Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats. Am J Physiol Renal Physiol 285: F303-F309, 2003. First published April 15, 2003 10.1152/ajprenal.00438.2002We tested whether the abundance of transport proteins involved in the urinary concentrating mechanism was altered in rats with uncontrolled diabetes mellitus (DM). Rats were injected with streptozotocin and killed 5, 10, 14, or 20 days later. Blood glucose in DM rats was 300-450 mg/dl (control: 70-130 mg/dl). Urine volume increased in DM rats from 41 Ϯ 7 ml/100 g body wt (BW) at 5 days to 69 Ϯ 3 ml/100 g BW at 20 days (control: 9 Ϯ 1). Urine osmolality of DM rats decreased at 5 days DM and remained low at 20 days. UT-A1 urea transporter protein in the inner medullary (IM) tip was 55% of control in 5-day DM rats but increased to 170, 220, and 280% at 10, 14, and 20 days DM, respectively, due to an increase in the 117-kDa glycoprotein form. UT-A1 in the IM base was increased to 325% of control at 5 days DM with no further increase at 20 days. Aquaporin-2 (AQP2) increased to 290% in the IM base at 5 days DM and 150% in the IM tip at 10 days; both showed no further increase at 20 days. NKCC2/ BSC1 increased to 240% in outer medulla at 20 days DM, but not at 5 or 10 days. UT-B and ROMK were unchanged at any time point. The increases in UT-A1, AQP2, and NKCC2/ BSC1 proteins during uncontrolled DM would tend to limit the loss of fluid and solute during uncontrolled diabetes. urea; water; urea transporter; aquaporin; sodium PATIENTS WITH UNCONTROLLED type I diabetes mellitus (DM) are polyuric due to the osmotic diuresis caused by glucosuria. The persistent osmotic diuresis frequently results in a serious degree of volume depletion, but these patients rarely present for medical attention with shock and cardiovascular collapse. Instead, they generally present with severe hyperglycemia and diabetic ketoacidosis. This clinical presentation suggests the hypothesis that compensatory changes occur within the kidney that permit sufficient solute and water reabsorption, despite the ongoing osmotic diuresis, to prevent hypovolemic shock.The goal of this study was to test this hypothesis using rats made diabetic by streptozotocin (STZ) injection. The STZ-treated rat is a commonly used animal model of type I diabetes. These rats rapidly develop hyperglycemia and polyuria, although they do not develop ketoacidosis. Because the renal medulla is responsible for the production of concentrated or dilute urine, we hypothesized that any compensatory mechanism to conserve water and solute may involve changes in the abundance of the medullary transport proteins involved in the urinary concentrating mechanism. Therefore, we measured the abundance of the UT-A1 and UT-B urea transporters, the aquaporin-2 (AQP2) water channel, the NKCC2/BSC1 NaϪ cotransporter, and the ROMK K ϩ channel from rats made diabetic for 5, 10, 14, or 20 days. METHODSAnimal preparation. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 125-200...

. Expression of urea transporters in the developing rat kidney. Am J Physiol Renal Physiol 282: F530-F540, 2002; 10.1152/ajprenal.00246.2001.-Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). Because newborn rats are not capable of producing concentrated urine, we examined the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunocytochemistry. Kidneys from 16-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were studied. In the adult kidney, UT-A was expressed intensely in the inner medullary collecting duct (IMCD) and terminal portion of the short-loop descending thin limb (DTL) and weakly in long-loop DTL in the outer part of the inner medulla. UT-A immunoreactivity was not present in the fetal kidney but was observed in the IMCD and DTL in 1-day-old pups. The intensity of UT-A immunostaining in the IMCD gradually increased during postnatal development. In 4-and 7-day-old pups, UT-A immunoreactivity was present in the DTL at the border between the outer and inner medulla. In 14-and 21-day-old pups, strong UT-A immunostaining was observed in the terminal part of short-loop DTL in the outer medulla, and weak labeling remained in long-loop DTL descending into the outer part of the inner medulla. In the adult kidney, there was intense staining for UT-B in descending vasa recta (DVR) and weak labeling of glomeruli. In the developing kidney, UT-B was first observed in the DVR of a 20-day-old fetus. After birth there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles. The intensity of immunostaining remained strong in the outer medulla but gradually decreased in the inner medulla. We conclude that the expression of urea transporters in short-loop DTL and DVR coincides with the development of the ability to produce a concentrated urine.

