Upregulation of Urea Transporter UT-A2 and Water Channels AQP2 and AQP3 in Mice Lacking Urea Transporter UT-B

Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl-or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.sodium-potassium-2 chloride cotransporter; diabetes mellitus THE RENAL MEDULLA IS THE LOCATION in which water excretion is controlled through the production of concentrated or dilute urine. Several solute transport proteins play a major role in the urinary concentrating mechanism, including urea transporters and the NaϪ cotransporter (NKCC2/BSC1). Among the urea transporters, UT-A1 is expressed in the inner medullary collecting duct and is important for vasopressin-regulated urea reabsorption (reviewed in Ref. 21). UT-A2 and UT-B are expressed in the thin descending limb and descending vasa recta, respectively, and are important for intrarenal urea recycling (reviewed in Ref. 21). NKCC2/BSC1 is expressed in the thick ascending limb of the loop of Henle and is responsible for the active reabsorption of NaCl that drives the single effect to concentrate urine (reviewed in Ref. 22).Several studies show that UT-A1, as well as NKCC2/BSC1 and aquaporin-2 (AQP2), protein abundances increase after 5 days of uncontrolled diabetes mellitus due to streptozotocin (2,10,11,19,27). We showed that UT-A1 protein abundance does not change in streptozotocin-treated Brattleboro rats, indicating that vasopressin is necessary for the increase in UT-A1 protein abundance because Brattleboro rats lack vasopressin (11). When we administered vasopressin to Brattleboro rats and then induced diabetes melli...

Section: Discussionmentioning

confidence: 99%

Urea may regulate urea transporter protein abundance during osmotic diuresis

Kim¹,

Klein²,

Racine³

et al. 2005

“…A portion of each region was placed in RNAlater (Qiagen). The remaining tissues were homogenized and sheared for protein lysate, as we previously described (26). The samples in RNAlater were stored at 4°C overnight and then stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis

Seshadri

Klein

et al. 2006

121

expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis. Am J Physiol Renal Physiol 290: F397-F408, 2006. First published September 6, 2005 doi:10.1152/ajprenal.00162.2005.-Chronic metabolic acidosis induces dramatic increases in net acid excretion that are predominantly due to increases in urinary ammonia excretion. The current study examines whether this increase is associated with changes in the expression of the renal ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Chronic metabolic acidosis was induced in Sprague-Dawley rats by HCl ingestion for 1 wk; control animals were pair-fed. After 1 wk, metabolic acidosis had developed, and urinary ammonia excretion increased significantly. Rhcg protein expression was increased in both the outer medulla and the base of the inner medulla. Intercalated cells in the outer medullary collecting duct (OMCD) and in the inner medullary collecting duct (IMCD) in acid-loaded animals protruded into the tubule lumen and had a sharp, discrete band of apical Rhcg immunoreactivity, compared with a flatter cell profile and a broad band of apical immunolabel in control kidneys. In addition, basolateral Rhcg immunoreactivity was observed in both control and acidotic kidneys. Cortical Rhcg protein expression and immunoreactivity were not detectably altered. Rhcg mRNA expression was not significantly altered in the cortex, outer medulla, or inner medulla by chronic metabolic acidosis. Rhbg protein and mRNA expression were unchanged in the cortex, outer and inner medulla, and no changes in Rhbg immunolabel were evident in these regions. We conclude that chronic metabolic acidosis increases Rhcg protein expression in intercalated cells in the OMCD and in the IMCD, where it is likely to mediate an important role in the increased urinary ammonia excretion. connecting segment; collecting duct AMMONIA IS THE PRINCIPAL component of net acid excretion, a critical function of the kidneys, both under basal conditions and in response to metabolic acidosis (13,20).

“…Fluorescence-based real-time RT-PCR was carried out using the LightCycler instrument (Roche Diagnostics, Indianapolis, IN) with a LightCycler FastStart DNA Master PLUS SYBR Green I kit. Primers were designed, and real-time PCR was carried out as described previously (11). ␤-Actin was used as the reference gene, and pooled wild-type cDNA as the calibrator.…”

Section: Methodsmentioning

confidence: 99%

Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2 gene deletion

Yang

Zhao²,

Qian³

2006

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 ...