusion of arginine with subsequent copeptin measurement was shown to be an even simpler and better tolerated test, but head to head comparison is still lacking., (© 2020 S. Karger AG, Basel.)

V2 vasopressin receptor (V2R) is a member of the G protein-coupled receptor (GPCR) family in which many disease-causing mutations have been identified and thus generated much interest. Loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI) whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). The mechanisms underlying a V2R loss-of-function can be theoretically classified as either protein expression, localization (ER retention) or functional disorders. Functional analyses have revealed however that these mechanisms are likely to be complex. Strikingly, V2R mutations at the same site can result in opposite phenotypes, e.g., R137H and R137L/C cause NDI and NSIAD, respectively. These findings support the notion that the constitutive activation of GPCRs might be often associated with their instability and denaturation. Thus, functional analysis of disease-causing V2R mutations may not only reveal potential new treatment strategies using pharmacochaperones for NDI and inverse agonists for NSIAD, but also provide a greater understanding of the physiological functions of GPCRs and highlight the new paradigms, i.e., biased agonism and protean agonism., (© 2020 Elsevier Inc. All rights reserved.)

Lithium salts, used for treating bipolar disorder, frequently induce nephrogenic diabetes insipidus (NDI) thereby limiting therapeutic success. NDI is associated with loss of expression of the gene coding for the molecular water channel, aquaporin-2, in the renal collecting duct (CD). Here, we use systems biology methods in a well-established rat model of lithium-induced NDI to identify signaling pathways activated at the onset of polyuria. Using single-tubule RNA-Seq, full transcriptomes were determined in microdissected cortical collecting ducts (CCDs) of rats after 72 hours without or with initiation of lithium chloride administration. Transcriptome-wide changes in mRNA abundances were mapped to gene sets associated with curated canonical signaling pathways, showing evidence for activation of NF-κB signaling with induction of genes coding for multiple chemokines and most components of the Major Histocompatibility Complex Class I antigen-presenting complex. Administration of anti-inflammatory doses of dexamethasone to lithium chloride-treated rats countered the loss of aquaporin-2. RNA-Seq also confirmed prior evidence of a shift from quiescence into the cell cycle with arrest. Time course studies demonstrated an early (12 hour) increase in multiple immediate early response genes including several transcription factors. Protein mass spectrometry in microdissected CCDs provided corroborative evidence and identified decreased abundance of several anti-oxidant proteins. Thus, in the context of prior observations, our study can be best explained by a model in which lithium increases ERK activation leading to induction of NF-κB signaling and an inflammatory-like response that represses Aqp2 transcription., (Published by Elsevier Inc.)

Background: Arginine-vasopressin (AVP) binding to vasopressin V2 receptors promotes redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. This pathway fine-tunes renal water reabsorption and urinary concentration, and its perturbation is associated with diabetes insipidus. Previously, we identified the antimycotic drug fluconazole as a potential modulator of AQP2 localization., Methods: We assessed the influence of fluconazole on AQP2 localization in vitro and in vivo as well as the drug's effects on AQP2 phosphorylation and RhoA (a small GTPase, which under resting conditions, maintains F-actin to block AQP2-bearing vesicles from reaching the plasma membrane). We also tested fluconazole's effects on water flow across epithelia of isolated mouse collecting ducts and on urine output in mice treated with tolvaptan, a VR2 blocker that causes a nephrogenic diabetes insipidus-like excessive loss of hypotonic urine., Results: Fluconazole increased plasma membrane localization of AQP2 in principal cells independent of AVP. It also led to an increased AQP2 abundance associated with alterations in phosphorylation status and ubiquitination as well as inhibition of RhoA. In isolated mouse collecting ducts, fluconazole increased transepithelial water reabsorption. In mice, fluconazole increased collecting duct AQP2 plasma membrane localization and reduced urinary output. Fluconazole also reduced urinary output in tolvaptan-treated mice., Conclusions: Fluconazole promotes collecting duct AQP2 plasma membrane localization in the absence of AVP. Therefore, it might have utility in treating forms of diabetes insipidus ( e.g. , X-linked nephrogenic diabetes insipidus) in which the kidney responds inappropriately to AVP., (Copyright © 2019 by the American Society of Nephrology.)

