Search

Hyponatremia (hypo-osmolality) is a disorder of water homeostasis due to abnormal renal diluting capacity. The body limits the degree to which serum sodium concentration falls through a mechanism called "vasopressin escape". Vasopressin escape is a process that prevents the continuous decrease in serum sodium concentration even under conditions of sustained high plasma vasopressin levels. Previous reports suggest that aldosterone may be involved in the vasopressin escape mechanism. The abilities of aldosterone synthase (Cyp11b2) knockout and wild-type mice to escape from vasopressin were compared. Wild-type mice escaped while the aldosterone synthase knockout mice did not. Both the water channel aquaporin 2 (AQP2) and the urea transporter UT-A1 protein abundances were higher in aldosterone synthase knockout than in wild-type mice at the end of the escape period. Vasopressin escape was also blunted in rats given spironolactone, a mineralocorticoid receptor blocker. Next, the role of the phosphatase, calcineurin (protein phosphatase 2B, PP2B), in vasopressin escape was studied since aldosterone activates calcineurin in rat cortical collecting ducts. Tacrolimus, a calcineurin inhibitor, blunted vasopressin escape in rats compared with the control rats, increased UT-A1, AQP2, and pS256-AQP2, and decreased pS261-AQP2 protein abundances. Our results indicate that aldosterone regulates vasopressin escape through calcineurin-mediated protein changes in UT-A1 and AQP2. [ABSTRACT FROM AUTHOR]

We previously showed that the phosphatases PP1/PP2A and PP2B dephosphorylate the water channel, AQP2, suggesting their role in water reabsorption. In this study, we investigated whether protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B or calcineurin), which are present in the inner medullary collecting duct (IMCD), are regulators of urea and water permeability. Inhibition of calcineurin by tacrolimus increased both basal and vasopressin-stimulated osmotic water permeability in perfused rat IMCDs. However, tacrolimus did not affect osmotic water permeability in the presence of aldosterone. Inhibition of PP2A by calyculin increased both basal and vasopressin-stimulated osmotic water permeability, and aldosterone reversed the increase by calyculin. Previous studies showed that adrenomedullin (ADM) activates PP2A and decreases osmotic water permeability. Inhibition of PP2A by calyculin prevented the ADM-induced decrease in water reabsorption. ADM reduced the phosphorylation of AQP2 at serine 269 (pSer269 AQP2). Urea is linked to water reabsorption by building up hyperosmolality in the inner medullary interstitium. Calyculin increased urea permeability and phosphorylated UT-A1. Our results indicate that phosphatases regulate water reabsorption. Aldosterone and adrenomedullin decrease urea or osmotic water permeability by acting through calcineurin and PP2A, respectively. PP2A may regulate water reabsorption by dephosphorylating pSer269, AQP2, and UT-A1. [ABSTRACT FROM AUTHOR]

Background: Our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) prevents muscle atrophy by attenuation of protein degradation in mice. The current study examines the impact of Acu/LFES on protein synthesis. Method: C57/BL6 mice received Acu/LFES treatment on hindlimb for 30 min once. Acu/LFES points were selected by WHO Standard Acupuncture Nomenclature and electric stimulation applied using an SDZ-II Electronic acupuncture instrument. Muscle protein synthesis was measured by the surface-sensing of translation (SUnSET) assay. Exosomes were isolated using serial centrifugation and concentration and size of the collected exosomes were measured using a NanoSight instrument. The mature microRNA library in serum exosomes was validated using a High Sensitivity DNA chip. Results: Protein synthesis was enhanced in the both hindlimb and forelimb muscles. Blocking exosome secretion with GW4869 decreased the Acu/LFES-induced increases in protein synthesis. MicroRNA-deep sequencing demonstrated that four members of the Let-7 miRNA family were significantly decreased in serum exosomes. Real time qPCR further verified Acu/LFES-mediated decreases of let-7c-5p in serum exosomes and skeletal muscles. In cultured C2C12 myotubes, inhibition of let-7c not only increased protein synthesis, but also enhanced protein abundance of Igf1 and Igf1 receptors. Using a luciferase reporter assay, we demonstrated that let-7 directly inhibits Igf1. Conclusion: Acu/LFES on hindlimb decreases let-7-5p leading to upregulation of the Igf1 signaling and increasing protein synthesis in both hindlimb and forelimb skeletal muscles. This provides a new understanding of how the electrical acupuncture treatment can positively influence muscle health. [ABSTRACT FROM AUTHOR]

