Tubular Diseases and Stones Seen From Pediatric and Adult Nephrology Perspectives.

The tubular system of the kidneys is a complex series of morphologic and functional units orchestrating the content of tubular fluid as it flows along the nephron and collecting ducts. Renal tubules maintain body water, regulate electrolytes and acid-base balance, reabsorb precious organic solutes, and eliminate specific metabolites, toxins, and drugs. In addition, decisive mechanisms to adjust blood pressure are governed by the renal tubules. Genetic as well as acquired disorders of these tubular functions may cause serious diseases that manifest both in childhood and adulthood. This article addresses a selection of tubulopathies and the underlying pathomechanisms, while highlighting the important differences in pediatric and adult nephrology care. These range from rare monogenic conditions such as nephrogenic diabetes insipidus, cystinosis, and Bartter syndrome that present in childhood, to the genetic and acquired tubular pathologies causing hypertension or nephrolithiasis that are more prevalent in adults. Both pediatric and adult nephrologists must be aware of these conditions and the age-dependent manifestations that warrant close interaction between the two subspecialties.

Molecular Characterization of an Aquaporin-2 Mutation Causing Nephrogenic Diabetes Insipidus.

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.

AQP2: Mutations Associated with Congenital Nephrogenic Diabetes Insipidus and Regulation by Post-Translational Modifications and Protein-Protein Interactions.

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.

Chloroquine attenuates lithium-induced NDI and proliferation of renal collecting duct cells.

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 ß-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.

Potassium depletion induces cellular conversion in the outer medullary collecting duct altering Notch signaling pathway.

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.

Molecular characterization of an aquaporin-2 mutation causing a severe form of nephrogenic diabetes insipidus.

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

Nephrogenic diabetes insipidus in a 15-year-old Hispanic female with a novel AQP2 mutation.

Nephrogenic diabetes insipidus (NDI) is a rare inherited disorder most often caused by mutations in the arginine-vasopressin receptors or aquaporin channels, which subsequently impairs the water reabsorption in the kidney. This case report describes a 15-year-old female diagnosed with NDI after an acute gastroenteritis and multiple fluid boluses leading to intractable emesis. Gene testing reveals our patient is compound heterozygous for novel AQP2 gene mutations with a cytosine-to-thymine substitution at nucleotide position 277 and adenine-to-cytosine substitution at nucleotide position 659. Therefore, we report a novel AQP2 gene mutation in an adolescent patient which is outside the common age for diagnosis.

Contiguous gene deletion in a Chinese family with X-linked nephrogenic diabetes insipidus: challenges in early diagnosis and implications for affected families.

Nephrogenic diabetes insipidus (NDI) is a rare disorder of the renal collecting tubules, characterized by an inability to concentrate urine due to an impaired response to arginine vasopressin (AVP), resulting in dilute urine and polyuria. Causes of NDI are heterogeneous and diagnosing congenital NDI (cNDI) in young infants is clinically challenging, as typical symptoms are often unappreciated or inconspicuous. Instead, young infants may present with non-specific signs such as vomiting, poor feeding, failure to thrive, unexplained fevers, irritability, constipation or diarrhea. We report a 37-day-old infant who presented with polyuria and severe hypernatremic dehydration that was unresponsive to vasopressin. The patient was treated with amiloride, indomethacin and hydrochlorothiazide. Genetic analysis revealed a novel contiguous deletion involving the entire AVPR2 gene and the last exon of the adjacent ARHGAP4 gene. A study of the family confirmed the carrier status in the mother. This case illustrates the importance of molecular testing in confirming the diagnosis in the index patient, as well as in identifying asymptomatic at-risk female carriers so that appropriate genetic counselling can be given for reproductive planning. All pediatric patients with suspected cNDI should undergo genetic analysis for a definitive diagnosis.

Severe congenital nephrogenic diabetes insipidus in a compound heterozygote with a new large deletion of the AQP2 gene. A case report.

BACKGROUND: Congenital nephrogenic diabetes insipidus (NDI) is a rare condition characterized by severe polyuria, due to the inability of the kidneys to concentrate urine in response to arginine vasopressin (AVP). In the majority of the cases, the disease shows an X-linked inherited pattern, although an autosomal recessive inheritance was also observed. METHODS: We report a patient with a severe NDI diagnosed during the neonatal period. Because the patient was female without a family history of congenital NDI, her disease was thought to exhibit an autosomal recessive form. RESULTS: A full mutation analysis of AVP receptor 2 (AVPR2; MIM#300538) gene showed no mutations. However, direct Sanger sequencing of the aquaporin 2 (AQP2) revealed an apparently homozygous mutation at nucleotide position NM_000486.5:c.374C>T (p.Thr125Met) in exon 2. Further customized multiplex ligation-dependent probe amplification (MLPA), single-nucleotide polymorphism (SNP) array analysis, and long-range polymerase chain reaction (PCR) followed by Sanger sequencing showed a heterozygous exonic deletion comprising exons 2, 3, and partially 4 of AQP2. CONCLUSION: This is the first case of a compound heterozygote patient with a missense mutation involving NM_000486.5:exon2:c.374C>T (p.Thr125Met) and a gross deletion of at least exons 2, 3, and partially 4 on the AQP2 to present with a severe NDI phenotype.