Aim: Therapeutic use of lithium in bipolar disorder is limited by the development of nephrogenic diabetes insipidus (NDI). We reported that pharmacological blockade of P2Y 12 receptor (R) with clopidogrel or prasugrel significantly ameliorated lithium-induced NDI in rodents. Using mice genetically lacking P2Y 12 -R we evaluated whether the observed amelioration is mediated through P2Y 12 -R METHODS: P2ry12 -/- mouse line (C57/BL6) was rederived from cryopreserved embryos of the knockout (KO) mice generated by Deltagen Inc. Syngeneic wild type (WT) mice obtained by heterozygous crossing were inbred. Groups of adult WT and KO mice were fed lithium-added (40 mmol LiCl/kg food) or regular diet, and euthanized after 2 or 4 weeks. Twenty-four hour urine samples and terminal blood and kidney samples were analyzed., Results: At both time points, lithium-induced polyuria and decrease in aquaporin-2 (AQP2) protein abundance in the kidney medulla were less marked in KO vs WT mice. Immunofluorescence microscopy revealed that lithium-induced alterations in the cellular disposition of AQP2 protein in the medullary collecting ducts of WT mice were blunted in KO mice. Serum lithium, sodium and osmolality were similar in both genotypes after lithium treatment. After 2 weeks, lithium induced marked increases in urinary excretion of Na, K, and arginine vasopressin in WT mice but not in KO mice., Conclusion: Taken together, our data show that similar to pharmacological blockade, deletion of P2Y 12 -R significantly ameliorates lithium-induced NDI, without reducing serum lithium levels. Hence, targeting P2Y 12 -R with currently available drugs in the market offers a novel and safer method for treating NDI., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms., Methods: We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout ( Tgr5 -/- ) mice., Results: Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5 -/- mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated Tgr5 -/- mice, LCA treatment failed to prevent reduction of AQP2 expression., Conclusions: TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney., (Copyright © 2018 by the American Society of Nephrology.)

While lithium remains the most efficacious treatment for bipolar disorder, it can cause significant nephrotoxicity. The molecular mechanisms behind both this process and the development of nephrogenic diabetes insipidus still remain to be fully elucidated but appear to involve alterations in glycogen synthase kinase 3 signalling, G2 cell cycle progression arrest, alterations in inositol and prostaglandin signalling pathways, and dysregulated trafficking and transcription of aquaporin 2 water channels. The end result of this is a tubulointerstitial nephropathy with microcyst formation and relative glomerular sparing, both visible on pathology specimens and increasingly noted on non-invasive imaging. This paper will elucidate the current evidence pertaining to the pathophysiology of lithium induced nephrotoxicity., (© 2018 Asian Pacific Society of Nephrology.)

A series of compounds formerly identified by high-throughput screening was studied for their ability to serve as pharmacoperones for the vasopressin type 2 receptor (V2R) mutant L83Q, which is known to cause nephrogenic diabetes insipidus (NDI). Three compounds were particularly effective in rerouting the mutant receptor in a concentration-dependent manner, were neither agonists nor antagonists, and displayed low cellular toxicity. Compound 1 was most effective and can be used as a molecular probe for future studies of how small molecules may affect NDI caused by mutant V2R. These compounds, however, failed to rescue the V2R Y128S mutant, indicating that the compounds described may not work in the rescue of all known mutants of V2R. Taken collectively, the present studies have now identified a promising lead compound that could function as a pharmacoperone to correct the trafficking defect of the NDI-associated mutant V2R L83Q and thus has the therapeutic potential for the treatment of NDI., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Congenital nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentrating ability. Symptomatic polyuria is present from birth, even with normal release of the antidiuretic hormone vasopressin by the pituitary. Over the last two decades, the aquaporin-2 (AQP2) gene has been cloned and the molecular mechanisms of urine concentration have been gradually elucidated. Vasopressin binds to the vasopressin type II receptor (V2R) in the renal collecting ducts and then activates AQP2 phosphorylation and trafficking to increase water reabsorption from urine. Most cases of congenital NDI are caused by loss-of-function mutations to V2R, resulting in unresponsiveness to vasopressin. In this article, we provide an overview of novel therapeutic molecules of congenital NDI that can activate AQP2 by bypassing defective V2R signaling with a particular focus on the activators of the calcium and cAMP signaling pathways.

