Purinergic signaling is enhanced in the absence of UT-A1 and UT-A3.

ATP is an important paracrine regulator of renal tubular water and urea transport. The activity of P2Y2 , the predominant P2Y receptor of the medullary collecting duct, is mediated by ATP, and modulates urinary concentration. To investigate the role of purinergic signaling in the absence of urea transport in the collecting duct, we studied wild-type (WT) and UT-A1/A3 null (UT-A1/A3 KO) mice in metabolic cages to monitor urine output, and collected tissue samples for analysis. We confirmed that UT-A1/A3 KO mice are polyuric, and concurrently observed lower levels of urinary cAMP as compared to WT, despite elevated serum vasopressin (AVP) levels. Because P2Y2 inhibits AVP-stimulated transport by dampening cAMP synthesis, we suspected that, similar to other models of AVP-resistant polyuria, purinergic signaling is increased in UT-A1/A3 KO mice. In fact, we observed that both urinary ATP and purinergic-mediated prostanoid (PGE2 ) levels were elevated. Collectively, our data suggest that the reduction of medullary osmolality due to the lack of UT-A1 and UT-A3 induces an AVP-resistant polyuria that is possibly exacerbated by, or at least correlated with, enhanced purinergic signaling.

Lack of urea transporters, UT-A1 and UT-A3, increases nitric oxide accumulation to dampen medullary sodium reabsorption through ENaC.

Although the role of urea in urine concentration is known, the effect of urea handling by the urea transporters (UTs), UT-A1 and UT-A3, on sodium balance remains elusive. Serum and urinary sodium concentration is similar between wild-type mice (WT) and UT-A3 null (UT-A3 KO) mice; however, mice lacking both UT-A1 and UT-A3 (UT-A1/A3 KO) have significantly lower serum sodium and higher urinary sodium. Protein expression of renal sodium transporters is unchanged among all three genotypes. WT, UT-A3 KO, and UT-A1/A3 KO acutely respond to hydrochlorothiazide and furosemide; however, UT-A1/A3 KO fail to show a diuretic or natriuretic response following amiloride administration, indicating that baseline epithelial Na+ channel (ENaC) activity is impaired. UT-A1/A3 KO have more ENaC at the apical membrane than WT mice, and single-channel analysis of ENaC in split-open inner medullary collecting duct (IMCD) isolated in saline shows that ENaC channel density and open probability is higher in UT-A1/A3 KO than WT. UT-A1/A3 KO excrete more urinary nitric oxide (NO), a paracrine inhibitor of ENaC, and inner medullary nitric oxide synthase 1 mRNA expression is ~40-fold higher than WT. Because endogenous NO is unstable, ENaC activity was reassessed in split-open IMCD with the NO donor PAPA NONOate [1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine)], and ENaC activity was almost abolished in UT-A1/A3 KO. In summary, loss of both UT-A1 and UT-A3 (but not UT-A3 alone) causes elevated medullary NO production and salt wasting. NO inhibition of ENaC, despite elevated apical accumulation of ENaC in UT-A1/A3 KO IMCD, appears to be the main contributor to natriuresis in UT-A1/A3 KO mice.

Chronic lithium treatment induces novel patterns of pendrin localization and expression.

Prolonged lithium treatment is associated with various renal side effects and is known to induce inner medullary collecting duct (IMCD) remodeling. In animals treated with lithium, the fraction of intercalated cells (ICs), which are responsible for acid-base homeostasis, increases compared with renal principal cells (PCs). To investigate the intricacies of lithium-induced IMCD remodeling, male Sprague-Dawley rats were fed a lithium-enriched diet for 0,1, 2, 3, 6, 9, or 12 wk. Urine osmolality was decreased at 1 wk, and from 2 to 12 wk, animals were severely polyuric. After 6 wk of lithium treatment, approximately one-quarter of the cells in the initial IMCD expressed vacuolar H+-ATPase, an IC marker. These cells were localized in portions of the inner medulla, where ICs are not normally found. Pendrin, a Cl-/[Formula: see text] exchanger, is normally expressed only in two IC subtypes found in the convoluted tubule, the cortical collecting duct, and the connecting tubule. At 6 wk of lithium treatment, we observed various patterns of pendrin localization and expression in the rat IMCD, including a novel phenotype wherein pendrin was coexpressed with aquaporin-4. These observations collectively suggest that renal IMCD cell plasticity may play an important role in lithium-induced IMCD remodeling.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3.

Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) have very low levels of urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability.

Smooth Muscle-Targeted Overexpression of Peroxisome Proliferator Activated Receptor-γ Disrupts Vascular Wall Structure and Function.

Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis.

Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct.

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.

Vasopressin regulation of multisite phosphorylation of UT-A1 in the inner medullary collecting duct.

Vasopressin signaling is critical for the regulation of urea transport in the inner medullary collecting duct (IMCD). Increased urea permeability is driven by a vasopressin-mediated elevation of cAMP that results in the direct phosphorylation of urea transporter (UT)-A1. The identification of cAMP-sensitive phosphorylation sites, Ser(486) and Ser(499), in the rat UT-A1 sequence was the first step in understanding the mechanism of vasopressin action on the phosphorylation-dependent modulation of urea transport. To investigate the significance of multisite phosphorylation of UT-A1 in response to elevated cAMP, we used highly specific and sensitive phosphosite antibodies to Ser(486) and Ser(499) to determine cAMP action at each phosphorylation site. We found that phosphorylation at both sites was rapid and sustained. Furthermore, the rate of phosphorylation of the two sites was similar in both mIMCD3 cells and rat inner medullary tissue. UT-A1 localized to the apical membrane in response to vasopressin was phosphorylated at Ser(486) and Ser(499). We confirmed that elevated cAMP resulted in increased phosphorylation of both sites by PKA but not through the vasopressin-sensitive exchange protein activated by cAMP pathway. These results elucidate the multisite phosphorylation of UT-A1 in response to cAMP, thus providing the beginning of understanding the intracellular factors underlying vasopressin stimulation of urea transport in the IMCD.

Genes and proteins of urea transporters.

A urea transporter protein in the kidney was first proposed in 1987. The first urea transporter cDNA was cloned in 1993. The SLC14a urea transporter family contains two major subgroups: SLC14a1, the UT-B urea transporter originally isolated from erythrocytes; and SLC14a2, the UT-A group originally isolated from kidney inner medulla. Slc14a1, the human UT-B gene, arises from a single locus located on chromosome 18q12.1-q21.1, which is located close to Slc14a2. Slc14a1 includes 11 exons, with the coding region extending from exon 4 to exon 11, and is approximately 30 kb in length. The Slc14a2 gene is a very large gene with 24 exons, is approximately 300 kb in length, and encodes 6 different isoforms. Slc14a2 contains two promoter elements: promoter I is located in the typical position, upstream of exon 1, and drives the transcription of UT-A1, UT-A1b, UT-A3, UT-A3b, and UT-A4; while promoter II is located within intron 12 and drives the transcription of UT-A2 and UT-A2b. UT-A1 and UT-A3 are located in the inner medullary collecting duct, UT-A2 in the thin descending limb and liver, UT-A5 in testis, UT-A6 in colon, UT-B1 primarily in descending vasa recta and erythrocytes, and UT-B2 in rumen.

Absence of PKC-alpha attenuates lithium-induced nephrogenic diabetes insipidus.

Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.

Lack of an effect of collecting duct-specific deletion of adenylyl cyclase 3 on renal Na+ and water excretion or arterial pressure.

cAMP is a key mediator of connecting tubule and collecting duct (CD) Na(+) and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na(+) or K(+) excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na(+) channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na(+) and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na(+) and water excretion in general or vasopressin regulation of CD function in particular.

miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export.

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or ß-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAß in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

Urine concentration in the diabetic mouse requires both urea and water transporters.

The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH(2)O. In contrast, urine osmolality was low (800 mosmol/kgH(2)O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.

Molecular mechanisms of urea transport in health and disease.

In the late 1980s, urea permeability measurements produced values that could not be explained by paracellular transport or lipid phase diffusion. The existence of urea transport proteins were thus proposed and less than a decade later, the first urea transporter was cloned. The family of urea transporters has two major subgroups, designated SLC14A1 (or UT-B) and Slc14A2 (or UT-A). UT-B and UT-A gene products are glycoproteins located in various extra-renal tissues however, a majority of the resulting isoforms are found in the kidney. The UT-B (Slc14A1) urea transporter was originally isolated from erythrocytes and two isoforms have been reported. In kidney, UT-B is located primarily in the descending vasa recta. The UT-A (Slc14A2) urea transporter yields six distinct isoforms, of which three are found chiefly in the kidney medulla. UT-A1 and UT-A3 are found in the inner medullary collecting duct (IMCD), while UT-A2 is located in the thin descending limb. These transporters are crucial to the kidney's ability to concentrate urine. The regulation of urea transporter activity in the IMCD involves acute modification through phosphorylation and subsequent movement to the plasma membrane. UT-A1 and UT-A3 accumulate in the plasma membrane in response to stimulation by vasopressin or hypertonicity. Long-term regulation of the urea transporters in the IMCD involves altering protein abundance in response to changes in hydration status, low protein diets, or adrenal steroids. Urea transporters have been studied using animal models of disease including diabetes mellitus, lithium intoxication, hypertension, and nephrotoxic drug responses. Exciting new genetically engineered mouse models are being developed to study these transporters.

