Evan C. Ray

Cutting It Out: ENaC Processing in the Human Nephron

Studies over the past decade have shed light on how proteases activate the epithelial sodium channel (ENaC). Two ENaC subunits ( α and γ ) have short stretches of embedded amino acid residues that inhibit the channel. Following protease-dependent cleavage of these subunits at sites flanking these

A Trapped Intracellular Cation Modulates K+ Channel Recovery From Slow Inactivation

Upon depolarization, many voltage-gated potassium channels undergo a time-dependent decrease in conductance known as inactivation. Both entry of channels into an inactivated state and recovery from this state govern cellular excitability. In this study, we show that recovery from slow inactivation is regulated by intracellular permeant cations. When inactivated channels are hyperpolarized, closure of the activation gate traps a cation between the activation and inactivation gates. The identity of the trapped cation determines the rate of recovery, and the ability of cations to promote recovery follows the rank order K+ > NH4 + > Rb+ > Cs+ >> Na+, TMA. The striking similarity between this rank order and that for single channel conductance suggests that these two processes share a common feature. We propose that the rate of recovery from slow inactivation is determined by the ability of entrapped cations to move into a binding site in the channels selectivity filter, and refilling of this site is required for recovery.

Sodium Retention and Volume Expansion in Nephrotic Syndrome: Implications for Hypertension

Sodium retention is a major clinical feature of nephrotic syndrome. The mechanisms responsible for sodium retention in this setting have been a subject of debate for years. Excessive sodium retention occurs in some individuals with nephrotic syndrome in the absence of activation of the renin-angiotensin-aldosterone system, suggesting an intrinsic defect in sodium excretion by the kidney. Recent studies have provided new insights regarding mechanisms by which sodium transporters are activated by factors present in nephrotic urine. These mechanisms likely have a role in the development of hypertension in nephrotic syndrome, where hypertension may be difficult to control, and provide new therapeutic options for the management of blood pressure and edema in the setting of nephrotic syndrome.

Trial of Amiloride in Type 2 Diabetes With Proteinuria

Introduction Renal sodium (Na+) retention and extracellular fluid volume expansion are hallmarks of nephrotic syndrome, which occurs even in the absence of activation of hormones that stimulate renal Na+ transporters. Plasmin-dependent activation of the epithelial Na+ channel has been proposed to have a role in renal Na+ retention in the setting of nephrotic syndrome. We hypothesized that the epithelial Na+ channel inhibitor amiloride would be an effective therapeutic agent in inducing a natriuresis and lowering blood pressure in individuals with macroscopic proteinuria. Methods We conducted a pilot double-blind randomized cross-over study comparing the effects of daily administration of either oral amiloride or hydrochlorothiazide to patients with type 2 diabetes and macroscopic proteinuria. Safety and efficacy were assessed by monitoring systolic blood pressure, kidney function, adherence, weight, urinary Na+ excretion, and serum electrolytes. Nine subjects were enrolled in the trial. Results No significant difference in systolic blood pressure or weight was seen between subjects receiving hydrochlorothiazide and those receiving amiloride (P ≥ 0.15). Amiloride induced differences in serum potassium (P < 0.001), with a 0.88 ± 0.30 mmol/l greater acute increase observed. Two subjects developed acute kidney injury and hyperkalemia when treated with amiloride. Four subjects had readily detectable levels of urinary plasminogen plus plasmin, and 5 did not. Changes in systolic blood pressure in response to amiloride did not differ between individuals with versus those without detectable urinary plasminogen plus plasmin. Discussion In summary, among patients with type 2 diabetes, normal renal function, and proteinuria, there were reductions in systolic blood pressure in groups treated with hydrochlorothiazide or amiloride. Acute kidney injury and severe hyperkalemia were safety concerns with amiloride.

Functional roles of clusters of hydrophobic and polar residues in the epithelial Na+ channel knuckle domain

Background: There are regulatory interactions between ENaC and extracellular factors. Results: Mutations of multiple α subunit knuckle residues activate ENaC by suppressing the inhibitory effect of Na+. Channels lacking the β or γ subunit knuckle have processing defects. Conclusion: Interactions between the α subunit knuckle and palm/finger domains regulate ENaC. Significance: Intrasubunit domain-domain interactions have important regulatory roles. The extracellular regions of epithelial Na+ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na+ (Na+ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na+ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na+ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na+ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na+ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na+ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.

