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

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

Inherited secondary nephrogenic diabetes insipidus: concentrating on humans.

The study of human physiology is paramount to understanding disease and developing rational and targeted treatments. Conversely, the study of human disease can teach us a lot about physiology. Investigations into primary inherited nephrogenic diabetes insipidus (NDI) have contributed enormously to our understanding of the mechanisms of urinary concentration and identified the vasopressin receptor AVPR2, as well as the water channel aquaporin-2 (AQP2), as key players in water reabsorption in the collecting duct. Yet, there are also secondary forms of NDI, for instance as a complication of lithium treatment. The focus of this review is secondary NDI associated with inherited human diseases, such as Bartter syndrome or apparent mineralocorticoid excess. Currently, the underlying pathophysiology of this inherited secondary NDI is unclear, but there appears to be true AQP2 deficiency. To better understand the underlying mechanism(s), collaboration between clinical and experimental physiologists is essential to further investigate these observations in appropriate experimental models.

Aquaporin 2 promotes cell migration and epithelial morphogenesis.

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin ß1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin ß1 by mutating the RGD motif led to reduced endocytosis, retention of integrin ß1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin ß1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

[Vasopressin receptor antagonists: the vaptans]. / Antagonistas del receptor de vasopresina: los vaptanes.

The non-peptide vasopressin antagonists (VPA), called vaptans, were developed in the 1990s to antagonize both the pressor and antidiuretic effects of vasopressin. There are three subtypes of VPA receptors: V1a, V1b and V2. V1a receptors are widely distributed in the body, mainly the blood vessels and myocardium. The V1b receptors are located mainly in the anterior pituitary gland and play a role in ACTH release. V2 receptors are located in the collecting tubular renal cells. Both V1a and V1b receptors act through the intracellular phosphoinositol signalling pathway, Ca(++) being the second messenger. V2 receptors work through AMPc generation, which promotes aquaporin 2 (AQP2) trafficking and allows water to enter the cell. The vaptans act competitively at the AVP receptor. The most important are mozavaptan, lixivaptan, satavaptan and tolvaptan, all of which are selective V2 antagonists and are administered through the oral route. In contrast, conivaptan is a dual V1 and V2 antagonist administered through the endovenous route. The main characteristics of vaptans are their effect on free water elimination without affecting electrolyte excretion. There are several studies on the effects of these drugs in hypervolemic hyponatremia (heart failure, hepatic cirrhosis) as well as in normovolemic hyponatremia (inappropriate secretion of ADH [SIADH]). Current studies show that the vaptans are effective and well tolerated, although knowledge of these drugs remains limited. There are no studies of the use of vaptans in severe hyponatremia. Osmotic demyelination syndrome due to excessively rapid correction of hyponatremia has not been described.

Hormone resistance at the clinical level.

This presentation introduces clinical aspects of hormone resistance through a number of case studies that illustrate how molecular defects at various steps in hormone production, signaling, or responsiveness can produce disease in humans. The presentation also shows how careful phenotyping of such patients has triggered decades of translational research.

Hsp90 inhibitor partially corrects nephrogenic diabetes insipidus in a conditional knock-in mouse model of aquaporin-2 mutation.

Mutations in aquaporin-2 (AQP2) that interfere with its cellular processing can produce autosomal recessive nephrogenic diabetes insipidus (NDI). Prior gene knock-in of the human NDI-causing AQP2 mutation T126M produced mutant mice that died by age 7 days. Here, we used a novel "conditional gene knock-in" strategy to generate adult, AQP2-T126M mutant mice. Mice separately heterozygous for floxed wild-type AQP2 and AQP2-T126M were bred to produce hemizygous mice, which following excision of the wild-type AQP2 gene by tamoxifen-induced Cre-recombinase gave AQP2(T126M/-) mice. AQP2(T126M/-) mice were polyuric (9-14 ml urine/day) compared to AQP2(+/+) mice (1.6 ml/day) and had reduced urine osmolality (400 vs. 1800 mosmol). Kidneys of AQP2(T126M/-) mice expressed core-glycosylated AQP2-T126M protein in an endoplasmic reticulum pattern. Screening of candidate protein folding "correctors" in AQP2-T126M-transfected kidney cells showed increased AQP2-T126M plasma membrane expression with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG increased urine osmolality in AQP2(T126M/-) mice by >300 mosmol but had no effect in AQP2(-/-) mice. Kidneys of 17-AAG-treated AQP2(T126M/-) mice showed partial rescue of defective AQP2-T126M cellular processing. Our results establish an adult mouse model of NDI and demonstrate partial restoration of urinary concentration function by a compound currently in clinical trials for other indications.

Severe urinary concentrating defect in renal collecting duct-selective AQP2 conditional-knockout mice.

Aquaporin-2 (AQP2) is the predominant vasopressin-regulated water channel in kidney connecting tubule (CNT) and collecting duct (CD) and is essential for renal regulation of body water balance. However, the relative role of AQP2 to urinary concentration in the CNT and CD segments is unknown. To examine this directly, transgenic mice expressing AQP2 selectively in CNT but lacking AQP2 expression in CD (AQP2-CD-KO) and mice lacking AQP2 globally (AQP2-total-KO) were generated by exploiting the Cre/loxP technology. LoxP sites were inserted into AQP2 introns 2 and 3, and transgenic mice were bred with strains expressing Cre recombinase under the control of CD-specific Hoxb7- or global EIIa promoter. Mice lacking AQP2 globally died postnatally (days 5-12). AQP2-CD-KO mice were viable to adulthood and showed decreased body weight, 10-fold increased urine production (0.96 +/- 0.11 vs. 0.10 +/- 0.01 ml/g of body weight), and decreased urinary osmolality (170 +/- 19 vs. 1,630 +/- 135 milliosmoles/kg of H(2)O). Immunohistochemical staining of AQP2-CD-KO kidneys (n = 12) revealed sustained, strong AQP2 expression in CNT cells, whereas >95% of CD principal cells were completely AQP2-negative. Water deprivation for 3 hours caused only marginal decreased urine output (87 +/- 7% of levels when mice had free water access; P = 0.04) with no change in urine osmolality, revealing an absence of compensatory mechanisms. These results demonstrate that AQP2 in CNT is sufficient for postnatal survival and that AQP2 in CD is essential for regulation of body water balance and cannot be compensated for by other mechanisms.