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Acute kidney injury (AKI) has been reported to be associated with excess risks of death, kidney disease progression and cardiovascular events although previous studies have important limitations. To further examine this, we prospectively studied adults from four clinical centers surviving three months and more after hospitalization with or without AKI who were matched on center, pre-admission CKD status, and an integrated priority score based on age, prior cardiovascular disease or diabetes mellitus, preadmission estimated glomerular filtration rate (eGFR) and treatment in the intensive care unit during the index hospitalization between December 2009-February 2015, with follow-up through November 2018. All participants had assessments of kidney function before (eGFR) and at three months and annually (eGFR and proteinuria) after the index hospitalization. Associations of AKI with outcomes were examined after accounting for pre-admission and three-month post-discharge factors. Among 769 AKI (73% Stage 1, 14% Stage 2, 13% Stage 3) and 769 matched non-AKI adults, AKI was associated with higher adjusted rates of incident CKD (adjusted hazard ratio 3.98, 95% confidence interval 2.51-6.31), CKD progression (2.37,1.28-4.39), heart failure events (1.68, 1.22-2.31) and all-cause death (1.78, 1.24-2.56). AKI was not associated with major atherosclerotic cardiovascular events in multivariable analysis (0.95, 0.70-1.28). After accounting for degree of kidney function recovery and proteinuria at three months after discharge, the associations of AKI with heart failure (1.13, 0.80-1.61) and death (1.29, 0.84-1.98) were attenuated and no longer significant. Thus, assessing kidney function recovery and proteinuria status three months after AKI provides important prognostic information for long-term clinical outcomes., Graphical abstract

Hypertension is a common complication of chronic kidney disease and persists among most patients with end-stage renal disease despite the provision of conventional thrice weekly hemodialysis (HD). We analyzed the effects of frequent HD on blood pressure in the randomized controlled Frequent Hemodialysis Network trials. The daily trial randomized 245 patients to 12 months of 6× (“frequent”) vs. 3× (“conventional”) weekly in-center hemodialysis; the nocturnal trial randomized 87 patients to 12 months of 6× weekly nocturnal HD vs. 3× weekly predominantly home-based hemodialysis. In the daily trial, compared with 3× weekly HD, 2 months of frequent HD lowered predialysis systolic blood pressure by −7.7 mmHg [95% confidence interval (CI): −11.9 to −3.5] and diastolic blood pressure by −3.9 mmHg [95% CI: −6.5 to −1.3]. In the nocturnal trial, compared with 3× weekly HD, 2 months of frequent HD lowered systolic blood pressure by −7.3 mmHg [95% CI: −14.2 to −0.3] and diastolic blood pressure by −4.2 mmHg [95% CI: −8.3 to −0.1]. In both trials, blood pressure treatment effects were sustained until month 12. Frequent HD resulted in significantly fewer antihypertensive medications (daily: −0.36 medications [95% CI: −0.65 to −0.08]; nocturnal: −0.44 mediations [95% CI: −0.89 to −0.03]). In the daily trial, the relative risk per dialysis session for intradialytic hypotension was lower with 6×/week HD but given the higher number of sessions per week, there was a higher relative risk for intradialytic hypotensive requiring saline administration. In summary, frequent HD reduces blood pressure and the number of prescribed antihypertensive medications.

Summary AKI is an important clinical problem that has become increasingly more common. Mortality rates associated with AKI remain high despite advances in supportive care. Patients surviving AKI have increased long-term mortality and appear to be at increased risk of developing CKD and progressing to ESRD. No proven effective pharmacologic therapies are currently available for the prevention or treatment of AKI. Advances in addressing this unmet need will require the development of novel therapeutic agents based on precise understanding of key pathophysiological events and the implementation of well designed clinical trials. To address this need, the National Institute of Diabetes and Digestive and Kidney Diseases sponsored the “Clinical Trials in Acute Kidney Injury: Current Opportunities and Barriers” workshop in December 2010. The event brought together representatives from academia, industry, the National Institutes of Health, and the US Food and Drug Administration. We report the discussions of workgroups that developed outlines of clinical trials for the prevention of AKI in two patient populations: patients undergoing elective surgery who are at risk for or who develop AKI, and patients who are at risk for contrast-induced AKI. In both of these populations, primary prevention or secondary therapy can be delivered at an optimal time relative to kidney injury. The workgroups detailed primary and secondary endpoints for studies in these groups, and explored the use of adaptive clinical trial designs for trials of novel preventive strategies to improve outcomes of patients with AKI.

Summary AKI remains an important clinical problem, with a high mortality rate, increasing incidence, and no Food and Drug Administration-approved therapeutics. Advances in addressing this clinical need require approaches for rapid diagnosis and stratification of injury, development of therapeutic agents based on precise understanding of key pathophysiological events, and implementation of well designed clinical trials. In the near future, AKI biomarkers may facilitate trial design. To address these issues, the National Institute of Diabetes and Digestive and Kidney Diseases sponsored a meeting, “Clinical Trials in Acute Kidney Injury: Current Opportunities and Barriers,” in December of 2010 that brought together academic investigators, industry partners, and representatives from the National Institutes of Health and the Food and Drug Administration. Important issues in the design of clinical trials for interventions in AKI in patients with sepsis or AKI in the setting of critical illness after surgery or trauma were discussed. The sepsis working group discussed use of severity of illness scores and focus on patients with specific etiologies to enhance homogeneity of trial participants. The group also discussed endpoints congruent with those endpoints used in critical care studies. The second workgroup emphasized difficulties in obtaining consent before admission and collaboration among interdisciplinary healthcare groups. Despite the difficult trial design issues, these clinical situations represent a clinical opportunity because of the high event rates, severity of AKI, and poor outcomes. The groups considered trial design issues and discussed advantages and disadvantages of several short- and long-term primary endpoints in these patients.

After initial reports suggesting that patients initiating peritoneal dialysis (PD) had better survival than patients on conventional dialysis in the first year, many investigators have scrutinized the existing databases to determine the reasons for this apparent difference. It is clear that patients starting on PD are selected – they are younger and have less comorbidity compared to the general population on dialysis. Despite improvements in the morbidities of patients on PD over the past 15 years, the survival of patients on hemodialysis over 5 years is greater than for patients on hemodialysis. The latest analysis points to the considerable survival difference in patients who start dialysis with an arteriovenous fistula or graft compared to patients who start dialysis with a cuffed catheter. Thus, the apparent early survival advantage for patients starting PD can be attributed to selection and comorbidities. Data are emerging that the best survival occurs when patients are dialyzed more frequently than the conventional three times a week. The questions regarding dialysis modality now can shift from whether to encourage PD to how to encourage frequent hemodialysis.

