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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]

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]

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]

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]

Renal urea handling is central to the urine concentrating mechanism, and as such the ability to image urea transport in the kidney is an important potential imaging biomarker for renal functional assessment. Glucagon levels associated with changes in dietary protein intake have been shown to influence renal urea handling; however, the exact mechanism has still to be fully understood. Here we investigate renal function and osmolite distribution using [13C,15N] urea dynamics and 23Na distribution before and 60 min after glucagon infusion in six female rats. Glucagon infusion increased the renal [13C,15N] urea mean transit time by 14%, while no change was seen in the sodium distribution, glomerular filtration rate or oxygen consumption. This change is related to the well‐known effect of increased urea excretion associated with glucagon infusion, independent of renal functional effects. This study demonstrates for the first time that hyperpolarized 13C‐urea enables monitoring of renal urinary excretion effects in vivo. Hyperpolarized 13C urea MRI during 60 min infusion of glucagon resulted in an increased 13C‐urea renal mean transit time for urea, without changing GFR, renal blood flow or renal oxygen tension. This change is related to the well‐known effect of increased urea excreation associated with the glucagon infusion, independent of renal functional effects. This study demonstrate that hyperpolarized 13C‐urea enables monitoring of renal urinary excretion effects in vivo. [ABSTRACT FROM AUTHOR]

Urea transport by the inner medullary collecting duct (IMCD) is critically important to the urine concentrating mechanism. Transepithelial urea transport requires urea transporters in both apical and basolateral membranes of the IMCD. It is generally agreed that urea moves across the apical membrane by UT-A1, but the identity of the basolateral urea transporter is controversial. One published account localizes UT-A3 at the basolateral membrane in mouse IMCD providing a mechanism for transepithelial urea transport. This study used immunohistochemistry (IHC) with an antibody to the mouse UT-A3 C-terminus. Another report shows UT-A3 localized to the apical membrane in rat IMCD by immunofluorescence (IF) with an antibody to the rat UT-A3 C-terminus. If UT-A3 is not in the basolateral membrane, then the basolateral urea transporter remains unidentified. The goal of this study: directly compare UT-A3 localization in rat and mouse. UT-A3 was identified by western blot using the mouse UT-A3 antibody, and verified using an N-terminal UT-A1 antibody that sees both UT-A1 and UT-A3. IHC analysis revealed that UT-A3, as well as UT-A1, is most abundant in IM tip. Co-immunoprecipitation did not reveal a UT-A1/ UT-A3 interaction. Both IHC and IF analysis of perfused rat and mouse kidneys showed UT-A3 was predominately expressed at the apical plasma membrane. We conclude that UT-A3 primarily localizes to the apical membrane in both rat and mouse IMCD. The mechanistic advantage of UT-A3 in the apical membrane, in addition to UT-A1, remains to be solved. The identity of the basolateral urea transporter remains unknown. [ABSTRACT FROM AUTHOR]

Diabetes is associated with increased activity of the renal renin angiotensin system (RAS). Previously, we demonstrated that streptozotocin (STZ)-induced diabetic rats have significantly increased abundances of renal distal sodium transporters and channels, e.g., the thiazide-sensitive Na-Cl cotransporter (NCC) and the epithelial sodium channel (ENaC). To determine the role of elevated RAS activity in these changes, male Sprague-Dawley rats (100-150 g) were made diabetic by intravenous injection of STZ (65 mg/kg·bw) or treated with vehicle (n = 12/group). Rats were fed standard 1% NaCl rodent chow and watered ad libitum. After 14 days, rats were further divided (n = 6/group) to receive candesartan in the drinking water (2 mg/kg/day) or plain water for 7 additional days. Immunoblotting revealed, in cortex, diabetes caused a modest increase in NCC, β- and γ-ENaC (85-kDa band) abundances (band density ∼ 150% of non-diabetic controls). Candesartan not only reversed these increases, but significantly reduced abundance of NCC and ENaC subunits to ∼50% of non-candesartan-treated rats in both diabetic and non-diabetic rats. In contrast, in the medulla, diabetes caused a much greater increase in ENaC subunits and α-1-Na-K-ATPase (band densities 200-600% of non-diabetic controls) and these increases were irreversible by candesartan. Moreover, candesartan increased β-and γ-ENaC in the non-diabetic rats, in the medulla tip. Thus the increased abundances of sodium transporters in the cortex, but not the medulla, with diabetes, are candesartan-sensitive, and therefore, likely due to enhanced ATl-mediated signaling. [ABSTRACT FROM AUTHOR]

Trafficking of the urea transporter UT-A1 to the plasma membrane is key in vasopressin-regulated urea transport, but the mechanisms remain unclear. Our experiments show UT-A1 involvement with several SNARE-mediated accessory proteins involved in orientation of proteins in the plasma membrane. One group of proteins involved in membrane orientation is the syntaxin family. We recently reported that snapin is important in the interaction of UT-A1 with syntaxin-4 and SNAP23 (t-SNARE). UT-A1-MDCK cells with adenoviral-mediated over-expression of both snapin and syntaxin-4, show that the association of UT-A1 with syntaxin-4 is enhanced snapin. Over-expression of syntaxin-4 alone showed minimal UT-A1/syntaxin-4 binding. Confocal microscopy of UT-A1 and snapin show colocalization in both cytoplasm and plasma membrane. Co-injection of UT-A1 with snapin cRNA (2 ug) in Xenopus oocytes increased urea influx. Co-injection of UT-A1 with t-SNARE without snapin, decreased urea influx. These results suggest that snapin promotes the initial docking of UT-A1 at the plasma membrane to form a SNARE complex. When we treated UT-A1-MDCK cells with vasopressin or forskolin, UT-A1 and snapin were redistributed to the plasma membrane. Collectively, our data support a role for a snapin syntaxin-4/SNAP23 mediated UT-A1 docking complex in vasopressin-regulated trafficking of UT-A1 in UT-A1-MDCK cells. In addition, we verified that the components of the snapin/t-SNARE/UT-A1 complex are present in rat inner medulla, suggesting that this mechanism may be physiologically important. [ABSTRACT FROM AUTHOR]