Aim: We have reported earlier that a high salt intake triggered an aestivation‐like natriuretic‐ureotelic body water conservation response that lowered muscle mass and increased blood pressure. Here, we tested the hypothesis that a similar adaptive water conservation response occurs in experimental chronic renal failure. Methods: In four subsequent experiments in Sprague Dawley rats, we used surgical 5/6 renal mass reduction (5/6 Nx) to induce chronic renal failure. We studied solute and water excretion in 24‐hour metabolic cage experiments, chronic blood pressure by radiotelemetry, chronic metabolic adjustment in liver and skeletal muscle by metabolomics and selected enzyme activity measurements, body Na+, K+ and water by dry ashing, and acute transepidermal water loss in conjunction with skin blood flow and intra‐arterial blood pressure. Results: 5/6 Nx rats were polyuric, because their kidneys could not sufficiently concentrate the urine. Physiological adaptation to this renal water loss included mobilization of nitrogen and energy from muscle for organic osmolyte production, elevated norepinephrine and copeptin levels with reduced skin blood flow, which by means of compensation reduced their transepidermal water loss. This complex physiologic‐metabolic adjustment across multiple organs allowed the rats to stabilize their body water content despite persisting renal water loss, albeit at the expense of hypertension and catabolic mobilization of muscle protein. Conclusion: Physiological adaptation to body water loss, termed aestivation, is an evolutionary conserved survival strategy and an under‐studied research area in medical physiology, which besides hypertension and muscle mass loss in chronic renal failure may explain many otherwise unexplainable phenomena in medicine. [ABSTRACT FROM AUTHOR]

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and mycBK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway. [ABSTRACT FROM AUTHOR]

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-β, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-β, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson’s trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO. [ABSTRACT FROM AUTHOR]

The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca2+ in control podocytes but not in TRPC6 knockdown podocytes. High glucose also elevated the expression of a tight junction protein, zonula occludens-1, and induced the redistribution of zonula occludens-1 and loss of podocyte processes. These data together suggest that high glucose reduces protein levels of podocin by activating TRPC6 and induces morphological changes of cultured podocytes. [ABSTRACT FROM AUTHOR]

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTβ), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels. [ABSTRACT FROM AUTHOR]

Acupuncture with low-frequency electrical stimulation (Acu/LFES) can prevent muscle atrophy by increasing muscle protein anabolism in mouse models of chronic kidney disease. During the treatment of muscle wasting, we found that Acu/LFES on the gastrocnemius muscle of the leg enhances renal blood flow. We also found that Acu/LFES increases exosome abundance and alters exosome-associated microRNA expression in the circulation. When exosome secretion was blocked using GW4869, the Acu/LFES-induced increase in renal blood flow was limited. This provided evidence that the increased renal blood flow is exosome mediated. To identify how exosomes regulate renal blood flow, we performed microRNA deep sequencing in exosomes isolated from treated and untreated mouse serum and found that the 34 microRNAs are altered by Acu/LFES. In particular, miR-181d-5p is increased in the serum exosome of Acu/ LFES-treated mice. In silico searching suggested that miR-181d-5p could target angiotensinogen. Using a luciferase reporter assay, we demonstrated that miR-181 directly inhibits angiotensinogen. When Acu/LFES-treated muscle was excised and incubated in culture medium, we found that the amount of exosomes and miR-181d-5p was increased in the medium providing evidence that Acu/LFES can increase miR-181 secretion. We conclude that Acu/LFES on leg hindlimb increases miR-181 in serum exosome leading to increased renal blood flow. This study provides important new insights about the mechanism(s) by which acupuncture may regulation of muscle-organ cross talk through exosome-derived microRNA. [ABSTRACT FROM AUTHOR]