The recently concluded Frequent Hemodialysis Network (FHN) trials have demonstrated some striking and unexpected results. Both the daily arm and the nocturnal arm of the trial clearly demonstrated that frequent (daily or nightly) dialysis reduced blood pressure, reduced the number of antihypertensive medications, and reduced serum phosphorous concentration. One of the major questions addressed by these studies was the extent to which left ventricular mass was reduced by frequent dialysis. While the daily FHN trial showed a clear effect of frequent dialysis to reduce left ventricular mass, the nocturnal FHN trial produced inconclusive results. These apparently contradictory results are probably influenced by inadequate power and the somewhat skewed patient selection in the nocturnal arm. Patients in the nocturnal FHN trial had a shorter time on dialysis prior to enrollment, and greater residual renal function than did patients in the daily FHN trial. From a general perspective, it appears that there is minimal difference in the effect on left ventricular mass between frequent daily dialysis and nocturnal dialysis. The FHN trial was not designed to determine the effects of frequent dialysis on mortality. The analyses of this question using retrospective data strongly suggest that frequent dialysis prolongs life. The nephrology community now has the task to develop new ways to deliver improved therapy to patients on dialysis. This task will be challenging as resources for health care are constrained. New approaches to the care of such patients will be needed to realize the important conceptual advances embedded in the results of the FHN trials.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

To assess whether endocrine dysfunction may cause derangement in energy homeostasis in patients undergoing hemodialysis (HD), we profiled hormones, during a 3-day period, from the adipose tissue and the gut and the nervous system around the circadian clock in 10 otherwise healthy HD patients and 8 normal controls. The protocol included a 40-h fast. We also measured energy-protein intake and output and assessed appetite and body composition. We found many hormonal abnormalities in HD patients: 1) leptin levels were elevated, due, in part, to increased production, and nocturnal surge in response to daytime feeding, exaggerated. 2) Peptide YY (PYY), an anorexigenic gut hormone, was markedly elevated and displayed an augmented response to feeding. 3) Acylated ghrelin, an orexigenic gut hormone, was lower and did not exhibit the premeal spike as observed in the controls. 4) neuropeptide Y (NPY), a potent orexigenic peptide, was markedly elevated and did not display any circadian variation. 5) Norepinephrine, marginally elevated, did not exhibit the normal nocturnal dip. By contrast, α-melanocyte-stimulating hormone and glucagon-like peptide-1 were not different between the two groups. Despite these hormonal abnormalities, HD patients maintained a good appetite and had normal body lean and fat mass, and there was no evidence of increased energy expenditure or protein catabolism. We explain the hormonal abnormalities as well as the absence of anorexia on suppression of parasympathetic activity (vagus nerve dysfunction), a phenomenon well documented in dialysis patients. Unexpectedly, we noted that the combination of high leptin, PYY, and NPY with suppressed ghrelin may increase arterial blood pressure, impair vasodilatation, and induce cardiac hypertrophy, and thus could predispose to adverse cardiovascular events that are the major causes of morbidity and mortality in the HD population. This is the first report attempting to link hormonal abnormalities associated with energy homeostasis to adverse cardiovascular outcome in the HD patients.

Background and objectives: Cognitive impairment is common among persons with ESRD, but the underlying mechanisms are unknown. This study evaluated the prevalence of cognitive impairment and association with modifiable ESRD- and dialysis-associated factors in a large group of hemodialysis patients. Design, setting, participants, & measurements: Cross-sectional analyses were conducted on baseline data collected from 383 subjects participating in the Frequent Hemodialysis Network trials. Global cognitive impairment was defined as a score

Recessive inheritance of mutations in ceroid neuronal lipofuscinosis type 3 ( CLN3) results in juvenile neuronal ceroid lipofuscinosis (JNCL), a childhood neurodegenerative disease with symptoms including loss of vision, seizures, and motor and mental decline. CLN3p is a transmembrane protein with undefined function. Using a Cln3 reporter mouse harboring a nuclear-localized bacterial β-galactosidase (β-Gal) gene driven by the native Cln3 promoter, we detected β-Gal most prominently in epithelial cells of skin, colon, lung, and kidney. In the kidney, β-Gal-positive nuclei were predominant in medullary collecting duct principal cells, with increased expression along the medullary osmotic gradient. Quantification of Cln3 transcript levels from kidneys of wild-type ( Cln3+/+) mice corroborated this expression gradient. Reporter mouse-derived renal epithelial cultures demonstrated a tonicity-dependent increase in β-Gal expression. RT-quantitative PCR determination of Cln3 transcript levels further supported osmoregulation at the Cln3 locus. In vivo, osmoresponsiveness of Cln3 was demonstrated by reduction of medullary Cln3 transcript abundance after furosemide administration. Primary cultures of epithelial cells of the inner medulla from Cln3lacZ/lacZ (CLN3p-null) mice showed no defect in osmolyte accumulation or taurine flux, arguing against a requirement for CLN3p in osmolyte import or synthesis. CLN3p-deficient mice with free access to water showed a mild urine-concentrating defect but, upon water deprivation, were able to concentrate their urine normally. Unexpectedly, we found that CLN3p-deficient mice were hyperkalemic and had a low fractional excretion of K+. Together, these findings suggest an osmoregulated role for CLN3p in renal control of water and K+ balance.

The haem oxygenase-1–biliverdin reductase system degrades haem and generates biliverdin and bilirubin, both of which possess antioxidant and anti-inflammatory properties. Biliverdin and bilirubin are protective in intestinal injury models, but little is known about their generation and fate in the intestine. In the present work, an in vitro intestinal epithelial cell model, Caco-2 cells, were exposed to haem from either the apical or the basolateral side, and bile pigment generation and transport were measured spectrophotometrically and with high-pressure liquid chromatography. The Caco-2 cells generated bilirubin and bilirubin glucuronides upon exposure to haem. Bilirubin appeared predominantly in the apical medium regardless of the side to which haem was applied. In contrast to an earlier report, significant bidirectional haem transport was not observed. We conclude that Caco-2 cells metabolize haem and export its metabolic product, bilirubin, principally to the lumen, where it may exert antioxidant and anti-inflammatory functions.

