The kidney's ability to concentrate urine is vitally important to our quality of life. In the hypertonic environment of the kidney, urea transporters must be regulated to optimize function. We previously showed that hypertonicity increases urea permeability and that the protein kinase C (PKC) blockers chelerythrine and rottlerin decreased hypertonicity-stimulated urea permeability in rat inner medullary collecting ducts (IMCDs). Because PKCα knockout (PKCα−/−) mice have a urine-concentrating defect, we tested the effect of hypertonicity on urea permeability in isolated perfused mouse IMCDs. Increasing the osmolality of perfusate and bath from 290 to 690 mosmol/kgH2O did not change urea permeability in PKCα−/− mice but significantly increased urea permeability in wild-type mice. To determine whether the response to protein kinase A was also missing in IMCDs of PKCα−/− mice, tubules were treated with vasopressin and subsequently with the PKC stimulator phorbol dibutyrate (PDBu). Vasopressin stimulated urea permeability in PKCα−/− mice. Like vasopressin, forskolin stimulated urea permeability in PKCα−/− mice. We previously showed that, in rats, vasopressin and PDBu have additive stimulatory effects on urea permeability. In contrast, in PKCα−/− mice, PDBu did not further increase vasopressin-stimulated urea permeability. Western blot analysis showed that expression of the UT-A1 urea transporter in IMCDs was increased in response to vasopressin in wild-type mice as well as PKCα−/− mice. Hypertonicity increased UT-A1 phosphorylation in wild-type mice but not in PKCα−/− mice. We conclude that PKCα mediates hypertonicity-stimulated urea transport but is not necessary for vasopressin stimulation of urea permeability in mouse IMCDs.
Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the inner medullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in the IMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases both the phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of a calcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigated whether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effect of PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolality from 290 to 690 mosmol/kgH2O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either 10 μM chelerythrine or 50 μM rottlerin. To determine the potential synergy between vasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated urea permeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeability was reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transport through a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of a cascade of activations remains to be determined.
BACKGROUND: Urea transporters (UTs) are important in urine concentration and in urea recycling, and UT-B has been implicated in both. In kidney, UT-B was originally localized to outer medullary descending vasa recta, and more recently detected in inner medullary descending vasa recta. Endogenously produced microRNAs (miRs) bind to the 3'UTR of genes and generally inhibit their translation, thus playing a pivotal role gene regulation. METHODS: Mice were dehydrated for 24 hours then sacrificed. Inner and outer medullas were analyzed by polymerase chain reaction (PCR) and quantitative PCR for miRNA expression and analyzed by western blotting for protein abundance. RESULTS: MiRNA sequencing analysis of mouse inner medullas showed a 40% increase in miRNA-200c in dehydrated mice compared with controls. An in silico analysis of the targets for miR-200c revealed that miRNA-200c could directly target the gene for UT-B. PCR confirmed that miR-200c is up-regulated in the inner medullas of dehydrated mice while western blot showed that UT-B protein abundance was down-regulated in the same portion of the kidney. However, in the outer medulla, miR-200c was reduced and UT-B protein was increased in dehydrated mice. CONCLUSIONS: This is the first indication that UT-B protein and miR-200c may each be differentially regulated by dehydration within the kidney outer and inner medulla. The inverse correlation between the direction of change in miR-200c and UT-B protein abundance in both the inner and outer medulla suggests that miR-200c may be associated with the change in UT-B protein in these 2 portions of the kidney medulla.
Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension.
The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [32P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.
AIM: This study evaluates the effect of dapagliflozin, a SGLT2 inhibitor, on fluid or electrolyte balance and its effect on urea transporter-A1 (UT-A1), aquaporin-2 (AQP2) and Na-K-2Cl cotransporter (NKCC2) protein abundance in diabetic rats. METHODS: Diabetes mellitus (DM) was induced by injection of streptozotocin into the tail vein. Serum Na+, K+, Cl- concentration, urine Na+, K+, Cl- excretion, blood glucose, urine glucose excretion, urine volume, urine osmolality and urine urea excretion were analyzed after the administration of dapagliflozin. UT-A1, AQP2 and NKCC2 proteins were detected by western blot. RESULTS: Dapagliflozin treatment decreased blood glucose concentration by 38% at day 7 and by 47% at day 14 and increased the urinary glucose excretion rate compared with the untreated diabetic animals. Increased 24-hour urine volume, decreased urine osmolality and hyponatremia, hypokalemia and hypochloremia observed in diabetic rats were attenuated by dapagliflozin treatment. Western blot analysis showed that UT-A1, AQP2 and NKCC2 proteins are upregulated in DM rats over control rats; dapagliflozin treatment results in a further increase in inner medulla tip UT-A1 protein abundance by 42% at day 7 and by 46% at day 14, but it did not affect the DM-induced upregulation of AQP2 and NKCC2 proteins. CONCLUSION: Dapagliflozin treatment augmented the compensatory changes in medullary transport proteins in DM. These changes would tend to conserve solute and water even with persistent glycosuria. Therefore, diabetic rats treated with dapagliflozin have a mild osmotic diuresis compared to nondiabetic animals, but this does not result in an electrolyte disorder or significant volume depletion.