David H. Ellison

Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.

Maintenance of fluid and electrolyte homeostasis is critical for normal neuromuscular function. Bartters syndrome is an autosomal recessive disease characterized by diverse abnormalities in electrolyte homeostasis including hypokalaemic metabolic alkalosis; Gitelmans syndrome represents the predominant subset of Bartters patients having hypomagnesemia and hypocalciuria. We now demonstrate complete linkage of Gitelmans syndrome to the locus encoding the renal thiazide-sensitive Na–Cl cotransporter, and identify a wide variety of non-conservative mutations, consistent with loss of function alleles, in affected subjects. These findings demonstrate the molecular basis of Gitelmans syndrome. We speculate that these mutant alleles lead to reduced sodium chloride reabsorption in the more common heterozygotes, potentially protecting against development of hypertension.

WNK kinases regulate thiazide-sensitive Na-Cl cotransport.

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis.

Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule.

The mechanisms that govern homeostasis of complex systems have been elusive but can be illuminated by mutations that disrupt system behavior. Mutations in the gene encoding the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and hyperkalemia. We show that physiology in mice transgenic for genomic segments harboring wild-type (TgWnk4WT) or PHAII mutant (TgWnk4PHAII) Wnk4 is changed in opposite directions: TgWnk4PHAII mice have higher blood pressure, hyperkalemia, hypercalciuria and marked hyperplasia of the distal convoluted tubule (DCT), whereas the opposite is true in TgWnk4WT mice. Genetic deficiency for the Na-Cl cotransporter of the DCT (NCC) reverses phenotypes seen in TgWnk4PHAII mice, demonstrating that the effects of the PHAII mutation are due to altered NCC activity. These findings establish that Wnk4 is a molecular switch that regulates the balance between NaCl reabsorption and K+ secretion by altering the mass and function of the DCT through its effect on NCC.

Diuretic Therapy and Resistance in Congestive Heart Failure

Treatment of congestive heart failure has changed dramatically during the past 20 years, but diuretic drugs remain an essential component. Diuretics are essential despite the fact that these drugs stimulate the renin-angiotensin-aldosterone (RAA) axis and lead to adaptive responses that may be counterproductive. In this paper, new diuretic drugs and new uses of older drugs are discussed. These approaches emphasize low-dose combination therapy and may prove superior to traditional approaches that rely exclusively on loop diuretics. Such approaches aim to prevent adverse compensatory processes that appear to result from chronic diuretic treatment. These include acute and chronic increases in plasma renin activity and stimulation of the sympathetic nervous system, both of which increase afterload and may tend to increase mortality. They also include adaptive changes in nephron structure and function resulting from diuretic-induced increases in distal sodium load and diuretic-induced neurohormonal stimulation. These adaptations blunt the effectiveness of diuretic therapy. Diuretic strategies that rely on combinations of diuretics are emphasized as a method to prevent resistance. If diuretic resistance does develop, higher-dose combination regimens, continuous diuretic infusions and mechanical ultrafiltration can be used to overcome diuretic adaptations and restore diuretic efficacy. The goal of reducing the extracellular fluid volume with the least stimulation of the RAA axis and minimal changes in nephron architecture can be achieved in many patients.

Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion.

We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake.

The Physiologic Basis of Diuretic Synergism: Its Role in Treating Diuretic Resistance

Diuretic drugs usually improve edema when used judiciously. Some patients, however, become resistant to their effects. Diuretic resistance may result from dietary indiscretion, poor compliance, impaired bioavailability, imparied diuretic secretion into the lumen of the renal tubule, or because other drugs interfere with diuretic activity. When easily treatable causes of diuretic resistance have been excluded, resistance often reflects the intensity of the stimuli to sodium retention. Recent experimental work has indicated ways in which the kidney adapts to chronic diuretic treatment and has indicated how these adaptations may limit diuretic effectiveness. First, nephron segments downstream from the site of diuretic action increase sodium-chloride (NaCl) reabsorption because the delivered NaCl load increases. Second, diuretic-induced contraction of the extracellular fluid volume stimulates kidney tubules to retain NaCl until the next dose of diuretic is administered. Third, kidney tubules themselves may become hypertrophic because they are chronically stimulated by diuretic-induced increases in NaCl delivery. These adaptations all increase the rate of NaCl reabsorption and blunt the effectiveness of diuretic therapy. When diuretic resistance is present, using a second diuretic drug that acts in a different nephron segment is often effective. Recent experimental results suggest that a second class of drug may act synergistically with the first by blocking the adaptive processes that limit diuretic effectiveness. On the basis of an understanding of the mechanisms of diuretic adaptation and resistance, treatment regimens can be designed to block specific adaptive mechanisms and to improve diuretic therapy.