Structural Basis for Mutations of Human Aquaporins Associated to Genetic Diseases.

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.

AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus.

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.

Amiloride modifies the progression of lithium-induced renal interstitial fibrosis.

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 ß-catenin activity through its effects on GSK-3ß.

Lithium induces aerobic glycolysis and glutaminolysis in collecting duct principal cells.

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 NH4+ 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.

Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus.

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.

Efficiency of Osmotic Concentration after Combined Treatment with Vasopressin and Blockage of Prostaglandin Synthesis.

We performed a complex functional study of the effects of prostaglandin synthesis blockage with diclofenac on manifestation of the hydroosmotic effect of vasopressin V2-receptor agonist desmopressin in the kidneys of Wistar rats with normal synthesis of endogenous vasopressin and homozygous Brattleboro rats with hereditary impaired synthesis of neurohypophyseal hormone vasopressin. Blockage of prostaglandin synthesis led to more pronounced increase in urine osmolality in Brattleboro rats than in Wistar rats due to elevation of not only urine but also sodium gradient at the expense of elimination of the inhibitory effect of prostaglandins on sodium reabsorption and membrane permeability for urine. During combined treatment, the effects of the hormone predominated: the increase in urine osmolality in Wistar and Brattleboro rats did not differ from that after desmopressin administration.

Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway.

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.

Epigallocatechin-3-gallate Attenuates Renal Damage by Suppressing Oxidative Stress in Diabetic db/db Mice.

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-ß1, 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.

Novel AVPR2 mutation causing partial nephrogenic diabetes insipidus in a Japanese family.

BACKGROUND: X-linked recessive congenital nephrogenic diabetes insipidus (NDI) is caused by mutations of the arginine vasopressin type 2 receptor gene (AVPR2). More than 200 mutations of the AVPR2 gene with complete NDI have been reported although only 15 mutations with partial NDI has been reported to date. METHODS: We herein report a Japanese kindred with partial NDI. The proband is an 8-year-old boy who was referred to our hospital for nocturnal enuresis. Water deprivation test and hypertonic saline test suggested partial renal antidiuretic hormone arginine vasopressin (AVP) resistance. RESULTS: Analysis of genomic DNA revealed a novel missense mutation (p.L161P) in the patient. The patient's mother was heterozygous for the mutation. Three-dimensional (3-D) modeling study showed that L161P possibly destabilizes the transmembrane domain of the V2 receptor, resulting in its misfolding or mislocalization. CONCLUSIONS: Distinguishing partial NDI from nocturnal enuresis is important. A clinical clue for diagnosis of partial NDI is an incompatibly high level of AVP despite normal serum osmolality.

An unusual case of hereditary nephrogenic diabetes insipidus (HNDI) affecting mother and daughter.

Hereditary nephrogenic diabetes iInsipidus (HNDI) is an uncommon disorder due to a resistance to anti-diuretic hormone leading to a reduced urinary concentrating ability. The X-linked form is fully expressed in hemizygous male patients, but diabetes insipidus may also present in heterozygous females where it must be distinguished from autosomal and other secondary causes. We report a mother and daughter in the same family with HNDI due to a heterozygous deletion in exon 1 of the AVPR2 gene, not previously described in the literature. A 5-year-old girl was referred for investigation of polyuria and polydipsia. The patient had a water deprivation test elsewhere at the age of 3 that was inconclusive. A degree of water restriction was imposed leading to headaches. The thyroid, cortisol, renal, and calcium profiles were normal. Her mother showed similar symptoms that had not been previously investigated. AQP2 (Aquaporin) and initial AVPR2 gene sequencing had not identified a mutation, but subsequent quantitative polymerase chain reaction analysis revealed a heterozygous large exon 1 deletion of the AVPR2 gene. The same deletion was also found in the child's mother. The patient's symptoms have significantly improved on appropriate treatment. Further analysis revealed skewed X inactivation in mother and daughter.

Folding and stability of the aquaglyceroporin GlpF: Implications for human aqua(glycero)porin diseases.

Aquaporins are highly selective polytopic transmembrane channel proteins that facilitate the permeation of water across cellular membranes in a large diversity of organisms. Defects in aquaporin function are associated with common diseases, such as nephrogenic diabetes insipidus, congenital cataract and certain types of cancer. In general, aquaporins have a highly conserved structure; from prokaryotes to humans. The conserved structure, together with structural dynamics and the structural framework for substrate selectivity is discussed. The folding pathway of aquaporins has been a topic of several studies in recent years. These studies revealed that a conserved protein structure can be reached by following different folding pathways. Based on the available data, we suggest a complex folding pathway for aquaporins, starting from the insertion of individual helices up to the formation of the tetrameric aquaporin structure. The consequences of some known mutations in human aquaporin-encoding genes, which most likely affect the folding and stability of human aquaporins, are discussed.