No abstract

Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable full-length AQP2 transcript in kidney and a Ͼ95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from ϳ2,000 to Ͻ500 mosmol/kgH2O after 4 -5 days, with urine output increasing from 2 to 25 ml/day. Urine osmolality did not increase after water deprivation. Interestingly, AQP3 protein expression in the collecting duct was increased by about fivefold after AQP2 gene excision. Mild renal damage was seen after 6 wk of polyuria, with collecting duct dilatation, yet normal creatinine clearance and serum chemistries. These results establish the first adult model of nephrogenic diabetes insipidus (NDI) caused by AQP2 deficiency, with daily urine output comparable to body weight, although remarkable preservation of renal function compared with non-inducible NDI models. water transport; water channel; transgenic mouse; NDI; polyuria AQUAPORIN-2 (AQP2) IS THE antidiuretic hormone (vasopressin)-regulated water channel expressed in the mammalian kidney collecting duct. Apical plasma membrane AQP2 targeting is regulated by a vesicular transport mechanism in which vasopressin acting through cAMP causes the exocytic insertion of AQP2 (1, 26). AQP2 is of clinical importance in acquired disorders of urinary concentrating function, both in nephrogenic diabetes insipidus (NDI), as produced by lithium therapy (20), and the syndrome of inappropriate antidiuretic hormone (7). Mutations in the AQP2 gene can cause the very rare disorder of hereditary NDI (5,8,12). Several AQP2 mutations causing autosomal recessive NDI, such as AQP2-T126M, are associated with defective AQP2 folding and retention at the endoplasmic reticulum (2, 23, 36). There is great interest in identifying small-molecule correctors of defective protein folding as potential therapy for human hereditary diseases, such as NDI caused by V2 receptor mutation (22) and cystic fibrosis caused by CFTR mutation (28). We found that small-molecule chemical chaperones correct the defective processing of AQP2 mutants in transfected cell models, including AQP2-T126M (35).Transgenic mouse models of NDI caused by AQP2 mutation are needed to evaluate the in vivo efficacy of small-molecule therapies that are active in cell culture models. To this end, we generated transgenic AQP2-T126M knock-in mice in which the T126M mutation was introduced into the mouse AQP2 gene by targeted ...

Abstract. The UT-B urea transporter is the major urea transporter in red blood cells and kidney descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect. Whether deletion of UT-B altered the expression of other transporter proteins involved in urinary concentration was tested. Fluorescence-based real-time reverse transcription-PCR and Northern blot analysis showed upregulation of the UT-A2 urea transporter and the aquaporin 2 (AQP2) and AQP3 water channel transcripts but no change in other urea transporters or AQP. Western blot analysis showed that UT-A2 protein abundance in the outer medulla of UT-B null mice increased to 122 Ϯ 6% of wild-type control. AQP2 protein abundance increased to 177 Ϯ 32% and 127 Ϯ 7% in the outer and inner medulla, respectively, of UT-B null versus wild-type mice. The abundance of UT-A1, AQP1, renal outer medullary potassium channel, and NKCC2/BSC1 proteins were not significantly different between UT-B null and wild-type mice. The increases in AQP2 and AQP3 would reduce water loss and improve concentrating ability. The lack of UT-B does not result in a change in expression of urea transporters involved in urea reabsorption from the inner medullary collecting duct (UT-A1 and UT-A3). However, UT-B null mice have a selective increase in UT-A2 protein abundance. This may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.

Aquaporin-1 (AQP1) and aquaporin-3 (AQP3) water channels expressed in the kidney play a critical role in the urine concentrating mechanism. Mice with AQP1 or AQP3 deletion have a urinary concentrating defect. To better characterize this defect, we studied the influence of an acute urea load (300 mumol ip) in conscious AQP1-null, AQP3-null, and wild-type mice. Urine was collected and assayed every 2 h, from 2 h before (baseline) to 8 h after the urea load. Mice of all genotypes excreted the urea load in approximately 4 h with the same time course. Interestingly, despite their low baseline, the AQP3-null mice raised their urine osmolality and urea concentration progressively after the urea load to values almost equal to those in wild-type mice at 8 h. In contrast, urine non-urea solute concentration did not change. Urine volume fell in the last 4 h to about one-fourth of basal values. AQP1-null mice increased their urine flow rate much more than AQP3-null mice and showed no change in urine osmolality and urea concentration. The urea load strongly upregulated urea transporter UT-A3 expression in all three genotypes. These observations show that the lack of AQP3 does not interfere with the ability of the kidney to concentrate urea but impairs its ability to concentrate other solutes. This solute-selective response could result from the capacity of AQP3 to transport not only water but also urea. The results suggest a novel role for AQP3 in non-urea solute concentration in the urine.