Abstract: Background: The treatment of muscle wasting is accompanied by benefits in other organs, possibly resulting from muscle–organ crosstalk. However, how the muscle communicates with these organs is less understood. Two microRNAs (miRs), miR‐23a and miR‐27a, are located together in a gene cluster and regulate proteins that are involved in the atrophy process. MiR‐23a/27a has been shown to reduce muscle wasting and act as an anti‐fibrotic agent. We hypothesized that intramuscular injection of miR‐23a/27a would counteract both muscle wasting and renal fibrosis lesions in a streptozotocin‐induced diabetic model. Methods: We generated an adeno‐associated virus (AAV) that overexpresses the miR‐23a∼27a∼24‐2 precursor RNA and injected it into the tibialis anterior muscle of streptozotocin‐induced diabetic mice. Muscle cross‐section area (immunohistology plus software measurement) and muscle function (grip strength) were used to evaluate muscle atrophy. Fibrosis‐related proteins were measured by western blot to monitor renal damage. In some cases, AAV‐GFP was used to mimic the miR movement in vivo, allowing us to track organ redistribution by using the Xtreme Imaging System. Results: The injection of AAV‐miR‐23a/27a increased the levels of miR‐23a and miR‐27a as well as increased phosphorylated Akt, attenuated the levels of FoxO1 and PTEN proteins, and reduced the abundance of TRIM63/MuRF1 and FBXO32/atrogin‐1 in skeletal muscles. It also decreased myostatin mRNA and protein levels as well as the levels of phosphorylated pSMAD2/3. Provision of miR‐23a/27a attenuates the diabetes‐induced reduction of muscle cross‐sectional area and muscle function. Curiously, the serum BUN of diabetic animals was reduced in mice undergoing the miR‐23a/27a intervention. Renal fibrosis, evaluated by Masson trichromatic staining, was also decreased as were kidney levels of phosphorylated SMAD2/3, alpha smooth muscle actin, fibronectin, and collagen. In diabetic mice injected intramuscularly with AAV‐GFP, GFP fluorescence levels in the kidneys showed linear correlation with the levels in injected muscle when examined by linear regression. Following intramuscular injection of AAV‐miR‐23a∼27a∼24‐2, the levels of miR‐23a and miR‐27a in serum exosomes and kidney were significantly increased compared with samples from control virus‐injected mice; however, no viral DNA was detected in the kidney. Conclusions: We conclude that overexpression of miR‐23a/27a in muscle prevents diabetes‐induced muscle cachexia and attenuates renal fibrosis lesions via muscle–kidney crosstalk. Further, this crosstalk involves movement of miR potentially through muscle originated exosomes and serum distribution without movement of AAV. These results could provide new approaches for developing therapeutic strategies for diabetic nephropathy with muscle wasting. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) causes loss of lean body mass by multiple mechanisms. This study examines whether autophagy-mediated proteolysis contributes to CKD-induced muscle wasting. We tested autophagy in the muscle of CKD mice with plantaris muscle overloading to mimic resistance exercise or with acupuncture plus low-frequency electrical stimulation (Acu/LFES) treatment. In CKD muscle, Bnip3, Beclin-1, and LC3II mRNAs and proteins were increased compared with those in control muscle, indicating autophagosome-lysosome formation induction. Acu/LFES suppressed the CKD-induced upregulation of autophagy. However, overloading increased autophagy-related proteins in normal and CKD muscle. Serum from uremic mice induces autophagy formation but did not increase the myosin degradation or actin break down in cultured muscle satellite cells. We examined mitochondrial biogenesis, copy number, and ATP production in cultured myotubes, and found all three aspects to be decreased by uremic serum. Inhibition of autophagy partially reversed this decline in cultured myotubes. In CKD mice, the mitochondrial copy number, biogenesis marker peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial transcription factor A (TFAM), and mitochondrial fusion marker Mitofusin-2 (Mfn2) are decreased. Both muscle overloading and Acu/LFES increased mitochondrial copy number, and reversed the CKD-induced decreases in PGC-1α, TFAM, and Mfn2. We conclude that the autophagy is activated in the muscle of CKD mice. However, myofibrillar protein is not directly broken down through autophagy. Instead, CKD-induced upregulation of autophagy leads to dysfunction of mitochondria and decrease of ATP production. [ABSTRACT FROM AUTHOR]