Chronic use of chloroquine disrupts the urine concentration mechanism by lowering cAMP levels in the inner medulla.

Chloroquine, a widely used anti-malaria drug, has gained popularity for the treatment of rheumatoid arthritis, systemic lupus erythematosus (SLE), and human immunodeficiency virus (HIV). Unfortunately, chloroquine may also negatively impact renal function for patients whose fluid and electrolyte homeostasis is already compromised by diseases. Chronic administration of chloroquine also results in polyuria, which may be explained by suppression of the antidiuretic response of vasopressin. Several of the transporters responsible for concentrating urine are vasopressin-sensitive including the urea transporters UT-A1 and UT-A3, the water channel aquaporin-2 (AQP2), and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). To examine the effect of chloroquine on these transporters, Sprague-Dawley rats received daily subcutaneous injections of 80 mg·kg(-1)·day(-1) of chloroquine for 4 days. Twenty-four hour urine output was twofold higher, and urine osmolality was decreased by twofold in chloroquine-treated rats compared with controls. Urine analysis of treated rats detected the presence chloroquine as well as decreased urine urea and cAMP levels compared with control rats. Western blot analysis showed a downregulation of AQP2 and NKCC2 transporters; however, UT-A1 and UT-A3 abundances were unaffected by chloroquine treatment. Immunohistochemistry showed a marked reduction of UT-A1 and AQP2 in the apical membrane in inner medullary collecting ducts of chloroquine-treated rats. In conclusion, chloroquine-induced polyuria likely occurs as a result of lowered cAMP production. These findings suggest that chronic chloroquine treatment would exacerbate the already compromised fluid homeostasis observed in diseases like chronic kidney disease.

The role of nitric oxide in the dysregulation of the urine concentration mechanism in diabetes mellitus.

Uncontrolled diabetes mellitus results in osmotic diuresis. Diabetic patients have lowered nitric oxide (NO) which may exacerbate polyuria. We examined how lack of NO affects the transporters involved in urine concentration in diabetic animals. Diabetes was induced in rats by streptozotocin. Control and diabetic rats were given L-NAME for 3 weeks. Urine osmolality, urine output, and expression of urea and water transporters and the Na-K-2Cl cotransporter were examined. Predictably, diabetic rats presented with polyuria (increased urine volume and decreased urine osmolality). Although metabolic parameters of control rats were unaffected by L-NAME, treated diabetic rats produced 30% less urine and osmolality was restored. UT-A1 and UT-A3 were significantly increased in diabetic rat inner medulla. While L-NAME treatment alone did not alter UT-A1 or UT-A3 abundance, absence of NO prevented the upregulation of both transporters in diabetic rats. Similarly, AQP2 and NKCC2 abundance was increased in diabetic animals however, expression of these transporters were unchanged by L-NAME treatment of diabetes. Increased expression of the concentrating transporters observed in diabetic rats provides a compensatory mechanism to decrease solute loss despite persistent glycosuria. Our studies found that although diabetic-induced glycosylation remained increased, total protein expression was decreased to control levels in diabetic rats treated with L-NAME. While the role of NO in urine concentration remains unclear, lowered NO associated with diabetes may be deleterious to the transporters' response to the subsequent osmotic diuresis.

Lack of protein kinase C-α leads to impaired urine concentrating ability and decreased aquaporin-2 in angiotensin II-induced hypertension.