Aquaporins (AQPs) are among the best structural-characterized membrane proteins, fulfilling the role of allowing water flux across cellular membranes. Thus far, 34 single amino acid polymorphisms have been reported in HUMSAVAR for human aquaporins as disease-related. They affect AQP2, AQP5 and AQP8, where they are associated with nephrogenic diabetes insipidus, keratoderma and colorectal cancer, respectively. For half of these mutations, although they are mostly experimentally characterized in their dysfunctional phenotypes, a structural characterization at a molecular level is still missing. In this work, we focus on such mutations and discuss what the structural defects are that they appear to cause. To achieve this aim, we built a 3D molecular model for each mutant and explored the effect of the mutation on all of their structural features. Based on these analyses, we could collect the structural defects of all the pathogenic mutations (here or previously analysed) under few main categories, that we found to nicely correlate with the experimental phenotypes reported for several of the analysed mutants. Some of the structural analyses we present here provide a rationale for previously experimentally observed phenotypes. Furthermore, our comprehensive overview can be used as a reference frame for the interpretation, on a structural basis, of defective phenotypes of other aquaporin pathogenic mutants.

Lithium is widely used in treatment of bipolar affective disorders but often causes nephrogenic diabetes insipidus (NDI), a disorder characterized by severe urinary-concentrating defects. Lithium-induced NDI is caused by lithium uptake by collecting duct principal cells and altered expression of aquaporin-2 (AQP2), which are essential for water reabsorption of tubular fluid in the collecting duct. Sex hormones have previously been shown to affect the regulation of AQP2, so we tested whether tamoxifen (TAM), a selective estrogen receptor modulator, would attenuate lithium-induced alterations on renal water homeostasis. Rats were treated for 14 days with lithium, and TAM treatment was initiated 1 wk after onset of lithium administration. Lithium treatment resulted in severe polyuria and reduced AQP2 expression, which were ameliorated by TAM. Consistent with this, TAM attenuated downregulation of AQP2 and increased phosphorylation of the cAMP-responsive element-binding protein, which induced AQP2 expression in freshly isolated inner-medullary collecting duct suspension prepared from lithium-treated rats. In conclusion, TAM attenuated polyuria dose dependently and impaired urine concentration and downregulation of AQP2 protein expression in rats with lithium-induced NDI. These findings suggest that TAM is likely to be a novel therapeutic option for lithium-induced NDI.

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

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

Aim: Long-term administration of lithium has been associated with the development of a chronic interstitial fibrosis in addition to nephrogenic diabetes insipidus (NDI). Earlier studies have demonstrated that amiloride, by blocking the epithelial sodium channel ENaC and thus preventing lithium uptake into the principal cells of the collecting ducts, can partially reverse lithium-induced NDI. However, there are no long-term studies examining whether or not amiloride also modifies the progressive chronic interstitial fibrosis and tubular atrophy often evident with long-term lithium exposure., Methods: Using an established animal model of lithium-induced chronic interstitial fibrosis, rats were treated with amiloride and lithium for 5 months following 1 month of exposure to lithium alone and compared with control animals and those given only lithium., Results and Conclusions: In this study, the 5 months of amiloride therapy partially mitigated the lithium-induced NDI and limited the further progression of lithium-induced kidney fibrosis. This improvement was associated with decreased expression of the pro-fibrotic connective tissue growth factor (CTGF), along with reduced myofibroblast infiltration and decreased collagen deposition around the distended cortical collecting ducts. This may, in part, be mediated by modifying lithium-induced alterations in β-catenin activity through its effects on GSK-3β., (© 2016 Asian Pacific Society of Nephrology.)