Vasopressin triggers the phosphorylation and apical plasma membrane accumulation of aquaporin 2 (AQP2), and it plays an essential role in urine concentration. Vasopressin, acting through protein kinase A, phosphorylates AQP2. However, the phosphorylation state of AQP2 could also be affected by the action of protein phosphatases (PPs). Rat inner medullas (IM) were incubated with calyculin (PP1 and PP2A inhibitor, 50 nM) or tacrolimus (PP2B inhibitor, 100 nM). Calyculin did not affect total AQP2 protein abundance (by Western blot) but did significantly increase the abundances of pS256-AQP2 and pS264-AQP2. It did not change pS261-AQP2 or pS269-AQP2. Calyculin significantly enhanced the membrane accumulation (by biotinylation) of total AQP2, pS256-AQP2, and pS264-AQP2. Likewise, immunohistochemistry showed an increase in the apical plasma membrane association of pS256-AQP2 and pS264-AQP2 in calyculin-treated rat IM. Tacrolimus also did not change total AQP2 abundance but significantly increased the abundances of pS261-AQP2 and pS264-AQP2. In contrast to calyculin, tacrolimus did not change the amount of total AQP2 in the plasma membrane (by biotinylation and immunohistochemistry). Tacrolimus did increase the expression of pS264-AQP2 in the apical plasma membrane (by immunohistochemistry). In conclusion, PP1/ PP2A regulates the phosphorylation and apical plasma membrane accumulation of AQP2 differently than PP2B. Serine-264 of AQP2 is a phosphorylation site that is regulated by both PP1/PP2A and PP2B. This dual regulatory pathway may suggest a previously unappreciated role for multiple phosphatases in the regulation of urine concentration. [ABSTRACT FROM AUTHOR]

Fundamental kidney physiology research can provide important insight into how the kidney works and suggest novel therapeutic opportunities to treat human diseases. This is especially true for nephrogenic diabetes insipidus (NDI). Over the past decade, studies elucidating the molecular physiology and signaling pathways regulating water transport have suggested novel therapeutic possibilities. In patients with congenital NDI due to mutations in the type 2 vasopressin receptor (V2R) or acquired NDI due to lithium (or other medications), there are no functional abnormalities in the aquaporin-2 (AQP2) water channel, or in another key inner medullary transport protein, the UT-A1 urea transporter. If it is possible to phosphorylate and/or increase the apical membrane accumulation of these proteins, independent of vasopressin or cAMP, one may be able to treat NDI. Sildenifil (through cGMP), erlotinib, and simvastatin each stimulate AQP2 insertion into the apical plasma membrane. Some recent human data suggest that sildenafil and simvastatin may improve urine concentrating ability. ONO-AE1-329 (ONO) stimulates the EP4 prostanoid receptor (EP4), which stimulates kinases that in turn phosphorylate AQP2 and UT-A1. Clopidogrel is a P2Y12-R antagonist that potentiates the effect of vasopressin and increases AQP2 abundance. Metformin stimulates AMPK to phosphorylate and activate AQP2 and UT-A1, and it increases urine concentrating ability in two rodent models of NDI. Since metformin, sildenafil, and simvastatin are commercially available and have excellent safety records, the potential for rapidly advancing them into clinical trials is high. [ABSTRACT FROM AUTHOR]

In humans, urea is excreted in sweat, largely through the eccrine sweat gland. The urea concentration in human sweat is elevated when compared to blood urea nitrogen. The sweat urea nitrogen ( UN) of patients with end-stage kidney disease ( ESRD) is increased when compared with healthy humans. The ability to produce sweat is maintained in the overwhelming majority of ESRD patients. A comprehensive literature review found no reports of sweat UN neither in healthy rodents nor in rodent models of chronic kidney disease ( CKD). Therefore, this study measured sweat UN concentrations in healthy and uremic rats. Uninephrectomy followed by renal artery ligation was used to remove 5/6 of renal function. Rats were then fed a high-protein diet to induce uremia. Pilocarpine was used to induce sweating. Sweat droplets were collected under oil. Sweat UN was measured with a urease assay. Serum UN was measured using a fluorescent ortho-pthalaldehyde reaction. Immunohistochemistry ( IHC) was accomplished with a horseradish peroxidase and diaminobenzidine technique. Sweat UN in uremic rats was elevated greater than two times compared to healthy pair-fed controls (220 ± 17 and 91 ± 15 mmol/L, respectively). Post hoc analysis showed a significant difference between male and female uremic sweat UN (279 ± 38 and 177 ± 11 mmol/L, respectively.) IHC shows, for the first time, the presence of the urea transporters UT-B and UT-A2 in both healthy and uremic rat cutaneous structures. Future studies will use this model to elucidate how rat sweat UN and other solute excretion is altered by commonly prescribed diuretics. [ABSTRACT FROM AUTHOR]

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 (UTA1). 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. [ABSTRACT FROM AUTHOR]