In rats with streptozotocin-induced diabetes mellitus for 10-20 days, we showed that the abundance of the major medullary transport proteins involved in the urinary concentrating mechanism, urea transporter (UT-A1), aquaporin-2 (AQP2), and the Na+-K+-2Cl- cotransporter (NKCC2/BSC1), is increased, despite the ongoing osmotic diuresis. To test whether vasopressin is necessary for these diabetes mellitus-induced changes in UT-A1, AQP2, or NKCC2/BSC1, we studied Brattleboro rats because they lack vasopressin. Brattleboro rats were given vasopressin (2.4 microg/day via osmotic minipump) for 5 or 12 days. At 5 days, vasopressin increased AQP2 protein abundance but decreased UT-A1 abundance compared with untreated Brattleboro rats. At 12 days, vasopressin increased the abundance of both UT-A1 and AQP2 proteins but did not alter NKCC2/BSC1. Next, untreated Brattleboro rats were made diabetic for 10 days by injecting them with streptozotocin (40 mg/kg). Diabetes mellitus increased the abundance of AQP2 and NKCC2/BSC1 proteins, but UT-A1 protein abundance did not increase. Third, vasopressin-treated Brattleboro rats were made diabetic with streptozotocin for 10 days. In vasopressin-treated Brattleboro rats, diabetes mellitus increased UT-A1, AQP2, and NKCC2/BSC1 protein abundances. Vasopressin significantly increased UT-A1 phosphorylation in vasopressin-treated diabetic Brattleboro rats but not in the other groups of Brattleboro rats. We conclude that 1) administering vasopressin to Brattleboro rats for 12 days, but not for 5 days, increases UT-A1 protein abundance and 2) vasopressin is necessary for the increase in UT-A1 protein in diabetic rats but is not necessary for the increase in AQP2 or NKCC2 proteins.

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...

Sands JM, Kim D. Urea and NaCl regulate UT-A1 urea transporter in opposing directions via TonEBP pathway during osmotic diuresis. Am J Physiol Renal Physiol 296: F67-F77, 2009. First published October 22, 2008 doi:10.1152/ajprenal.00143.2008.-In our previous studies of varying osmotic diuresis, UT-A1 urea transporter increased when urine and inner medullary (IM) interstitial urea concentration decreased. The purposes of this study were to examine 1) whether IM interstitial tonicity changes with different urine urea concentrations during osmotic dieresis and 2) whether the same result occurs even if the total urinary solute is decreased. Rats were fed a 4% high-salt diet (HSD) or a 5% high-urea diet (HUD) for 2 wk and compared with the control rats fed a regular diet containing 1% NaCl. The urine urea concentration decreased in HSD but increased in HUD. In the IM, UT-A1 and UT-A3 urea transporters, CLC-K1 chloride channel, and tonicity-enhanced binding protein (TonEBP) transcription factor were all increased in HSD and decreased in HUD. Next, rats were fed an 8% low-protein diet (LPD) or a 0.4% low-salt diet (LSD) to decrease the total urinary solute. Urine urea concentration significantly decreased in LPD but significantly increased in LSD. Rats fed the LPD had increased UT-A1 and UT-A3 in the IM base but decreased in the IM tip, resulting in impaired urine concentrating ability. The LSD rats had decreased UT-A1 and UT-A3 in both portions of the IM. CLC-K1 and TonEBP were unchanged by LPD or LSD. We conclude that changes in CLC-K1, UT-A1, UT-A3, and TonEBP play important roles in the renal response to osmotic diuresis in an attempt to minimize changes in plasma osmolality and maintain water homeostasis.UT-A3 urea transporter; CLC-K1 chloride channel; TonEBP transcription factor THE UT-A1 UREA TRANSPORTER plays a key role in urine concentration in mammals (21). In our previous studies, SpragueDawley rats made diabetic with streptozotocin had a great increase in UT-A1 abundance in both inner medullary (IM) base and tip to conserve water despite the ongoing osmotic diuresis (2, 12, 13). In contrast, diabetic Brattleboro rats (which lack vasopressin) did not increase UT-A1 protein abundance, and vasopressin-treated diabetic Brattleboro rats increased UT-A1 protein abundance more than nondiabetic vasopressintreated Brattleboros (14). This suggests that vasopressin and another factor (or factors) work together to increase UT-A1 abundance.Rats with NaCl diuresis induced by feeding of a high-salt diet also had a significant increase in UT-A1 abundance in both portions of the IM. However, rats with urea diuresis induced by feeding of urea did not increase UT-A1 abundance in either portion of IM, regardless of the severity of the osmotic diuresis (12). Rats with diabetes or NaCl diuresis have relatively decreased urea in the total urinary solute since the excretion of other solutes (glucose or NaCl) is increased, whereas rats fed a high-protein or -urea diet have an increase of urea in the total urinary solute, suggestin...