Classic urea modeling assumes that both urea generation rate (G) and residual renal urea clearance (Kru) are constant throughout the week, but this may not be true. Reductions in intradialysis G could be caused by lower plasma amino acid levels due to predialysis/intradialysis fasting and also to losses of amino acids into the dialysate. Intradialytic reductions in Kru could be due to lower intravascular volume, blood pressure, or osmotic load. To determine the possible effects of reduced G or Kru during dialysis on the calculation of the volume of distribution (V) and Kt/Vurea, we modeled 3 and 6/week nocturnal, 6/week short daily, and 3/week conventional hemodialysis. A modified 2-pool mathematical model of urea mass balance with a constant time-averaged G was used, but the model was altered to allow adjustment of the ratio of dialytic/interdialytic G (Gd/Gid) and dialytic/total Kru (Krud/Kru) to vary from 1.0 down to near zero. In patients dialyzed six times per week for 400 minutes per session, when Gd/Gid was decreased from 1.0 to 0.05, the predicted urea reduction ratio (URR) increased from 68.9% to 80.2%. To achieve an increased URR of this magnitude under conditions of constant G (Gd/Gid=1.0) required a decrease in modeled urea volume (V) of 36%. At Gd/Gid ratios of 0.8 or 0.6 (corresponding to 20% or 40% reductions in intradialysis G), the modeled URR was increased to 71.0% or 73.3%, causing a 7% or 15% factitious decrease in V. The error was intermediate for the 3/week nocturnal schedule, and was much less pronounced for the 6/week daily and 3/week conventional treatments. Reductions in intradialytic Kru had the opposite effect, lowering the predicted URR and increasing the apparent V, but here the errors were of much lesser amplitude. The results suggest that, particularly for nocturnal dialysis, the standard "constant G" urea kinetic model may need to be modified.

The federal government, foundations, associations, and the Institute of Medicine (IOM) are examining the relationships between physicians and “commercial interests,” particularly biotechnology, medical device, and pharmaceutical companies (Table 1). Discovering a lack of “systematic information” about conflicts of interest (COI) and commercial bias, IOM recently recommended that the federal government study the effect of such conflicts “on the quality of medical research, education, and practice.”1 View this table: Table 1. Recent activity concerning COI and commercial bias The American Society of Nephrology (ASN) shares IOM's concern over the lack of data related to COI and agrees that more research is needed. However, even in the absence of concrete information, it is the responsibility of a professional society to try to understand the extent to which COI interface with its missions. Perhaps no single aspect of COI for societies and those who represent them is as visible and subject to scrutiny as continuing medical education (CME). The longest phase of the educational continuum for physicians, CME is also a critical element of the country's health care system. “Given the accelerating pace of change in clinical information and technology, continuing education has never been more important,” observed the Josiah Macy, Jr., Foundation.2 By providing a preponderance of CME, professional societies such as ASN improve the quality of health care.3 In addition to CME, societies like ASN provide access to new knowledge by publishing peer-reviewed journals, support basic and clinical investigation through research grants, and advocate for the development of national health policies. To support these missions, many societies—including ASN—solicit funding from commercial interests. Although some have argued that professional societies should begin to eliminate such funding, others have suggested that such a restriction “would marginalize critical roles that industry, and many physicians working in industry, play in critical medical research alliances.”3,4 …

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.

Tyrosine kinase inhibitors (TKIs) directed against the Bcr-Abl kinase have revolutionized the treatment of chronic myelogenous leukemia (CML). Relatively little is known regarding the effects of these agents on the kidney. Clinically, there have been a handful of reports associating imatinib with acute renal failure. Preclinical reports indicate that imatinib inhibits signaling pathways which may play a role in renal injury. We report the case of a patient with imatinib-resistant CML who developed renal failure after being placed on dasatinib. When she later became resistant to dasatinib she was switched to nilotinib. Shortly thereafter, she became dialysis-independent. Second-generation Bcr-Abl TKIs may influence renal function based on differential inhibition of related tyrosine kinases.

In renal distal nephron and airway epithelial cells, adrenocortical steroids increase epithelial Na+ channel (ENaC) activity and also markedly increase the expression of the alpha-subunit. The present experiments were designed to reconstitute this steroid effect in ENaC-expressing cells by overexpressing the subunits whose expression is enhanced by corticosteroids. In renal collecting duct monolayers, corticosteroids increased ENaC activity 5- to 8-fold, endogenous alpha-ENaC mRNA and protein approximately 10-fold, and beta-ENaC protein and mRNA 1.2- to 2-fold. gamma-ENaC expression was unchanged. To determine whether this increase in expression was sufficient to increase ENaC activity, we used a regulated adenovirus system to increase expression of each subunit alone and in combination. Unexpectedly, increased expression of the alpha- and/or beta-subunit had no effect on ENaC activity in collecting duct cells or lung epithelial cells. In contrast, a small increase in gamma-ENaC expression increased ENaC activity about threefold. This increase in activity was additive to the effect of steroids. Thus, even though corticosteroids strongly increase alpha-ENaC expression and moderately increase beta-ENaC expression, these effects are not, by themselves, sufficient to increase ENaC activity. Knockdown experiments are consistent with the idea that the increased expression of alpha-ENaC is necessary for the full steroid effect on ENaC. Increased expression of gamma-ENaC and corticosteroid treatment enhances ENaC activity by parallel, noninteracting pathways. These results underscore the importance of other actions of steroid hormones for long-term enhancement of ENaC activity and raise new possibilities for regulation of ENaC activity by gamma-ENaC expression.

Heme induces Cl−secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl−secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents ( Isc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced Iscwas caused by Cl−secretion because it was inhibited in Cl−-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl−secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced Isc. SnPP-induced Iscwas inhibited by the antioxidant vitamins, α-tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl−secretion can be regulated by the redox environment of the cell.

Mutations that disrupt a PY motif in epithelial Na+channel (ENaC) subunits increase surface expression of Na+channels in the collecting duct, resulting in greater Na+reabsorption. Nedd4 and Nedd4–2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. We recently reported that human Nedd4–2 (hNedd4–2) is expressed as many isoforms because of alternative promoter usage and/or variable splicing. To understand the relevance of hNedd4–2 isoforms for collecting duct Na+transport, we studied the interaction with ENaC and the intracellular localization and function of the following three naturally occurring hNedd4–2 isoforms: full-length Nedd4–2 (Nedd4–2), Nedd4–2 lacking the NH2-terminal C2 domain (Nedd4–2ΔC2), and Nedd4–2 lacking the C2 domain and WW domains 2 and 3 (Nedd4–2ΔWW2,3). Nedd4–2 and Nedd4–2ΔC2 associate with ENaC and robustly reduce Na+transport in Xenopus oocytes, whereas the interaction with and functional effect of Nedd4–2ΔWW2,3 on ENaC is weak. Nedd4–2 is expressed in the mouse collecting duct, and overexpression of Nedd4–2 reduces endogenous ENaC activity in a collecting duct cell line. This reduction in ENaC activity can be reversed early with exposure to dexamethasone, an effect that is associated with an increase in sgk1 abundance. The C2 domain is required to target Nedd4–2 to the plasma membrane in response to elevation of intracellular Ca2+concentration ([Ca2+]i) in MDCK cells, although it does not appear to mediate the inhibitory effect of [Ca2+]ion Na+transport. Our data illustrate that naturally occurring hNedd4–2 isoforms differentially associate with ENaC to regulate its activity.

