John B. Stokes

The effects of frequent nocturnal home hemodialysis: the Frequent Hemodialysis Network Nocturnal Trial

Prior small studies have shown multiple benefits of frequent nocturnal hemodialysis compared to conventional three times per week treatments. To study this further, we randomized 87 patients to three times per week conventional hemodialysis or to nocturnal hemodialysis six times per week, all with single-use high-flux dialyzers. The 45 patients in the frequent nocturnal arm had a 1.82-fold higher mean weekly stdKt/V(urea), a 1.74-fold higher average number of treatments per week, and a 2.45-fold higher average weekly treatment time than the 42 patients in the conventional arm. We did not find a significant effect of nocturnal hemodialysis for either of the two coprimary outcomes (death or left ventricular mass (measured by MRI) with a hazard ratio of 0.68, or of death or RAND Physical Health Composite with a hazard ratio of 0.91). Possible explanations for the left ventricular mass result include limited sample size and patient characteristics. Secondary outcomes included cognitive performance, self-reported depression, laboratory markers of nutrition, mineral metabolism and anemia, blood pressure and rates of hospitalization, and vascular access interventions. Patients in the nocturnal arm had improved control of hyperphosphatemia and hypertension, but no significant benefit among the other main secondary outcomes. There was a trend for increased vascular access events in the nocturnal arm. Thus, we were unable to demonstrate a definitive benefit of more frequent nocturnal hemodialysis for either coprimary outcome.

Inhibition of the Epithelial Na+ Channel by Interaction of Nedd4 with a PY Motif Deleted in Liddle’s Syndrome

The epithelial Na+ channel (ENaC) plays a critical role in Na+ absorption in the kidney and other epithelia. Mutations in the C terminus of the β or γENaC subunits increase renal Na+ absorption, causing Liddle’s syndrome, an inherited form of hypertension. These mutations delete or disrupt a PY motif that was recently shown to interact with Nedd4, a ubiquitin-protein ligase expressed in epithelia. We found that Nedd4 inhibited ENaC when they were coexpressed in Xenopusoocytes. Liddle’s syndrome-associated mutations that prevent the interaction between Nedd4 and ENaC abolished inhibition, suggesting that a direct interaction is required for inhibition by Nedd4. Inhibition also required activity of a ubiquitin ligase domain within the C terminus of Nedd4. Nedd4 had no detectable effect on the single channel properties of ENaC. Rather, Nedd4 decreased cell surface expression of both ENaC and a chimeric protein containing the C terminus of the β subunit. Decreased surface expression resulted from an increase in the rate of degradation of the channel complex. Thus, interaction of Nedd4 with the C terminus of ENaC inhibits Na+ absorption, and loss of this interaction may play a role in the pathogenesis of Liddle’s syndrome and other forms of hypertension.

Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity

Rats on a low-NaCl diet have a high Na+ channel activity in colon and kidney. To address the mechanism of this increased activity, we measured mRNA levels of three Na+channel subunits in epithelial tissue (rENaC) from rats having been fed either a low (0.13%)- or high (8%)-NaCl diet for 2-3 wk. The size of the mRNA for each of the rENaC subunits as determined by Northern blot was unaffected by diet. RNase protection assay showed heterogeneity of response by organs and subunit. In lung, there was no effect of diet on any of the three subunits. In descending colon, the low-NaCl diet increased β- and γ-rENaC mRNA, with no effect on α-rENaC mRNA. In the kidney, the response to dietary NaCl was dependent on the region. In cortex and outer medulla, diet had no effect on any of the subunits. Rats fed the low-NaCl diet had greater α-rENaC in inner medulla but not β- or γ-rENaC mRNA. We next asked whether acute administration of pure glucocorticoid (GC) or mineralocorticoid (MC) hormones to adrenalectomized rats reproduced the effects of a low-NaCl diet. Six hours after administration of GC or MC, a somewhat different heterogeneity occurred. In lung, α-rENaC mRNA was increased but only in response to GC. In colon, either GC or MC increased β- or γ-rENaC, and there was no effect on α-rENaC. In kidney, either GC or MC increased α-rENaC, without an effect on β- or γ-rENaC. In contrast to the response to a low-NaCl diet, all three regions were similarly affected by acute steroids. These results demonstrate a striking heterogeneity in response to physiological stimuli that regulate ENaC function. The mRNA levels of each of the rENaC subunits can be determined by the type of steroid and by factors unique to the organ and even to the specific region of the kidney.