Muscle wasting occurs in a variety of clinical situations, including denervation. There is no effective pharmacological treatment for muscle wasting. In this study, we used a tibial nerve denervation model to test acupuncture plus low-frequency electric stimulation (Acu-LFES) as a therapeutic strategy for muscle atrophy. Acupuncture needles were connected to an SDZ-II electronic acupuncture device delivering pulses at 20 Hz and 1 mA; the treatment was 15 min daily for 2 wk. Acu-LFES prevented soleus and plantaris muscle weight loss and increased muscle cross-sectional area in denervated mice. The abundances of Pax7, MyoD, myogenin, and embryonic myosin heavy chain were significantly increased by Acu-LFES in both normal and denervated muscle. The number of central nuclei was increased in Acu-LFES-treated muscle fibers. Phosphorylation of Akt was downregulated by denervation leading to a decline in muscle mass; however, Acu-LFES prevented the denervation-induced decline largely by upregulation of the IGF-1 signaling pathway. Acu-LFES reduced the abundance of muscle catabolic proteins forkhead O transcription factor and myostatin, contributing to the attenuated muscle atrophy. Acu-LFES stimulated the expression of macrophage markers (F4/80, IL-1b, and arginase-1) and inflammatory cytokines (IL-6, IFNγ, and TNFα) in normal and denervated muscle. Acu-LFES also stimulated production of the muscle-specific microRNAs miR-1 and miR-206. We conclude that Acu-LFES is effective in counteracting denervation-induced skeletal muscle atrophy and increasing muscle regeneration. Upregulation of IGF-1, downregulation of myostatin, and alteration of microRNAs contribute to the attenuation of muscle atrophy in denervated mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vasopressin increases urine concentration through activation of aquaporin-2 (AQP2) in the collecting duct. Nonsteroidal anti-inflammatory drugs (NSAIDs) block prostaglandin E2 synthesis, and may suppress AQP2 producing a urine concentrating defect. There are four serines in AQP2 that are phosphorylated by vasopressin. To determine if chronic use of NSAIDs changes AQP2’s phosphorylation at any of these residues, the effects of a non-selective NSAID, ibuprofen, and a COX-2-selective NSAID, meloxicam, were investigated. Daily ibuprofen or meloxicam increased the urine output and decreased the urine osmolality significantly by days 7 through 14. Concomitantly, meloxicam significantly reduced total AQP2 protein abundance in inner medulla (IM) tip to 64% of control and base to 63%, respectively. Ibuprofen significantly decreased total AQP2 in IM tip to 70% of control, with no change in base. Meloxicam significantly increased the ratios of p256-AQP2 and p261-AQP2 to total AQP2 in IM tip (to 44% and 40%, respectively). Ibuprofen increased the ratio of p256-AQP2 to total AQP2 in IM tip but did not affect p261-AQP2/total AQP2 in tip or base. Both ibuprofen and meloxicam increased p264-AQP2 and p269-AQP2 ratios in both tip and base. Ibuprofen increased UT-A1 levels in IM tip, but not in base. We conclude that NSAIDs reduce AQP2 abundance, contributing to decreased urine concentrating ability. They also increase some phosphorylated forms of AQP2. These changes may partially compensate for the decrease in AQP2 abundance, thereby lessening the decrease in urine osmolality. [ABSTRACT FROM AUTHOR]

Mortality and morbidity are increased in patients with muscle atrophy resulting from catabolic diseases such as diabetes. At present there is no pharmacological treatment that successfully reverses muscle wasting from catabolic conditions. We hypothesized that acupuncture plus low frequency electric stimulation (Acu-LFES) would mimic the impact of exercise and prevent diabetes-induced muscle loss. Streptozotocin (STZ) was used to induce diabetes in mice. The mice were then treated with Acu-LFES for 15 minutes daily for 14 days. Acupuncture points were selected according to the WHO Standard Acupuncture Nomenclature guide. The needles were connected to an SDZ-II electronic acupuncture device delivering pulses at 20Hz and 1mA. Acu-LFES prevented soleus and EDL muscle weight loss and increased hind-limb muscle grip function in diabetic mice. Muscle regeneration capacity was significantly increased by Acu-LFES. The expression of Pax7, MyoD, myogenin and embryo myosin heavy chain (eMyHC) was significantly decreased in diabetic muscle vs. control muscle. The suppressed levels in diabetic muscle were reversed by Acu-LFES. The IGF-1 signaling pathway was also upregulated by Acu-LFES. Phosphorylation of Akt, mTOR and p70S6K were downregulated by diabetes leading to a decline in muscle mass, however, Acu-LFES countered the diabetes-induced decline. In addition, microRNA-1 and -206 were increased by Acu-LFES after 24 days of treatment. We conclude that Acu-LFES is effective in counteracting diabetes-induced skeletal muscle atrophy by increasing IGF-1 and its stimulation of muscle regeneration. [ABSTRACT FROM AUTHOR]