Regulation of water and urea transport in the inner medullary collecting duct is essential for urine concentration. Aquaporin (AQP)2 water channels and urea transporter (UT)-A1 are inserted into the apical membrane upon phosphorylation of the channels to allow the transcellular movement of water and urea. Since ANG II activates PKC in many cell types, we tested the hypothesis that ANG II-induced regulation of water and urea transport is mediated by PKC. Osmotic minipumps delivered ANG II to wild-type (WT) or PKC-α(-/-) mice for 7 days. Inner medullas were harvested, and protein abundance was determined by immunoblot. ANG II increased systolic blood pressure to a similar degree in WT and PKC-α(-/-) mice. ANG II had no effect on the urine output of WT mice but increased that of PKC-α(-/-) mice. In accordance with observed differences in urine output, AQP2 abundance was unchanged in ANG II-treated WT animals but was decreased in PKC-α(-/-) mice. No change in membrane accumulation was seen. Phosphorylation of the cAMP-induced transcription factor CREB was decreased in PKC-α(-/-) mice in response to ANG II with no change in overall CREB abundance. ANG II did not alter the abundance of UT-A1 protein in WT or PKC-α(-/-) mice. Phosphorylation and overall abundance of tonicity-responsive enhancer-binding protein, a transcription factor that regulates UT-A1, were also unaltered by ANG II in either group. We conclude that PKC-α protects against ANG II-induced decreases in urine concentrating ability by maintaining AQP2 levels through CREB phosphorylation.

Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice.

Collecting duct (CD) adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption. To evaluate the role of CD-derived AC6 in regulating water homeostasis, mice were generated with CD-specific knockout (KO) of AC6 using the Cre/loxP system. CD AC6 KO and controls were studied under normal water intake, chronically water loaded, or water deprived; all of these conditions were repeated in the presence of continuous administration of 1-desamino-8-d-arginine vasopressin (DDAVP). During normal water intake or after water deprivation, urine osmolality (U(osm)) was reduced in CD AC6 KO animals vs. controls. Similarly, U(osm) was decreased in CD AC6 KO mice vs. controls after water deprivation+DDAVP administration. Pair-fed (with controls) CD AC6 KO mice also had lower urine osmolality vs. controls. There were no detectable differences between KO and control animals in fluid intake or urine volume under any conditions. CD AC6 KO mice did not have altered plasma AVP levels vs. controls. AVP-stimulated cAMP accumulation was reduced in acutely isolated inner medullary CD (IMCD) from CD A6 KO vs. controls. Medullary aquaporin-2 (AQP2) protein expression was lower in CD AC6 KO mice vs. controls. There were no differences in urinary urea excretion or IMCD UT-A1 expression; however, IMCD UT-A3 expression was reduced in CD AC6 KO mice vs. controls. In summary, AC6 in the CD regulates renal water excretion, most likely through control of AVP-stimulated cAMP accumulation and AQP2.

Acute calcineurin inhibition with tacrolimus increases phosphorylated UT-A1.

UT-A1, the urea transporter present in the apical membrane of the inner medullary collecting duct, is crucial to the kidney's ability to concentrate urine. Phosphorylation of UT-A1 on serines 486 and 499 is important for plasma membrane trafficking. The effect of calcineurin on dephosphorylation of UT-A1 was investigated. Inner medullary collecting ducts from Sprague-Dawley rats were metabolically labeled and treated with tacrolimus to inhibit calcineurin or calyculin to inhibit protein phosphatases 1 and 2A. UT-A1 was immunoprecipitated, electrophoresed, blotted, and total UT-A1 phosphorylation was assessed by autoradiography. Total UT-A1 was determined by Western blotting. A phospho-specific antibody to pser486-UT-A1 was used to determine whether serine 486 can be hyperphosphorylated by inhibiting phosphatases. Inhibition of calcineurin showed an increase in phosphorylation per unit protein at serine 486. In contrast, inhibition of phosphatases 1 and 2A resulted in an increase in UT-A1 phosphorylation but no increase in pser486-UT-A1. In vitro perfusion of inner medullary collecting ducts showed tacrolimus-stimulated urea permeability consistent with stimulated urea transport. The location of phosphorylated UT-A1 in rats treated acutely and chronically with tacrolimus was determined using immunohistochemistry. Inner medullary collecting ducts of the acutely treated rats showed increased apical membrane association of phosphorylated UT-A1 while chronic treatment reduced membrane association of phosphorylated UT-A1. We conclude that UT-A1 may be dephosphorylated by multiple phosphatases and that the PKA-phosphorylated serine 486 is dephosphorylated by calcineurin. This is the first documentation of the role of phosphatases and the specific site of phosphorylation of UT-A1, in response to tacrolimus.