Glucocorticoid Induction of Epithelial Sodium Channel Expression in Lung and Renal Epithelia Occurs via trans-Activation of a Hormone Response Element in the 5′-Flanking Region of the Human Epithelial Sodium Channel α Subunit Gene

In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel α subunit (αENaC). In H441 lung cells, 100 nm dexamethasone increases amiloride-sensitive short-circuit current (3.3 μA/cm2 to 7.5 μA/cm2), correlating with a 5-fold increase in αENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of αENaC, the human αENaC 5′-flanking region was cloned and tested in H441 cells. By deletion analysis, a ∼150-base pair region 5′ to the upstream promoter was identified that, when stimulated with 100 nm dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, mαENaC expression and luciferase expression from αENaC genomic fragments were also increased by 100 nm dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated αENaC gene transcription. We conclude that glucocorticoids stimulate αENaC expression in kidney and lung via activation of a hormone response element in the 5′-flanking region of hαENaC and this response, in part, is the likely basis for the up-regulation of Na+transport in these sites.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

Nedd4-2 Modulates Renal Na+-Cl− Cotransporter via the Aldosterone-SGK1-Nedd4-2 Pathway

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 channels 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.

Prevalence and Correlates of Cognitive Impairment in Hemodialysis Patients: The Frequent Hemodialysis Network Trials

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 <80 on the Modified Mini-Mental State Exam, and impaired executive function was defined as a score >or=300 seconds on the Trailmaking B test. Five main categories of explanatory variables were examined: urea clearance, nutritional markers, hemodynamic measures, anemia, and central nervous system (CNS)-active medications. RESULTS Subjects had a mean age of 51.6 +/- 13.3 years and a median ESRD vintage of 2.6 years. Sixty-one subjects (16%) had global cognitive impairment, and 110 subjects (29%) had impaired executive function. In addition to several nonmodifiable factors, the use of H1-receptor antagonists and opioids were associated with impaired executive function. No strong association was found between several other potentially modifiable factors associated with ESRD and dialysis therapy, such as urea clearance, proxies of dietary protein intake and other nutritional markers, hemodynamic measures, and anemia with global cognition and executive function after adjustment for case-mix factors. CONCLUSIONS Cognitive impairment, especially impaired executive function, is common among hemodialysis patients, but with the exception of CNS-active medications, is not strongly associated with several ESRD- and dialysis-associated factors.

The assessment, serial evaluation, and subsequent sequelae of acute kidney injury (ASSESS-AKI) study: design and methods

BackgroundThe incidence of acute kidney injury (AKI) has been increasing over time and is associated with a high risk of short-term death. Previous studies on hospital-acquired AKI have important methodological limitations, especially their retrospective study designs and limited ability to control for potential confounding factors.MethodsThe Assessment, Serial Evaluation, and Subsequent Sequelae of Acute Kidney Injury (ASSESS-AKI) Study was established to examine how a hospitalized episode of AKI independently affects the risk of chronic kidney disease development and progression, cardiovascular events, death, and other important patient-centered outcomes. This prospective study will enroll a cohort of 1100 adult participants with a broad range of AKI and matched hospitalized participants without AKI at three Clinical Research Centers, as well as 100 children undergoing cardiac surgery at three Clinical Research Centers. Participants will be followed for up to four years, and will undergo serial evaluation during the index hospitalization, at three months post-hospitalization, and at annual clinic visits, with telephone interviews occurring during the intervening six-month intervals. Biospecimens will be collected at each visit, along with information on lifestyle behaviors, quality of life and functional status, cognitive function, receipt of therapies, interim renal and cardiovascular events, electrocardiography and urinalysis.ConclusionsASSESS-AKI will characterize the short-term and long-term natural history of AKI, evaluate the incremental utility of novel blood and urine biomarkers to refine the diagnosis and prognosis of AKI, and identify a subset of high-risk patients who could be targeted for future clinical trials to improve outcomes after AKI.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

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.

rENaC Is the Predominant Na + Channel in the Apical Membrane of the Rat Renal Inner Medullary Collecting Duct

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.