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Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter-driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions.

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS).

The American Society of Nephrology, the European Renal Association–European Dialysis and Transplant Association and the International Society of Nephrology Joint Working Group on Ethical Issues in Nephrology have identified ten broad areas of ethical concern as priority challenges that require collaborative action. Here, we describe these challenges — equity in access to kidney failure care, avoiding futile dialysis, reducing dialysis costs, shared decision-making in kidney failure care, living donor risk evaluation and decision-making, priority setting in kidney disease prevention and care, the ethical implications of genetic kidney diseases, responsible advocacy for kidney health and management of conflicts of interest — with the aim of highlighting the need for ethical analysis of specific issues, as well as for the development of tools and training to support clinicians who treat patients with kidney disease in practising ethically and contributing to ethical policy-making.Here, the ASN-ERA-EDTA-ISN Joint Working Group on Ethical Issues in Nephrology highlights ten areas of ethical concern as priority challenges that require collaborative action and discusses the need for development of ethical training and guidance tools to manage these issues.

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

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.

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  Urea plays a key role in the urine-concentrating mechanism. Physiologic and molecular data demonstrate that urea transport in kidney and red blood cells occurs by specific urea transporter proteins. Two gene families for facilitated urea transporters, UT-A and UT-B, and several urea transporter cDNA isoforms have been cloned from human, rat, mouse, and several non-mammalian species. Polyclonal antibodies have been generated to many of the urea transporter proteins, and several novel findings have resulted from their use in integrative animal studies. For example, (a) vasopressin increases the phosphorylation of UT-A1 in rat inner medullary collecting duct; (b) UT-A1 protein abundance is increased in the rat inner medulla during conditions in which urine-concentrating ability is reduced; and (c) urea transporters are expressed in non-renal tissues, and UT-A protein abundance is up-regulated in uremia in both liver and heart. In addition to the facilitated urea transporters, functional evidence exists for active urea transport in the kidney collecting duct. This review summarizes the physiologic evidence for the existence of facilitated and active urea transporters, the molecular biology of the facilitated urea transporter gene families and cDNAs, and integrative studies into urea transporter protein regulation, both in the kidney and in other organs.

The Clinical and Translational Science Awards (CTSA) program of the National Center for Advancing Translational Sciences (NCATS) seeks to improve population health by accelerating the translation of scientific discoveries in the laboratory and clinic into practices for the community. CTSAs achieve this goal, in part, through their pilot project programs that fund promising early career investigators and innovative early-stage research projects across the translational research spectrum. However, there have been few reports on individual pilot projects and their impacts on the investigators who receive them and no studies on the long-term impact and outcomes of pilot projects. The Georgia CTSA funded 183 pilot projects from 2007 to 2015. We used a structured evaluation framework, the payback framework, to document the outcomes of 16 purposefully-selected pilot projects supported by the Georgia CTSA. We used a case study approach including bibliometric analyses of publications associated with the selected projects, document review, and investigator interviews. These pilot projects had positive impact based on outcomes in five \"payback categories\": (1) knowledge; (2) research targeting, capacity building, and absorption; (3) policy and product development; (4) health benefits; and (5) broader economic benefits. Results could inform our understanding of the diversity and breadth of outcomes resulting from Georgia CTSA-supported research and provide a framework for evaluating long-term pilot project outcomes across CTSAs.

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.