The kidney plays a central role in the regulation of the body water balance. The process of targeting the water channel aquaporin-2 (AQP2) on the apical plasma membrane of the collecting duct (CD) principal cells is mainly regulated by the antidiuretic peptide hormone arginine vasopressin (AVP), which is responsible for the maintenance of water homeostasis. Recently, much attention has been focused on the local factors modulating renal water reabsorption by AQP2 in the collecting ducts, especially prostaglandin E2 (PGE₂). PGE₂ is a lipid mediator involved in a variety of physiological and pathophysiological processes in the kidney. The biological function of PGE₂ is mainly mediated by four G-protein-coupled receptors, namely EP1-4, which couple to drive separate intracellular signaling pathways. Increasing evidence demonstrates that PGE₂ is essential for renal water transport regulation via multiple mechanisms. Each EP receptor plays a unique role in regulating water reabsorption in renal collecting ducts. This brief review highlights the role of PGE₂ in the regulation of water reabsorption and discusses the involvement of each EP receptor subtype in renal collecting duct. A better understanding of the role of PGE₂ in renal water transport process may improve disease management strategies for water balance disorders, including nephrogenic diabetes insipidus., Competing Interests: The authors declare no conflict of interest.

Earlier we reported that the recombinant soluble (pro) renin receptor sPRR-His upregulates renal aquoporin-2 (AQP2) expression, and attenuates polyuria associated with nephrogenic diabetes insipidus (NDI) induced by vasopressin type 2 receptor (V2R) antagonism. Patients that receive lithium therapy develop polyuria associated NDI that might be secondary to downregulation of renal AQP2. We hypothesized that sPRR-His attenuates indices of NDI associated with lithium treatment. Eight-week-old male C57/BL6 mice consumed chow supplemented with LiCl (40 mmol/kg diets) for 14 days. For the last 7 days mice received either sPRR-His [30  μ g/(kg day), i.v.; sPRR] or vehicle (Veh) via minipump. Control (Con) mice consumed standard chow for 14 days. Compared to Con mice, 14-d LiCl treatment elevated water intake and urine volume, and decreased urine osmolality, regardless of sPRR-His or Veh administration. These data indicate that sPRR-His treatment does not attenuate indices of NDI evoked by lithium. Unexpectedly, epididymal fat mass was lower, adipocyte UCP1 mRNA and protein expression were higher, and multilocular lipid morphology was enhanced, in LiCl-fed mice treated with sPRR-His versus vehicle. The beiging of white adipose tissue is a novel metabolic benefit of manipulating the sPRR in the context of lithium-induced NDI., (© 2017 University of Utah. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

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.)

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Fundamental kidney physiology research can provide important insight into how the kidney works and suggest novel therapeutic opportunities to treat human diseases. This is especially true for nephrogenic diabetes insipidus (NDI). Over the past decade, studies elucidating the molecular physiology and signaling pathways regulating water transport have suggested novel therapeutic possibilities. In patients with congenital NDI due to mutations in the type 2 vasopressin receptor (V2R) or acquired NDI due to lithium (or other medications), there are no functional abnormalities in the aquaporin-2 (AQP2) water channel, or in another key inner medullary transport protein, the UT-A1 urea transporter. If it is possible to phosphorylate and/or increase the apical membrane accumulation of these proteins, independent of vasopressin or cAMP, one may be able to treat NDI. Sildenifil (through cGMP), erlotinib, and simvastatin each stimulate AQP2 insertion into the apical plasma membrane. Some recent human data suggest that sildenafil and simvastatin may improve urine concentrating ability. ONO-AE1-329 (ONO) stimulates the EP4 prostanoid receptor (EP4), which stimulates kinases that in turn phosphorylate AQP2 and UT-A1. Clopidogrel is a P2Y12-R antagonist that potentiates the effect of vasopressin and increases AQP2 abundance. Metformin stimulates AMPK to phosphorylate and activate AQP2 and UT-A1, and it increases urine concentrating ability in two rodent models of NDI. Since metformin, sildenafil, and simvastatin are commercially available and have excellent safety records, the potential for rapidly advancing them into clinical trials is high., (Copyright © 2016 the American Physiological Society.)