Human epithelial Na+ channel missense variants identified in the GenSalt study alter channel activity

Mutations in genes encoding subunits of the epithelial Na+ channel (ENaC) can cause early onset familial hypertension, demonstrating the importance of this channel in modulating blood pressure. It remains unclear whether other genetic variants resulting in subtler alterations of channel function result in hypertension or altered sensitivity of blood pressure to dietary salt. This study sought to identify functional human ENaC variants to examine how these variants alter channel activity and to explore whether these variants are associated with altered sensitivity of blood pressure to dietary salt. Six-hundred participants of the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study with salt-sensitive or salt-resistant blood pressure underwent sequencing of the genes encoding ENaC subunits. Functional effects of identified variants were examined in a Xenopus oocyte expression system. Variants that increased channel activity included three in the gene encoding the α-subunit (αS115N, αR476W, and αV481M), one in the β-subunit (βS635N), and one in the γ-subunit (γL438Q). One α-subunit variant (αA334T) and one γ-subunit variant (βD31N) decreased channel activity. Several α-subunit extracellular domain variants altered channel inhibition by extracellular Na+ (Na+ self-inhibition). One variant (αA334T) decreased and one (αV481M) increased cell surface expression. Association between these variants and salt sensitivity did not reach statistical significance. This study identifies novel functional human ENaC variants and demonstrates that some variants alter channel cell surface expression and/or Na+ self-inhibition.

ENaC blockade in proteinuria-associated extracellular fluid volume overload - effective but risky

Renal Na retention and edema represent cardinal features of proteinuric kidney disease. Clinical data suggest that, in some patients with heavy proteinuria, nephrotic syndrome, renal Na retention, and extracellular fluid volume overload occur in spite of a suppressed renin-aldosterone system (Meltzer et al. 1979). Although systemic factors may contribute to urinary Na retention, a rat unilateral kidney model of nephrotic syndrome demonstrated that mechanisms intrinsic to the diseased kidney contribute to Na retention (Ichikawa et al. 1983). Proteinuria stimulates Na re-absorption in the aldosterone-sensitive distal nephron. This Na-reabsorption is sensitive to blockers of the epithelial sodium channel (ENaC), suggesting ENaC plays a role in proteinuria-associated Na-retention (Deschenes et al. 2001). The last decade has led to significant advances in our understanding of the regulation of ENaC and insights regarding mechanisms by which proteinuria may enhance ENaC activity. Ten years ago, researchers at the University of Pittsburgh and at the University of Southern Denmark independently described the ability of the serine protease, plasmin, to activate ENaC (Passero et al. 2008; Svenningsen et al. 2009). Plasmin’s precursor, plasminogen, can be leaked from the bloodstream into the tubular ultrafiltrate through damaged glomeruli and may be activated by urokinase expressed in the tubule. In channels where the c subunit has already undergone cleavage by the intracellular protease, furin, secondary cleavage by plasmin allows removal of an inhibitory tract between the cleavage sites. Loss of the inhibitory tract increases the channel’s open probability (Ray et al. 2015). A host of other secondary proteases, if present, could also perform this role and activate ENaC. However, for extracellular proteases to access the channel, ENaC must be present at the cell surface. Mechanisms that maintain ENaC expression and cell surface delivery in the context of proteinuria-associated extracellular fluid volume overload remain unclear. Interestingly, subjects with membranous nephropathy are unresponsive to mineralocorticoid antagonism (Usberti and Gazzotti 1998). This observation suggests that activation of ENaC in these patients may be independent of canonical mineralocorticoid signaling. Mechanistically, this distinguishes at least some types of nephrotic syndrome from other disorders characterized by total body Na overload, such as cirrhosis and heart failure. In these disorders, aldosterone signaling plays a key pathophysiologic role, and mineralocorticoid receptor antagonism represents an important therapeutic modality. In this issue of Physiological Reports, Gitte Hinrichs and colleagues at the University of Southern Denmark revisit activation of ENaC in proteinuria. They describe their experience using the ENaC antagonist, amiloride, in a patient with diabetes and nephrotic-range proteinuria (Hinricks et al. 2018). Although amiloride is generally considered a rather poor anti-hypertensive agent, prescription of 5 mg of amiloride per day to their patient was associated with significant diuresis and a 25 mmHg reduction in systolic blood pressure. The authors point out that others have reported brisk diuresis in patients with proteinuria in response to ENaC blockade, but their observations further support an important role for ENaC in proteinuria-associated Na retention. Hinricks et al. (2018) further observed that this patient’s hypertension and edema failed to respond to spironolactone. Although patient adherence to this medication prescription was not proven, this finding is consistent with mineralocorticoid-independent enhancement of renal Na retention, similar to previous observations in membranous nephropathy. Unfortunately, the patient also experienced acute kidney injury and dramatic hyperkalemia. Because he was concurrently prescribed an angiotensin-converting enzyme inhibitor, a mineralocorticoid antagonist, and a loop diuretic, these adverse effects may not be solely attributable to amiloride. Nonetheless, the findings are consistent with observations from a randomized crossover controlled trial that compared amiloride and hydrochlorothiazide in subjects with type 2 diabetes and proteinuria (Unruh et al. 2017). This study was