The epithelial Na+ channel (ENaC) has three subunits; the expression of each can be regulated. Liddle's syndrome is caused by an activating mutation in the C terminus of either the β or γ subunit. We used a doxycycline-regulated adenovirus system to express varying levels of human γENaC in renal collecting duct (M1 cell) monolayers. Increasing levels of wild type human γ ENaC (γhENaC) produced a 2.5-fold enhancement of Na+ transport. Expression of a truncated C terminus produced less protein than wild type or a γY627A missense mutation. However, either of these mutations produced a ∼4-fold increase in Na+ transport despite the different levels of protein expression. Unexpectedly, overexpression of a marginally detectable amount of γhENaC was sufficient to produce a full increase in Na+ transport; a further increase in protein expression produced no further increase in Na+ transport. Steroid treatment increased Na+ transport to a similar absolute magnitude in control monolayers and in monolayers expressing all types of γhENaC. Withdrawal of steroids after 24 h produced a decline in Na+ transport over 8 h in monolayers expressing wild type but not the Liddle's mutation. Using treatment with brefeldin A to estimate the disappearance rate constants, we found progressively slower disappearance rates in monolayers overexpressing γhENaC and the Liddle's mutant. Calculated insertion rates were slower for the Liddle's mutant than for wild type despite increasing rates of Na+ transport. These results raise questions regarding previously held assumptions about the behavior of ENaC.

Background We have previously shown that human myeloid cell types can acquire large amounts of iron (Fe)3+ from low-molecular-weight chelates by a process that is independent of adenosine triphosphate and dramatically increased by gallium (Ga) and other multivalent cationic metals. Methods To provide further insight into the mechanism responsible and its relevance to other cellular systems, we investigated Fe acquisition from nitrilotriacetic acid (NTA) by several myeloid and nonmyeloid cell lines in the presence and absence of Ga. Results Most nonmyeloid cells examined exhibited similar ability to acquire Fe from NTA. Ga increased the apparent maximum velocity (Vmax), with minimal changes in apparent Michaelis constant ( Km), of all cell lines. Both erythrocytes and erythrocyte ghosts acquired Fe from NTA, which increased with Ga exposure, analogous to nucleated cells. However, liposomes made from phospholipids did not exhibit Ga-inducible Fe association. Enzymes that modify surface proteins and carbohydrates did not alter HL-60 cell Fe acquisition. Modifying HL-60 membrane fatty acid content had only a minimal effect. Ga exposure did not change membrane potential or fluidity. However, electron microscopy suggested that Ga alters plasma membrane physical properties. Conclusion Multivalent cations appear to induce changes in cell membranes that may alter their interaction with Fe3+ and probably other multivalent cations.

Serum- and glucocorticoid-induced kinases (SGK) are members of the serine-threonine kinase family. SGK1, the isoform identified first, is rapidly induced by aldosterone. In this study, we determined that the two recently described isoforms, SGK2 and SGK3 are also expressed in renal cortical collecting duct (CCD) cells; however, their expression is not induced by aldosterone or glucocorticoids. SGK1 increases the activity of the epithelial sodium channel (ENaC) in oocytes but its cellular targets in native mineralocorticoid target cells and its mechanism of action are still unknown. We studied the role of SGK1 in corticosteroid-regulated Na transport in M-1 mouse CCD cell lines that stably over-express or down-regulate SGK1. Basal rates of transepithelial Na transport were significantly lower in CCD cells in which SGK1 expression or activity was down-regulated than in SGK1 overexpressing cells. Importantly, corticosteroid treatment failed to stimulate Na transport in cells with down-regulated SGK1 while it significantly increased Na transport in parent and SGK1 overexpressing M-1 cells. To determine if C-terminal PDZ interactions are important for SGK's effect on ENaC activity or trafficking, we examined the effects of mutant SGK1 in which the conserved PDZ binding domain has been eliminated. However, such mutations did not decrease its stimulatory effect on ENaC current in Xenopus oocytes. Fluorescence confocal microscopy revealed that the intracellular localization of full-length and PDZ binding mutated SGK1 was identical: they both localize to intracellular vesicular structures. On the other hand, N-terminally truncated (delta 60)-SGK1 did not increase ENaC activity. We conclude that SGK1 is a critical component in corticosteroid-regulated Na transport in mammalian CCD cells. Our data also indicate that the N-terminal of SGK1 is necessary for its stimulatory effect on Na transport while elimination of the C-terminal PDZ binding domain did not change its function.

Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11β-HSD2 abundance in DS rats. Consistent with the view that an elevated 11β-HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11β-HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.

Hypertension is a common complication of chronic kidney disease and persists among most patients with end-stage renal disease despite the provision of conventional thrice weekly hemodialysis (HD). We analyzed the effects of frequent HD on blood pressure in the randomized controlled Frequent Hemodialysis Network trials. The daily trial randomized 245 patients to 12 months of 6× ("frequent") vs. 3× ("conventional") weekly in-center hemodialysis; the nocturnal trial randomized 87 patients to 12 months of 6× weekly nocturnal HD vs. 3× weekly predominantly home-based hemodialysis. In the daily trial, compared with 3× weekly HD, 2 months of frequent HD lowered predialysis systolic blood pressure by -7.7 mmHg [95% confidence interval (CI): -11.9 to -3.5] and diastolic blood pressure by -3.9 mmHg [95% CI: -6.5 to -1.3]. In the nocturnal trial, compared with 3× weekly HD, 2 months of frequent HD lowered systolic blood pressure by -7.3 mmHg [95% CI: -14.2 to -0.3] and diastolic blood pressure by -4.2 mmHg [95% CI: -8.3 to -0.1]. In both trials, blood pressure treatment effects were sustained until month 12. Frequent HD resulted in significantly fewer antihypertensive medications (daily: -0.36 medications [95% CI: -0.65 to -0.08]; nocturnal: -0.44 mediations [95% CI: -0.89 to -0.03]). In the daily trial, the relative risk per dialysis session for intradialytic hypotension was lower with 6×/week HD but given the higher number of sessions per week, there was a higher relative risk for intradialytic hypotensive requiring saline administration. In summary, frequent HD reduces blood pressure and the number of prescribed antihypertensive medications.