The WNKs: Atypical Protein Kinases With Pleiotropic Actions

WNKs are serine/threonine kinases that comprise a unique branch of the kinome. They are so-named owing to the unusual placement of an essential catalytic lysine. WNKs have now been identified in diverse organisms. In humans and other mammals, four genes encode WNKs. WNKs are widely expressed at the message level, although data on protein expression is more limited. Soon after the WNKs were identified, mutations in genes encoding WNK1 and -4 were determined to cause the human disease familial hyperkalemic hypertension (also known as pseudohypoaldosteronism II, or Gordons Syndrome). For this reason, a major focus of investigation has been to dissect the role of WNK kinases in renal regulation of ion transport. More recently, a different mutation in WNK1 was identified as the cause of hereditary sensory and autonomic neuropathy type II, an early-onset autosomal disease of peripheral sensory nerves. Thus the WNKs represent an important family of potential targets for the treatment of human disease, and further elucidation of their physiological actions outside of the kidney and brain is necessary. In this review, we describe the gene structure and mechanisms regulating expression and activity of the WNKs. Subsequently, we outline substrates and targets of WNKs as well as effects of WNKs on cellular physiology, both in the kidney and elsewhere. Next, consequences of these effects on integrated physiological function are outlined. Finally, we discuss the known and putative pathophysiological relevance of the WNKs.

Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport

With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt.

The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension

Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use. These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, hydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment.