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity. [ABSTRACT FROM AUTHOR]

High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser591 on Src homology 2 domaincontaining phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591. [ABSTRACT FROM AUTHOR]

The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKCɑ influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKC-/- mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKCɑ, the open probability (Po) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 ± 0.04 vs. 0.17 ± 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of ɑ-, β-, and y-subunits of ENaC in principal cells in the cortical collecting ducts of PKCɑ-/- mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKCɑ-/- mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and Po by PKCɑ in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet. [ABSTRACT FROM AUTHOR]

Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/ CTGF/Nov). CCN1 mRNA was upregulated 113-fold in muscle of aged vs. young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1-treated MPCs did express a senescence marker Ink4A (SA-gal). Interestingly, we found CCN1 increased p53, p16, and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by sevenfold and protein increased by threefold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time- and dose-dependent manner. We conclude that in aging muscle, the circulating factor wnt-3a acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins. [ABSTRACT FROM AUTHOR]

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity. [ABSTRACT FROM AUTHOR]

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 hypertonicitystimulated urea permeability in rat inner medullary collecting ducts (IMCDs). Because PKCα knockout (PKCα-/-) mice have a urineconcentrating 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 hypertonicitystimulated urea transport but is not necessary for vasopressin stimulation of urea permeability in mouse IMCDs. [ABSTRACT FROM AUTHOR]

Nephrotic syndrome is associated with up-regulation of the heparin-binding epidermal growth factor (HB-EGF). Erlotinib blocks the activation of the epidermal growth factor receptor (EGFR) in response to HB-EGF. This study investigates the effect of Erlotinib on the progression of proteinuria, renal dysfunction, and salt retention in doxorubicin treated nephrotic rats. Male rats were divided into 3 pair-fed groups (n = 13/group) as follows: Control rats (Ctrl); rats receiving intravenous doxorubicin (Dox); and rats receiving intravenous doxorubicin followed by daily oral Erlotinib (Dox + Erl). Upon establishment of high grade proteinuria, urine sodium and creatinine clearance were measured. Kidney tissue was dissected and analyzed for γ-epithelial sodium channel (γENaC), sodium-potassium -chloride co-transporter 2 (NKCC2), sodium chloride co-transporter (NCC), aquaporin 2 (AQP2), and EGFR abundances using western blot. Creatinine clearance was preserved in the Dox + Erl rats as compared to the Dox group (in ml/min: Ctrl: 5.2±.5, Dox: 1.9±0.3, Dox + Erl: 3.6±0.5). Despite a minimal effect on the degree of proteinuria, Erlotinib prevented salt retention (Urinary Na in mEq/d: Ctrl: 2.2±0.2, Dox: 1.8±0.3, Dox + Erl: 2.2±0.2). The cleaved/uncleaved γENaC ratio was increased by 41±16% in the Dox group but unchanged in the Dox + Erl group when compared to Ctrl. The phosphorylated EGFR/total EGFR ratio was reduced by 74±7% in the Dox group and by 77±4% in the Dox + Erl group. In conclusion, Erlotinib preserved renal function and prevented salt retention in nephrotic rats. The observed effects do not appear to be mediated by direct blockade of EGFR. [ABSTRACT FROM AUTHOR]

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/kgH2O. In contrast, urine osmolality was low (800 mosmol/ kgH2O) 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. [ABSTRACT FROM AUTHOR]

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 abndance. 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. [ABSTRACT FROM AUTHOR]

Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats (n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

UTA1, 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. [ABSTRACT FROM AUTHOR]

The UT-A1 urea transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature P6acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation by tunicamycin or elimination of the glycosylation sites by mutation significantly reduced UT-A1 activity in oocytes. Taken together, our study reveals a new role of N-linked glycosylation in regulating UT-A1 activity by promoting UT-A1 trafficking into membrane lipid raft subdomains. [ABSTRACT FROM AUTHOR]

N-methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ∼80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a-/- mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a-/- mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways. [ABSTRACT FROM AUTHOR]

Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its NH2-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central pan, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these pbosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1. [ABSTRACT FROM AUTHOR]

The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves Na+ and HCO3- into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HC1-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hip-pocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-D-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl- D-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excito-toxicity. [ABSTRACT FROM AUTHOR]