We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq · 200 g body wt−1· day−1), rats were switched to a NaCl-deficient diet (0.02 meq · 200 g body wt−1· day−1). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease

Lung liquid absorption at birth is crucial for the successful onset of respiration. Na absorption by the renal collecting duct plays an important role in renal fluid and electrolyte homeostasis during the early postnatal period. The epithelial Na channel (ENaC) plays a central role in mediating these functions, and its subunit expression is developmentally regulated in a temporal and tissue specific pattern. Several lines of evidence suggest that the prenatal increase in circulating glucocorticoids may play an important role in increasing ENaC expression during maturation. We tested the role of the prenatal surge using corticotropin-releasing hormone (CRH) knockout (KO) mice. Relative ENaC expression in lungs of KO mice increased at the same rate as in wild-type (WT) mice, but absolute expression was only 20-30% of WT. In contrast, relative and absolute expression of all three subunits in kidneys was not different between KO and WT mice. Dexamethasone (Dex) increased alpha-ENaC mRNA in fetal lung and kidney explants within 24 h but had different effects on beta- or gamma-ENaC. Dex increased beta- and gamma-ENaC in lung, but only after48 h of exposure, and had no effect on kidney. The results suggest that the kidney metabolizes endogenous glucocorticoids, but the lung does not. Furthermore, the marked difference between lung and kidney responsiveness to glucocorticoids in beta- and gamma-ENaC expression suggests that factors other than steroids may be important in regulating functional ENaC expression during development.

The epithelial sodium channel (ENaC) is a heteromultimer composed of three subunits, each having two membrane-spanning domains with intracellular amino and carboxyl termini. Several hormones and proteins regulate channel activity, but the molecular nature of this regulation is unknown. We conducted experiments to determine a possible new site within the carboxyl terminus of the α-subunit involved in enhanced channel activity through endogenous kinases. When an α-subunit that was truncated to remove a PY motif was expressed in Xenopus oocytes with wild type human β- and γ-ENaC subunits, channel activity was greatly enhanced. The removal of the entire intracellular carboxyl terminus of the α-subunit eliminated this enhanced basal activity. Using several point mutations, we localized this site to two amino acid residues (Pro595-Gly596) near the second membrane-spanning domain. The nonspecific kinase inhibitor staurosporine inhibits basal channel activity of wild type ENaC but was ineffective in inhibiting channels mutated at this site. The major effect of these mutations was not on channel kinetics but was largely, if not entirely, on the number of active channels on the cell surface. This region is potentially important in effecting kinase-mediated increases in ENaC activity.

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

In an effort to gain insight into how kinases might regulate epithelial Na+ channel (ENaC) activity, we expressed human ENaC (hENaC) in Xenopus oocytes and examined the effect of agents that modulate the activity of some kinases. Activation of protein kinase C (PKC) by phorbol ester increased the activity of ENaC, but only in oocytes with a baseline current of

Physiologic resistance to the action of aldosterone. The collecting duct is one of the major targets for aldosterone's action. Experiments conducted several years ago suggested that the major site of action on Na+ and K+ transport was the cortical portion, the cortical collecting duct (CCD). Subsequent studies have shown that the entire collecting duct is capable of responding to aldosterone, but does so differently according to the region. The inner medullary collecting duct (IMCD), while exhibiting a relatively low rate of Na+ transport in isolated, perfused tubules, can develop substantial rates of Na+ transport when put in primary culture. The IMCD, in contrast to the CCD, usually secretes little K+. Investigations into the mechanisms for the lower rates of Na+ transport have revealed that transforming growth factor-β (TGF-β), which is endogenously produced in the inner medulla, can markedly reduce the natriferric action of aldosterone. This action of TGF-β is not apparent within the first few hours of exposure, but its effects, even after removal, last for over 48 hours. The mechanism of this antagonism appears to involve pathways that are parallel and independent of the major transcriptional effects of aldosterone.

The purpose of these experiments was to investigate the mechanisms whereby transforming growth factor-β (TGF-β) antagonizes the action of adrenocorticoid hormones on Na+transport by the rat inner medullary collecting duct in primary culture. Steroid hormones 1) increased Na+transport by three- to fourfold, 2) increased the maximum capacity of the Na+-K+pump by 30–50%, 3) increased the steady-state levels of the α1-subunit of the Na+-K+-ATPase by ∼30%, and 4) increased the steady-state levels of the α-subunit of the rat epithelial Na+channel (α-rENaC) by nearly fourfold. TGF-β blocked the effects of steroids on the increase in Na+transport and the stimulation of the Na+-K+-ATPase and pump capacity. However, there was no effect of TGF-β on the steroid-induced increase in mRNA levels of α-rENaC. The effects of TGF-β were not secondary to the decrease in Na+transport per se, inasmuch as benzamil inhibited the increase in Na+transport but did not block the increase in pump capacity or Na+-K+-ATPase mRNA. The results indicate that TGF-β does not inactivate the steroid receptor or its translocation to the nucleus. Rather, they indicate complex pathways involving interruption of the enhancement of pump activity and activation/inactivation of pathways distal to the steroid-induced increase in the transcription of α-rENaC.

Recent allegations have accused “respected experts” for purportedly promulgating information and opinions to physician-consumers while concealing their conflicts of interest (COIs) and commercial bias.1 The news blitz stemming from these transgressions has led to intense examination of the relationship between physicians and companies that sell medical products and devices. Universities, professional societies, and government agencies uniformly condemn such practice and endorse a variety of methods to reduce the likelihood of commercial influence on activities that are designed to be objective.2,3 In 2008, the American Society of Nephrology (ASN) created a committee to review the Society’s policies and practices regarding its interface with commercial interests. The committee submitted its ensuing report to the ASN Council and published a summary of its final recommendations—which were unanimously approved by council—in this issue of JASN.4 There are four largely separate but contiguous processes whereby commercial interests could influence how physicians provide treatment to patients: conducting research, publishing scientific information, educating practitioners, and developing policy. ASN s educational efforts comprise the majority of the Society’s interactions with commercial interests and, not incidentally, draw the most national attention from the perspective of potential COI and commercial bias. Thus, ASN conducts its major activity squarely in the sights of those who seek to eliminate the possibility of commercial influence in the process of educating practitioners.5 The ASN Committee on Corporate Relations struggled with the meaning and definition of COI. It is widely recognized that everyone has some form of conflict. Although we did not develop a unique definition, we generally agree with the definition proposed by the Institute of Medicine (IOM).6 The practical consequence of this definition is that identifying COI should focus on financial conflicts because they are more objective and better suited to regulatory scrutiny. However, although the committee focused its analysis on financial entanglements, we recognize that professional conflicts should be considered when implementing the proposed recommendations. This emphasis on financial conflicts transforms the focus of bias. We considered one definition of bias as “a conclusion or recommendation based on opinion unsupported by facts.” According to this definition as applied to medicine, almost no recommendation can be totally unbiased. The practical resolution of this matter is to focus on commercial bias, which the committee defines as “an inadequately supported judgment or recommendation about the use of a commercial product.” The committee made no recommendation regarding the highly controversial issue of continuing medical education (CME) funding. However, the sentiment of the committee is more closely aligned with IOM’s recommendations rather than other recommendations directed at eliminating industry support for CME.5,6 The IOM report makes two important points. First, CME is essential to providing high-quality care to patients and, therefore, its importance is increasing. Second, there is no information on the consequences of making a dramatic shift in funding for CME. Making a major change in how CME is funded in the absence of data will have unforeseen consequences.6 The committee generally agrees that any changes should be subject to careful review and testing. The committee strongly endorses the concept that there should be clear separation between the development of the content of educational programs and the generation of financial support. We are impressed with how well ASN has implemented this policy during the past few years. Although virtually everyone can agree that there should be no commercial bias in CME activities, there is surprisingly little understanding of the practical implications of this policy. There are two processes for uncovering potential COI and commercial bias. The first is the process of disclosure. The second is the evaluation of presentations by designated observers and participants. Disclosure is strongly endorsed by all groups. The Accreditation Council for Continuing Medical Education (ACCME) requires disclosure of accredited providers, including ASN. However, what should a speaker disclose? There is wide agreement that a speaker should disclose all financial relationships with commercial entities. In addition, some interPublished online ahead of print. Publication date available at www.jasn.org.