Heritable nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentration mechanisms in the kidney, which are mainly caused by loss-of-function mutations in the vasopressin type 2 receptor. For the treatment of heritable NDI, novel strategies that bypass the defective vasopressin type 2 receptor are required to activate the aquaporin-2 (AQP2) water channel. Here we show that Wnt5a regulates AQP2 protein expression, phosphorylation and trafficking, suggesting that Wnt5a is an endogenous ligand that can regulate AQP2 without the activation of the classic vasopressin/cAMP signalling pathway. Wnt5a successfully increases the apical membrane localization of AQP2 and urine osmolality in an NDI mouse model. We also demonstrate that calcineurin is a key regulator of Wnt5a-induced AQP2 activation without affecting intracellular cAMP level and PKA activity. The importance of calcineurin is further confirmed with its activator, arachidonic acid, which shows vasopressin-like effects underlining that calcineurin activators may be potential therapeutic targets for heritable NDI.

Purpose: Lithium is an essential treatment in bipolar disorder and treatment-resistant depression; however, its use has been limited by concerns regarding its renal adverse effects. An improved understanding of potential molecular mechanisms can help develop prevention and treatment strategies for lithium-associated renal disease., Methods: We conducted a systematic literature search using MEDLINE, Embase, and PsychINFO including English-language original research articles published prior to November 2015 that specifically investigated lithium's effects on nephrogenic diabetes insipidus (NDI) and chronic kidney disease (CKD), using molecular markers., Results: From a total of 3510 records, 71 pre-clinical studies and two relevant clinical studies were identified. Molecular alterations were reported in calcium signaling, inositol monophosphate, extracellular-regulated, prostaglandin, sodium/solute transport, G-protein-coupled receptors, nitric oxide, vasopressin/aquaporin, and inflammation-related pathways in lithium-associated renal disease. The majority of studies found that these mechanisms were implicated in NDI, while few studies had examined CKD., Discussion: Future studies will have to focus on (1) validating the present findings in human subjects and (2) examining CKD, which is the most clinically relevant lithium-associated renal effect. This will improve our understanding of lithium's biological effects, as well as inform a personalized medicine approach, which could lead to safer lithium prescribing and less renal adverse events.

Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/β-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Endoplasmic reticulum (ER) stress has been implicated in some types of glomerular and tubular disorders. The objectives of this study were to elucidate the role of ER stress in lithium-induced nephrogenic diabetes insipidus (NDI) and to investigate whether attenuation of ER stress by 4-phenylbutyric acid (4-PBA) improves urinary concentrating defect in lithium-treated rats. Wistar rats received lithium (40 mmol/kg food), 4-PBA (320 mg/kg body wt by gavage every day), or no treatment (control) for 2 wk, and they were dehydrated for 24 h before euthanasia. Lithium treatment resulted in increased urine output and decreased urinary osmolality, which was significantly improved by 4-PBA. 4-PBA also prevented reduced protein expression of aquaporin-2 (AQP2), pS256-AQP2, and pS261-AQP2 in the inner medulla of kidneys from lithium-treated rats after 24-h dehydration. Lithium treatment resulted in increased expression of ER stress markers in the inner medulla, which was associated with dilated cisternae and expansion of ER in the inner medullary collecting duct (IMCD) principal cells. Confocal immunofluorescence studies showed colocalization of a molecular chaperone, binding IgG protein (BiP), with AQP2 in principal cells. Immunohistochemistry demonstrated increased intracellular expression of BiP and decreased AQP2 expression in IMCD principal cells of kidneys from lithium-treated rats. 4-PBA attenuated expression of ER stress markers and recovered ER morphology. In IMCD suspensions isolated from lithium-treated rats, 4-PBA incubation was also associated with increased AQP2 expression and ameliorated ER stress. In conclusion, in experimental lithium-induced NDI, 4-PBA improved the urinary concentrating defect and increased AQP2 expression, likely via attenuating ER stress in IMCD principal cells., (Copyright © 2016 the American Physiological Society.)