Urinary Plasmin(ogen) as a Prognostic Factor for Hypertension

Introduction Plasmin and its precursor, plasminogen, are detectable in urine from patients with glomerular disease. Urinary plasmin(ogen) levels correlate with blood pressure (BP) and may contribute to renal Na+ retention by activating the epithelial Na+ channel (ENaC). In a longitudinal nested-cohort study, we asked whether urinary plasmin(ogen) levels predict subsequent increase in BP, incident hypertension, or mortality in subjects with type I diabetes, who often develop proteinuria. Methods The Pittsburgh Epidemiology of Diabetes Complications (EDC) study followed up type I diabetic subjects for 25 years. Urine specimens from 70 subjects with a spectrum of baseline urinary albumin levels were examined. Outcomes included increased BP after 2 years (≥1 SD over baseline systolic or diastolic BP, examined via logistic regression), 25-year incident hypertension (≥140/90 mm Hg or initiating BP-lowering medications), and all-cause or cardiovascular mortality, examined using Cox regression. Results Subjects experiencing a 2-year increase in BP had higher baseline urinary plasmin(ogen)/creatinine levels (uPl/Cr) than other subjects (P = 0.04); the difference in baseline urinary albumin/creatinine levels (uAlb/Cr) was similar (P = 0.07). Baseline uPl/Cr was associated with increased 25-year hypertension incidence (hazard ratio = 2.05, P = 0.001), all-cause mortality (HR = 2.05, P = 0.01) and cardiovascular mortality (HR = 3.30, P = 0.005), although not independent of uAlb/Cr. Conclusion This is the first long-term prospective study addressing clinical outcomes associated with increased urinary plasmin(ogen). Findings are consistent with a role for plasmin(ogen) in promoting increased BP, but also demonstrate the difficulty in distinguishing effects due to plasmin(ogen) from those of albuminuria.

Why Diuretics Fail Failing Hearts

Loop diuretics represent a key component of the therapeutic armamentarium used to reduce extracellular fluid volume in heart failure. Unfortunately, resistance to loop diuretics commonly occurs, complicating clinical management. In general, diuretic resistance refers to the inability to reduce

An Increasingly Complex Relationship Between Salt and Water

Address correspondence to Thomas R. Kleyman, MD, RenalElectrolyte Division, University of Pittsburgh, 3550 Terrace St, Pittsburgh, PA 15261. E-mail: kleyman@pitt.edu 2017 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2017.07.007 One hundred fifty years ago this year, physiologist Moritz Schiff proclaimed from a pulpit at the University of Florence that the sensation of thirst, rather than being secondary to dryness of the throat, is a “general sensation, stemming from lack of water in the blood.” Expounding, he declared “the dryness of the throat that normally accompanies [thirst] is merely a secondary phenomenon, analogous to the weight of the eyelids that accompanies sleepiness.” Fifty years later, Erich Leschke would demonstrate that intravenous infusion of hypertonic saline in humans provoked a “sudden and violent thirst.” Subsequent studies revealed that increased dietary salt stimulates fluid consumption in experimental animals, and the role of increased oral fluid intake in mitigating dietary salt–induced hypertonicity while expanding extracellular fluid volume became established renal canon. However, studies of humans in the 1980s suggested that the relationship between dietary salt and fluid intake may not be so simple. Delving deeper into this subject, Rakova and colleagues now present findings in support of the heretical notion that increased salt intake over the long term decreases fluid consumption in humans. An accompanying report by the same group further explores the metabolic effects of increased salt consumption in mouse models and the mechanisms that allow reduced fluid intake in the context of increased sodium consumption. These findings expand our understanding of the metabolic effects of salt consumption and test our faith in the dogma surrounding the role of water consumption in controlling extracellular tonicity.