Renal Mechanism of Trimethoprim-induced Hyperkalemia

Table. SI Units Hyperkalemia is increasingly being recognized in patients with human immunodeficiency virus (HIV) infection and the acquired immunodeficiency syndrome (AIDS) [1-7]. In this setting, it is likely that hyperkalemia is the result of inadequate renal potassium excretion. Three mechanisms could be responsible for renal potassium retention: adrenal insufficiency with inadequate production of aldosterone; acute renal failure with reduced glomerular filtration and damage to tubule cells; and inhibition of potassium secretion. Most attention has focused on the first two mechanisms [1-8]. We are aware, however, of three reports of reversible hyperkalemia [4, 9, 10] that suggest that a therapeutic agent may have a direct action on renal tubules to suppress potassium transport. A common factor in the three reports was the administration of trimethoprim for treatment of Pneumocystis carinii pneumonia. The purpose of our study was to test the hypothesis that trimethoprim causes hyperkalemia by a direct action on the distal nephron cells responsible for secreting potassium. Methods Study in Humans All patients receiving high-dose trimethoprim (20 mg/kg per day), with either sulfamethoxazole or dapsone, at the Yale-New Haven Hospital during a 4-month period, were included. As a part of the clinical management of these patients, serum measurements of sodium, potassium, and creatinine levels were recorded before, during, and after trimethoprim treatment. In some patients, increased serum potassium levels (>5.0 mmol/L) were identified while patients were receiving trimethoprim. In a group of these patients, we reviewed the clinical course and recommended further evaluation to search for causes of hyperkalemia. This evaluation included measurements of serum glucose, renin, aldosterone, and cortisol levels as well as osmolality; measurements of urinary sodium, potassium, chloride, glucose, and protein levels as well as osmolality; and examination of the urinary sediment. The tubule fluid/plasma concentration ratio for potassium in the cortical collecting duct (transtubular potassium gradient) [11, 12] was calculated from urine and serum values for potassium and osmolality as (K)urine/[K]serum)/([Osm]urine/[Osm]serum). The ability of cosyntropin to stimulate cortisol secretion was determined in patients whose cortisol level was < 552 nmol/L (<20 g/dL). Studies in Rats Male Sprague-Dawley rats, allowed free access to standard rat chow and tap water up to the time of the experiment, were anesthetized before surgical exposure of one kidney, as previously described [13]. The ureter was cannulated. Intravenous Infusion of Trimethoprim A salt solution (140 mmol/L sodium chloride, 4 mmol/L potassium chloride) was infused at 15 mL/h per kg body weight, and 45 minutes was allowed for equilibration after surgical preparation was completed. Subsequently, six 30-minute urine collections were obtained. The first 90 minutes was period I (collections 1 to 3), and the subsequent 90 minutes was period II (collections 4 to 6). The control and experimental groups of animals differed only in that at 90 minutes (after period I), 0.64 g/L of trimethoprim was added to the intravenous infusate (the trimethoprim infusion rate was 9.6 mg/h per kg body weight) in the experimental group and was maintained throughout period II. Sodium, potassium, and chloride concentrations were measured in urine [13]. The urine flow rate was measured, and the excretion rates for fluid, sodium, potassium, and chloride were calculated. Microperfusion of Distal Tubules Microperfusion experiments were done as described previously [13, 14]. Distal tubules were perfused with an artificial tubule fluid with or without trimethoprim (the composition of the perfusion solutions is given in Table 1. A perfusion pipette was positioned at the upstream end of the tubule, and a collection pipette was positioned at the downstream end. The perfusion pump was set to deliver 15 nL/min. A paired design was used as follows: After the first tubule fluid sample was collected, both the collection pipette and the perfusion pipette were removed: Then a second perfusion pipette containing a different solution [the order of solutions was alternated] and a second collection pipette were used to collect a second tubule fluid sample. The volume of collected samples was measured. Sodium, potassium, chloride, and inulin concentrations in perfused and collected fluids were measured, as described previously [14]. Osmolality and pH of bulk solutions were measured [14]. The perfusion rate was calculated from the collection rate and the inulin concentrations. Net fluid transport was calculated as the difference between perfusion and collection rates. Net transport rates for sodium, potassium, and chloride levels by each distal tubule were determined. Transepithelial voltage across the wall of the late distal tubule was measured, as previously described [15]. In experiments designed to test the effect of different concentrations of trimethoprim on transepithelial voltage, a higher perfusion rate (30 compared with 15 nL/min) was used to minimize changes in luminal ion composition along the length of the perfused tubule. Table 1. Distal Tubule Flow Rates, Collected Ion Concentrations, and Transport Rates with Control and Trimethoprim Solutions* Statistical Analysis Results were analyzed using the t-statistic. A P value of less than 0.05 (95% CI) was statistically different. Results Human Studies Records from 30 consecutive inpatients who were treated with high-dose trimethoprim at Yale-New Haven Hospital between 10 July and 18 November 1992 were reviewed. No patients were excluded from this study. All patients were HIV positive and were treated for presumed or confirmed P. carinii pneumonia. Twenty-three of the patients were treated with trimethoprim-sulfamethoxazole, and seven were treated with trimethoprim-dapsone. On average, the length of the trimethoprim treatment period was 5.3 2.79 days (mean SD; range, 1 to 13 days). Figure 1 shows that the serum potassium concentration increased by 0.6 mmol/L (CI, 0.29 to 0.95 mmol/L) during treatment with trimethoprim. When the drug was discontinued, the potassium concentration decreased to pretreatment values. In 15 of 30 patients (50%), the serum potassium concentration was more than 5.0 mmol/L on at least 1 day during trimethoprim treatment. Severe and potentially life-threatening hyperkalemia (potassium > 6.0 mmol/L) occurred in three patients (10%). The serum creatinine concentration was slightly higher during trimethoprim treatment than during recovery (mean difference, 15.9 mmol/L; CI, 5.3 to 26.5 mmol/L [0.18 mg/dL; CI, 0.06 to 0.30 mg/dL]). None of the patients were taking nonsteroidal anti-inflammatory drugs, converting-enzyme inhibitors, or potassium-sparing diuretics. Figure 1. Effect of trimethoprim on serum potassium concentration in patients with AIDS. Renal and adrenal function were evaluated in seven patients during hyperkalemia (potassium > 5 mmol/L). The mean serum potassium concentration was 5.9 0.9 mmol/L, and the urinary potassium concentration was 11.3 5.8 mmol/L (mean SD). Oliguria was not present in any of the patients, and the serum creatinine concentration was not increased above baseline (mean difference, 17.7 mmol/L; CI, 9.72 to 43.3 mmol/L [0.2 mg/dL; CI, 0.11 to 0.49 mg/dL]). The transtubular potassium gradient calculated for the cortical collecting duct [11, 12] was 1.9 1.1 (mean SD). This value was low (expected range was 6 to 11) for a plasma potassium concentration of >5 mmol/L. In three patients, the transtubular potassium gradient was calculated both during and after treatment with trimethoprim. After discontinuation of trimethoprim, the transtubular potassium gradient increased to normal values in all three patients (mean difference, 4.5; CI, 1.4 to 7.5). The urinary sodium concentration was 103 65.7 mmol/L, and there was mild hyponatremia (132 2.8 mmol/L). The plasma cortisol (497 152 nmol/L, supine [18.0 5.5 g/dL, supine]); renin (0.667 0.25 ng/[L x s], supine [2.4 0.9 ng/mL per hour, supine]); and aldosterone (535 264 pmol/L, supine [19.3 9.5 ng/dL, supine]) levels were all high normal or increased during hyperkalemia. In two patients with borderline serum cortisol levels (221 and 469 nmol/L [8 and 17 g/dL]), cosyntropin stimulation test results were normal. Glucose levels were all within the normal range. Rat Studies The effect of trimethoprim on potassium and sodium excretion rates in the whole kidney is shown in Figure 2. Compared with the control group, trimethoprim decreased potassium excretion by 572 nmol/min (CI, 299 to 845 nmol/min). The reduction in potassium excretion during trimethoprim infusion was 40% (CI, 21% to 60%) of the control rate measured in period II. Although sodium excretion in the control group increased with time, trimethoprim caused a twofold larger increase in sodium excretion. The difference between these changes, 1192 nmol/min (CI, 240 to 2142), was statistically significant. The increase in sodium excretion during trimethoprim infusion was 46% (CI, 9% to 83%) of the control rate measured in period II. There was no effect of trimethoprim on urine flow or chloride excretion rate (Appendix 1). Appendix Table 1. Figure 2. The scales depict the change () in ion excretion rate with time (period II minus period I) in control and experimental (Trimethoprim) animals. Panel A Panel B Table 1 gives flow rates, lumen ion concentrations, and ion transport rates from in vivo microperfusion of distal tubules of rats. Figure 3 shows that 1 mmol/L of trimethoprim inhibited net potassium secretion by 59% (CI, 26% to 92%). The rate of net sodium absorption did not decrease. Rates of net chloride and water absorption were also not affected. Figure 3. Net potassium transport during perfusion of 14 distal tubules with control and trimethoprim (TMP) solutions. Figure 4 shows the effect of trimethoprim concent