The epithelial Na + channel (ENaC) is composed of three homologous subunits: α, β and γ. We used gene targeting to disrupt the β subunit gene of ENaC in mice. The βENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the −/− mice, we found an increased urine Na + concentration despite hyponatremia and a decreased urine K + concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to αENaC-deficient mice, which die because of defective lung liquid clearance, neonatal βENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo , the β subunit is required for ENaC function in the renal collecting duct, but, in contrast to the α subunit, the β subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the βENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.

Stretching the renal pelvic wall increases ipsilateral afferent renal nerve activity (ARNA). This response is enhanced by inhibiting Na+-K+-ATPase with ouabain, suggesting a modulatory role for intracellular Na+in the activation of mechanosensitive neurons. The messenger RNA for α-, β-, and γ-subunits of epithelial Na+channels (ENaC) is found in collecting duct cells. Because ENaC subunits show homology with genes involved in mechanosensation, we examined whether ENaC mRNA could be found in the pelvic wall and whether the ARNA response to increased renal pelvic pressure was modulated by blockers of the Na+channel. α-, β-, and γ-subunits are present in the pelvis. The messenger RNA for the β- and γ-subunits is readily detected by in situ hybridization throughout the uroepithelium. The ARNA response to increased renal pelvic pressure was reduced by 53 ± 10% and 40 ± 10% ( P < 0.01) by renal pelvic perfusion with the inhibitors amiloride and benzamil, respectively. Amiloride inhibited the ouabain-induced enhancement of the ARNA response to increased renal pelvic pressure. The magnitude of this inhibition was inversely correlated with the magnitude of the amiloride-mediated blockade of the ARNA response to increased renal pelvic pressure ( P < 0.001). Amiloride also reduced the ARNA response to renal pelvic administration of substance P, a mediator of the ARNA response to increased renal pelvic pressure. We conclude that the ENaC complex in the pelvic uroepithelium participates in the activation of renal pelvic mechanosensitive neurons.

Na+absorption via amiloride-sensitive Na+channels is of critical importance in the transition between fetal and neonatal life in several tissues, including the colon, lung, and kidney. To characterize and contrast the mRNA expression of each of the three epithelial Na+channel complex (ENaC) subunits, we conducted RNase protection assays (RPA) and in situ hybridization in colon and lung in fetal (17, 19, 20, and 21 days) and postnatal (1, 3, 9, 15, and 30 days) rats (r). In the colon the α-, β-, and γ-rENaC subunits showed quantitatively different but qualitatively similar expression. All three subunits gradually increased in abundance from fetal day 19 through day 30 of life. The amount of each subunit on day 30 was approximately three times the amount at day 1. In situ hybridization showed that each subunit was localized to the surface epithelial cells with minimal expression in the crypts. The lung showed a completely different pattern. In contrast to the colon, the total amount of α-rENaC mRNA (by RPA) in the lung increased dramatically from fetal day 19 to 21, whereas β- and γ-rENaC showed modest prenatal increases. The amounts of all three mRNAs fell after birth through day 9 (to about 75% of the day 1 value). On days 15 and 30 the amount of mRNA rose to approach the values on day 1. α-rENaC mRNA abundance always exceeded β- and γ-rENaC, and the quantitative expression was different for α- than for β- and γ-rENaC. In situ hybridization studies showed that all three subunits were expressed in epithelial cells of the bronchi, bronchioles, and alveoli and not in blood vessels. These studies show striking developmental heterogeneity in rENaC mRNA expression between lung and colon, probably reflecting different developmental regulatory mechanisms in these organs.

The amiloride-sensitive epithelial sodium channel (ENaC) is composed of three subunits: alpha, beta, and gamma. The human alpha-ENaC subunit is expressed as at least two transcripts (N. Voilley, E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. Proc. Natl. Acad. Sci. USA 91: 247-251, 1994). To determine the origin of these transcripts, we characterized the 5' end of the alpha-ENaC gene. Four transcripts that differ at their first exon were identified. Exon 1A splices to exon 2 to form the 5' end of alpha-ENaC1, whereas exon 1B arises separately and continues into exon 2 to form alpha-ENaC2. Other variant mRNAs, alpha-ENaC3 and alpha-ENaC4, are formed by activating 5' splice sites within exon 1B. Although alpha-ENaC3 and -4 did not change the open reading frame for alpha-ENaC, alpha-ENaC2 contains upstream ATGs that add 59 amino acids to the previous (alpha-ENaC1) protein. To address the significance of these isoforms, both proteins were expressed in Xenopus oocytes. The cRNA for each alpha-ENaC transcript when combined with beta- and gamma-ENaC cRNA reconstituted a low-conductance ion channel with amiloride-sensitive currents of similar characteristics. We have thus identified variant alpha-ENaC mRNAs that lead to functional ENaC peptides.

The trivalent metals iron, aluminum, and gallium greatly increase the rate of iron acquisition from low molecular weight chelates by human myeloid cells. The present study explores the mechanism responsible. Gallium-induced iron acquisition was shown to lead to stable cellular association of iron, the magnitude of which varied with the chelate to which the iron was bound. The majority of this iron initially associated with the plasma membrane. Cellular depletion of ATP did not affect the response to gallium nor did it require the continued presence of extracellular gallium. However, continued cell association of gallium was needed as subsequent cellular exposure to metal chelators resulted in a rapid loss of the “induced” phenotype. Other trivalent metals (lanthanum and gadolinium) and tetravalent metals (tin and zirconium) but not divalent metals also induced iron acquisition. Neither enhanced iron reduction nor protein kinase C or tyrosine kinases appeared involved in gallium-mediated induction of iron acquisition. Exposure of HL-60 cells to polyvalent cationic metals results in a dramatic and sustained increase in the rate of iron acquisition from low molecular weight chelating agents. This could be important for the rapid clearance of iron by phagocytes from the extracellular environment at sites of local tissue damage.