The involvement of secretin (SCT) and secretin receptor (SCTR) in regulating body water homeostasis is well established. Identified as one of the vasopressin (Vp)-independent mechanisms in fluid balance, SCT regulates aquaporin 2 (AQP2) in the kidney distal collecting duct cells through activating intracellular cAMP production. This ability to bypass Vp-mediated water reabsorption in kidney implicates SCT's potential to treat nephrogenic diabetes insipidus (NDI). Research on NDI in the past has largely been focused on the searching for mutations in vasopressin receptor 2 (AVPR2), while the functional relationship between SCTR, AVPR2 and NDI remains unclear. Here, we demonstrate the interaction between SCTR and AVPR2 to modulate cellular signaling in vitro. Interestingly, we show in this report that upon heteromer formation with SCTR, R137H, a NDI-causing AVPR2 mutant that is defective in trafficking to cell surface, can functionally be rescued. Our data may provide an explanation for this clinically mild case of NDI, and insights into the pathological development of NDI in the future., Competing Interests: The authors have declared that no competing interests exist.

Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling., (Copyright © 2016 by the American Society of Nephrology.)

Unlabelled: X-linked nephrogenic diabetes insipidus (NDI, OMIM#304800) is caused by mutations in the arginine vasopressin (AVP, OMIM*192340) receptor type 2 (AVPR2, OMIM*300538) gene. A 20-month-old boy and his 8-year-old brother presented with polyuria, polydipsia, and failure to thrive. Both boys demonstrated partial DDAVP (1-desamino-8-D AVP or desmopressin) responses; thus, NDI diagnosis was delayed. While routine sequencing of AVPR2 showed a potential splice site variant, it was not until exome sequencing confirmed the AVPR2 splice site variant and did not reveal any more likely candidates that the patients' diagnosis was made and proper treatment was instituted. Both patients were hemizygous for two AVPR2 variants predicted in silico to affect AVPR2 messenger RNA (mRNA) splicing. A minigene assay revealed that the novel AVPR2 c.276A>G mutation creates a novel splice acceptor site leading to 5' truncation of AVPR2 exon 2 in HEK293 human kidney cells. Both patients have been treated with high-dose DDAVP with a remarkable improvement of their symptoms and accelerated linear growth and weight gain., Conclusion: We present here a unique case of partial X-linked NDI due to an AVPR2 splice site mutation; patients with diabetes insipidus of unknown etiology may harbor splice site mutations that are initially underestimated in their pathogenicity on sequence analysis., What Is Known: • X-linked nephrogenic diabetes insipidus is caused by AVPR2 mutations, and disease severity can vary depending on the functional effect of the mutation. What is New: • We demonstrate here that a splice site mutation in AVPR2 leads to partial X-linked NDI in two brothers. • Treatment with high-dose DDAVP led to improvement of polyuria and polydipsia, weight gain, and growth.

Epigallocatechin-3-gallate (EGCG), extracted from green tea, has been shown to have antioxidative activity. In the present study, we evaluated the effect of EGCG on the kidney function in db/db mice and also tried to investigate the underlying mechanism of the renoprotective effects of EGCG in both animals and cells. EGCG treatment could decrease the level of urinary protein, 8-iso-PGF2a, and Ang II. Moreover, EGCG could also change the level of several parameters associated with oxidative stress. In addition, the protein expression levels of AT-1R, p22-phox, p47-phox, p-ERK1/2, p-p38 MAPK, TGF- β 1, and α -SMA in diabetic db/db mice were upregulated, and all of these symptoms were downregulated with the treatment of EGCG at 50 and 100 mg/kg/d. Furthermore, the pathological changes were ameliorated in db/db mice after EGCG treatment. HK-2 cell-based experiments indicated that EGCG could inhibit the expression of MAPK pathways, which is the downstream effector of Ang II mediated oxidative stress. All these results indicated that EGCG treatment could ameliorate changes of renal pathology and delay the progression of DKD by suppressing hyperglycemia-induced oxidative stress in diabetic db/db mice.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI., (Copyright © 2015 by the American Society of Nephrology.)