In order to study the vasopressin regulation of urea transport in the inner medullary collecting duct (IMCD), we have developed the MDCK-UTA1 cell culture model which permits measurements of transepithelial tracer-urea fluxes. This requires one to confirm that the regulation of urea transport in the MDCK-UTA1 cells occurs, as in the IMCD, in the apical membrane. We permeabilized either the apical or basolateral side of the epithelial monolayer using amphotericin. Basolateral amphotericin had no effect on trans-epithelial tracer urea flux in non-stimulated cells (basal flux: 2 nmol cm-2 min-1). In contrast, apical amphotericin greatly increased urea flux to about 20 nmol cm-2 min-1. This indicates that the apical membrane is the side with the limiting urea permeability. In, the presence of forskolin, flux is stimulated approximately 10-fold by activating the urea transporter UT-A1. Again, basolateral amphotericin had no effect on the urea flux whereas apical amphotericin occasionally increased it a bit further. The apical urea permeabilities induced by amphotericin and forskolin were similar in magnitude. To confirm that the basolateral membrane indeed had the high urea permeability, we performed basolateral urea influx experiments. We measured influx rates of 10-20 nmol cm-2 min-1, with near-complete equilibration of basolateral and intracellular urea concentrations in 1-2 min. There was no significant stimulation of basolateral fluxes by forskolin. As MDCK cells do not appear to express native urea transporter, it is not clear which transport pathways might underlie the high basolateral urea permeability. [ABSTRACT FROM AUTHOR]

The 14-3-3 proteins are a group of highly conserved 30-kD regulatory proteins that mainly bind to phosphorylated residues in target proteins. 14-3-3 proteins interact with numerous cellular proteins and influence a large number of cellular processes. UT-A1 urea transporter contains a potential 14-3-3 recognition motif in the large cytoplasmic loop, suggesting a potential role of 14-3-3 in the regulation of UT-A1. To test this model, we examined the expression pattern of seven 14-3-3 protein isoforms in kidney cortex, outer medulla (OM) and inner medulla (IM). Zeta, beta and tau are highly and widely expressed in kidney cortex, OM and IM, as well as in brain, heart and liver. Gamma, eta, and epsilon are found in cortex, OM, and IM, but mainly expressed in IM. There was no detectable sigma in kidney tissue. We then prepared seven 14-3-3 isoform-specific GST fusion proteins for 14-3-3/UT-Al interaction studies. GST pull-down assays with UT-A1-MDCK cell lysate demonstrated that UT-A1 is in a protein complex with selected 14-3-3 isoforms: gamma eta and zeta. R18 peptide, a high-affinity competitive inhibitor of 14-3-3 interactions, prevented UT-A1 from binding to 14-3-3. To examine whether phosphorylation regulates 14-3-3 and UT-A1 binding, UT-A1-MDCK cells were treated with cAMP/adenylyl cyclase stimulator forskolin (10 µM) for 5, 15, and 30 min. Cell lysates were prepared and used for GST 14-3-3 gamma, eta and zeta pull-down assay. Phosphorylation significantly induced UT-A1 binding to 14-3-3. Our study identified 14-3-3 as a novel and potentially important regulator of urea transport activity. [ABSTRACT FROM AUTHOR]

Agonist (ATP/UTP) activation of P2Y2 receptor (P2Y2-R) in the medullary collecting duct (mCD) decreases arginine vasopressin (AVP)-induced water flow, and releases PGE2, a potent inhibitor of AVP action on mCD. Hence, we analyzed age and sex matched P2Y2-R null (KO) and wild (WT) mice (N = 6/genotype; courtesy of Dr. Beverly Koller) after subjecting them to metabolic balance by feeding a gel diet containing fixed (70%) water content for 10 days. The water balance data obtained on the last three days were averaged. As compared to the WT mice the KO mice showed significantly less water consumption (-11%; P < 0.04) and urine output (-32%; P < 0.02), and higher urinary osmolality (+ 38%; P < 0.002). There were no significant differences in total osmolar excretion or serum osmolality or AVP levels. Western analysis of renal medullas showed significant increases in mean protein abundances of AQP2 water channel (1.8-fold; P < 0.006), UT-A (∼2-fold; P < 0.02), and a tendency for UT-B (+14%; P = 0.09) urea transporter isoforms. These data indicate that deletion of P2Y2-R apparently sensitizes mCD to the action of circulating AVP, resulting in direct increase in protein abundances of AQP2 and indirect increase in the UT-A, the key players in urinary concentration. Thus these findings underscore our hypothesis that in vivo P2Y2-R plays a significant role in modulating the mCD function. [ABSTRACT FROM AUTHOR]