Background. End-stage renal disease is associated with reduced heart rate variability (HRV), components of which generally are associated with advanced age, diabetes mellitus and left ventricular hypertrophy. We hypothesized that daily in-center hemodialysis (HD) would increase HRV. Methods. The Frequent Hemodialysis Network (FHN) Daily Trial randomized 245 patients to receive 12 months of six versus three times per week in-center HD. Two hundred and seven patients had baseline Holter recordings. HRV measures were calculated from 24-h Holter electrocardiograms at both baseline and 12 months in 131 patients and included lowfrequency power (LF, a measure of sympathetic modulation), high-frequency power (HF, a measure of parasympathetic modulation) and standard deviation (SD) of the R–R interval (SDNN, a measure of beat-to-beat variation). Results. Baseline to Month 12 change in LF was augmented by 50% [95% confidence interval (95% CI) 6.1–112%, P = 0.022] and LF + HF was augmented by 40% (95% CI 3.3–88.4%, P = 0.03) in patients assigned to daily hemodialysis (DHD) compared with conventional HD. Changes in HF and SDNN were similar between the randomized groups. The effects of DHD on LF were attenuated by advanced age and diabetes mellitus (predefined subgroups). Changes in HF (r= −0.20, P =0.02) and SDNN (r= −0.18, P =0.04) were inversely associated with changes in left ventricular mass (LVM). Conclusions. DHD increased the LF component of HRV. Reduction of LVM by DHD was associated with increased vagal modulation of heart rate (HF) and with increased beatto-beat heart rate variation (SDNN), suggesting an important functional correlate to the structural effects of DHD on the heart in uremia.

Localization and regulation of renal Na + /myo-inositol cotransporter in diabetic rats. We have examined theeffect of diabetes on sodium/myo-inositol cotransporter (SMIT) mRNA levels and myo-inositol content in the kidney to test the hypothesis that diabetes-induced changes in renal myo-inositol levels are due to the regulation of SMIT mRNA levels.In streptozotocin-induced diabetic rats, after 3, 7 and 28 days of diabetes, SMIT mRNA levels in the whole kidney were increased three- to fivefold, and remained increased by about twofold after six months of diabetes. Insulin treatment of diabetic rats normalized blood glucose levels and prevented the increase in SMIT mRNA levels. Treating diabetic rats with sorbinil, an aldose reductase inhibitor,corrected the abnormal accumulation of sorbitol but had no effect on the diabetes-induced increase in renal SMIT mRNA levels. The regional distribution of SMIT mRNA from normal rats showed a relative abundance in cortex, outer medulla, and inner medulla of 1.0:3.4:7.0. After seven days of diabetes, the levels of SMIT mRNA and myo-inositol content were significantly increased only in the outer medulla. In situ hybridization studies revealed that SMIT mRNA in the outer medulla was predominately localized to the medullary thick ascending limbs of Henle's loop and was not localized to any specific cell in the inner medulla. This distribution pattern was unchanged in diabetic rats. These studies show that diabetes causes an increase in renal SMIT mRNA, which is primarily localized to the outer medulla. Accumulation of myo-inositol by the thick ascending limb of Henle's loop may account for most of the increase caused by diabetes.

The amiloride-sensitive epithelial sodium channel (ENaC) complex is made up of at least three different subunits alpha, beta, and gamma, which are developmentally regulated, selectively expressed, and variously up-regulated by steroid hormones. To understand mechanisms involved in regulation of the gamma subunit, we have determined the structure of the human gammaENaC gene. By 5' rapid amplification of cDNA ends, primer extension analysis, and nuclease protection assay, we identified transcription start sites in human brain, kidney, and lung. A human genomic library was screened and overlapping cosmid clones that span approximately 50 kilobases and contain the hgammaENaC gene were identified. The 5'-untranslated region is 141 bases long, and the translation start codon is contained within the second exon. The human gene spans at least 35 kilobases. The 5' end of the gene including portions of 5' flanking genomic DNA and the first intron are G + C rich and contain several CpG dinucleotides, consistent with a CpG island. The 5' flanking region contains no CCAAT or TATA-like elements but does contain two GC boxes as well as several putative transcription factor binding sites including AP-2, Sp1, CRE, PEA-3, and NF-IL6. This is the first description of the structural organization and the 5' flanking region of a member of the epithelial sodium channel complex.

The hypertonic environment of the renal medulla can change rapidly according to the state of hydration of the animal. We used primary cultures of rat inner medullary collecting duct (IMCD) cells to investigate the characteristics of Cl- currents activated by an acute reduction in osmolarity (ICl(osm)). Using the whole cell patch-clamp technique, we identified an outwardly rectifying current that decayed slowly at strongly depolarizing voltages. The onset of ICl(osm) began 6.7 min after the fall in bath osmolarity, a delay longer than reported in other cell types. Hypotonicity did not induce an increase in intracellular Ca2+ concentration, and activation of ICl(osm) did not require the presence of Ca2+. Intracellular ATP was needed to evoke ICl(osm) when the hypotonic stimulus was modest (50 mosmol/l or less) but was not necessary when the stimulus was stronger (100 mosmol/ l). ICl(osm) was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by tamoxifen or glibenclamide. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid produced a voltage-dependent block. Acute reduction in osmolarity using cells grown on filters did not induce a Cl- secretory current. The ICl(osm) of IMCD cells appears to be on the basolateral membrane and displays some unique features.

The transport of Na+ through amiloride-sensitive sodium channels (ENaC) plays a major role in the absorption of fluid across the pulmonary epithelium. The proteins forming the ENaC channel are encoded by three genes in the rat (alpha-, beta-, and gamma-rENaC). According to Northern blot, all three subunit mRNAs were expressed in adult rat lung. Each subunit was expressed as a single transcript of approximately 3.7, 2.2, and 3.2 kb for alpha-, beta-, and gamma-rENaC, respectively. To localize the alpha-, beta-, and gamma-rENaC subunit mRNAs, we used in situ hybridization. Frozen and paraffin-embedded tissues were hybridized with sense and antisense 35S-labeled riboprobes. The alpha-rENaC mRNA was most abundant and was expressed diffusely in epithelia of the trachea, bronchi, bronchioles, and alveoli. At the alveolar level, alpha-rENaC was expressed in type II cells. The beta- and gamma-rENaC mRNAs were most abundant in the bronchial and bronchiolar epithelia. All three subunits were expressed in the renal cortical collecting duct in a pattern similar to that previously reported by other investigators. Thus the rENaC subunit mRNAs are expressed in regions of the lung where functional Na+ absorption is found. These results are consistent with an important role for ENaC in the absorption of Na+ and fluid across the pulmonary epithelium in all regions of the lung.

We have investigated some of the factors known or suspected to influence ion transport by the rat inner medullary collecting duct and have analyzed their actions on active Na+ absorption and active anion secretion by primary cultures. Cells from the terminal 1-2 mm (tip) of the papilla had a lower basal rate of Na+ absorption (2.0 microA/cm2) than cells from the more proximal portions (6.5 microA/cm2). Aldosterone increased Na+ transport approximately sevenfold in the tip cells and approximately threefold in the proximal cells. The magnitude of anion secretion in response to adenosine 3',5'-cyclic monophosphate (cAMP) agonists was similar in the two regions and was unaffected by aldosterone. The morphology of monolayers from both regions was also similar. In monolayers cultured from the entire inner medulla, hypertonic (100 mosM) urea, NaCl, or sucrose reduced Na+ transport but had no significant effect on anion secretion. Transforming growth factor-beta 1, known to blunt the effect of steroids on Na+ transport, had no effect on anion secretion. Finally, cAMP had no effect on Na+ transport, a result that contrasts with its effect on Na+ transport by other epithelial cells demonstrating steroid-responsive, electrogenic Na+ transport. These results demonstrate some potential differences in the magnitude of Na+ transport by position along the inner medulla. They further demonstrate separate regulation of Na+ and anion transport.

Abstract In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (S HSD ). To evaluate the genetic integrity and salt sensitivity of these new S HSD , we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 S HSD , 16 “authentic” SS/Jr from the inbred colony of John Rapp (S Rapp ), 9 Harlan Sprague Dawley salt-resistant SR/Jr (R HSD ), and (genotyping only) 6 known “contaminated” Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between S Rapp and S* and 18 were polymorphic between S Rapp and R Rapp , but none of the 22 markers revealed polymorphisms between S Rapp and the new S HSD . The phenotyping studies showed that during an ultra–low salt diet, mean arterial pressure was higher ( P Rapp (129±2 mm Hg; mean±SE) and new S HSD (120±2 mm Hg) than in R HSD (93±1 mm Hg). A high salt diet increased mean arterial pressure in every S HSD and S Rapp . Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly ( P Rapp (+51±3 mm Hg) than in new S HSD (+39±3 mm Hg). In addition, salt-induced mortality was significantly greater in S Rapp (62.5%) than S HSD (8.3%) after 8 weeks ( P HSD were genotypically indistinguishable from S Rapp , had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new S HSD supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in S HSD compared with S Rapp . This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the S HSD and S Rapp may now constitute distinct substrains of Dahl SS/Jr.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high- Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK. Refereed/Peer-reviewed

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

We have previously demonstrated that the Na(+)-K+ pump on the basolateral membrane of the rabbit cortical collecting duct can function in the K+/K+ exchange mode. Increasing intracellular phosphate in red blood cells inhibits the Na+ pump and increases K+/K+ exchange. We found that maneuvers designed to increase intracellular phosphate in collecting duct cells caused an increase in K+/K+ exchange. Subjecting the cells to a metabolic insult (cyanide) increased K+/K+ exchange by the pump as judged by its ouabain sensitivity and lack of electrogenic or conductive characteristics. The results demonstrate that the rate of K+/K+ exchange by the Na(+)-K+ pump can be altered by changes in intracellular phosphate over a range that is physiologically or pathologically achievable. The results also suggest a mechanism for inhibition of vectorial Na+ transport during metabolic stress.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

Background Cognitive impairment is common in patients with end-stage renal disease receiving hemodialysis 3 times per week. Study Design Randomized clinical trial. Setting & Participants 218 individuals participating in the Frequent Hemodialysis Network (FHN) Daily Trial and 81 participating in the FHN Nocturnal Trial. Intervention The Daily Trial tested in-center hemodialysis 6 times per week versus 3 times per week. The Nocturnal Trial tested home nocturnal hemodialysis 6 times per week versus home or in-center hemodialysis 3 times per week. Outcomes Cognitive function was measured at baseline, month 4, and month 12. The primary outcome was performance on the Trail-Making Test, Form B, a measure of executive function, and a secondary outcome was performance on the Modified Mini-Mental State Examination, a measure of global cognition. The domains of attention, psychomotor speed, memory, and verbal fluency were assessed in 59 participants in the Daily Trial and 19 participants in the Nocturnal Trial. Results We found no benefit of frequent hemodialysis in either trial for the primary cognitive outcome (Daily Trial: OR for improvement, 0.99; 95% CI, 0.59-1.66; Nocturnal Trial: OR, 1.19; 95% CI, 0.48-2.96). Similarly, there was no benefit of frequent hemodialysis in either trial on global cognition, the secondary cognitive outcome. Exploratory analyses in the Daily Trial suggested possible benefits of frequent hemodialysis for memory and verbal fluency, but not for attention and psychomotor speed. Exploratory analyses in the Nocturnal Trial suggested no benefit of frequent hemodialysis on attention, psychomotor speed, memory, or verbal fluency. Limitations Unblinded intervention, small sample. Conclusions Frequent hemodialysis did not improve executive function or global cognition.

Background— An increase in left ventricular mass (LVM) is associated with mortality and cardiovascular morbidity in patients with end-stage renal disease. Methods and Results— The Frequent Hemodialysis Network (FHN) Daily Trial randomized 245 patients to 12 months of 6 times per week daily in-center hemodialysis or conventional hemodialysis; the FHN Nocturnal Trial randomized 87 patients to 12 months of 6 times per week nocturnal hemodialysis or conventional hemodialysis. The main cardiac secondary outcome was change in LVM. In each trial, we examined whether several predefined baseline demographic or clinical factors as well as change in volume removal, blood pressure, or solute clearance influenced the effect of frequent hemodialysis on LVM. In the Daily Trial, frequent hemodialysis resulted in a significant reduction in LVM (13.1 g; 95% CI, 5.0–21.3 g; P =0.002), LVM index (6.9 g/m 2 ; 95% CI, 2.4–11.3 g/m 2 ; P =0.003), and percent change in geometric mean of LVM (7.0%; 95% CI, 1.0%–12.6; P =0.02). Similar trends were noted in the Nocturnal Trial but did not reach statistical significance. In the Daily Trial, a more pronounced effect of frequent hemodialysis on LVM was evident among patients with left ventricular hypertrophy at baseline. Changes in LVM were associated with changes in blood pressure (conventional hemodialysis: R =0.28, P =0.01, daily hemodialysis: R =0.54, P Conclusions— Frequent in-center hemodialysis reduces LVM. The benefit of frequent hemodialysis on LVM may be mediated by salutary effects on blood pressure. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00264758.