WNK Kinases Orchestrate the Balance of Diverse Electrolyte Flux Pathways
Complex biological systems integrate diverse physiologic processes to produce global homeostasis. Genetic analysis can identify genes that perturb these integrating functions. Aldosterone is secreted by the adrenal gland in two distinct states: decreased intravascular volume or high plasma K+ levels. This raises the question of how the kidney differentially responds to aldosterone to increase salt reabsorption without increasing K+ secretion in the former state, but to increase K+ secretion without excessive salt reabsorption in the latter.
Pseudohypoaldosteronism type II (PHAII) is an autosomal-dominant disease featuring hypertension with hyperkalemia, despite otherwise normal renal function; the kidneys of these patients appear to be stuck in a physiologic state in which the kidney is always reabsorbing salt at the expense of impaired K+ secretion. By positional cloning, we identified mutations in two novel serine-threonine kinases, WNK1 and WNK4, as the cause of this disease. Missense mutations in WNK4 cluster within a few amino acids in the protein.
To gain insight into the functions of WNK4, we have used biochemistry, cell biology, and engineered animal models. We have shown that WNK4 is a switch that differentially modulates the activity of the major salt and K+ flux pathways in the distal nephron. WNK4 has three states: an equilibrium state that inhibits the thiazide-sensitive Na-Cl cotransporter (NCC), the epithelial Na+ channel (ENaC), and the K+ channel ROMK; a second state, induced by angiotensin II signaling, in which inhibition of salt-reabsorbing pathways is abrogated while ROMK inhibition is enhanced; and a third state, induced by aldosterone, in which ROMK and ENaC inhibition are alleviated while NCC inhibition is maintained. The mutations that cause PHAII in humans mimic the effect of angiotensin II, accounting for the phenotype of hypertension and hyperkalemia seen in affected patients. These observations characterize a previously unrecognized layer of regulation that orchestrates the balance between salt reabsorption and K secretion. Importantly, they identify the mechanism for the long-recognized blood pressurelowering effects of high dietary K+the secretion of K+ must occur at the expense of reduced salt reabsorption, leading to reduced blood pressure.
We have also shown that WNK kinases are major determinants of intracellular Cl levels via regulation of the balance between Cl entry via Na-K-Cl cotransporters and Cl exit via K-Cl cotransporters. These effects are likely determinants of the dynamic regulation of intracellular Cl in response to osmotic stress, as well as the regulation of the neuronal response to GABA, which is determined by the resting intracellular Cl level.
New Genes for Cardiovascular Disease
LRP6 in a new Mendelian form of early coronary disease. Arya Mani in the laboratory identified a unique family with an extremely high prevalence of death before the age of 55 from cardiovascular disease; this trait segregated as an autosomal-dominant trait. Positional cloning led to identification of a novel missense mutation that alters the function of low-density lipoprotein (LDL) receptor related protein 6 (LRP6) as the cause. Compared to noncarriers of the mutation in the family, carriers had uniformly and significantly elevated levels of LDL cholesterol, triglycerides, blood pressure, and glucose, but did not have altered HDL (high-density lipoprotein) levels or body mass index. This mutation thus induces a number of features of the metabolic syndrome, implicating LRP6 and its role in Wnt signaling in these traits.
KCNJ10 in SeSAME, a new salt-wasting syndrome.Ute Scholl in the lab recently identified a new syndrome featuring the renal features of Gitelman syndrome (salt wasting with low K+, low Mg2+, and low urinary Ca2+) with early-onset seizures, deafness, ataxia, mental retardation, and lower extremity weakness in five patients from four families. She demonstrated that all patients had loss-of-function mutations in both alleles of the K+ channel KCNJ10. KCNJ10 is expressed in astrocytes and sets the resting potential of these cells. Active neurons repolarize by releasing K+, and astrocytes clear this local rise in K+ level. Absence of this spatial buffering of K+ likely accounts for the observed seizure disorder and other neurologic features. KCNJ10 is expressed on the basolateral membrane of the distal nephron, where it likely is required for recycling out of the cell K+ that enters via the Na+/K+-ATPase, the driver of Na+ reabsorption. Loss of KCNJ10 would thus be expected to impair salt reabsorption in nephron segments that are most dependent on high Na+/K+-ATPase activity, which proves to be the distal convoluted tubule. These findings define a new syndrome and its molecular basis and establish the functional role of basolateral K+ channels in renal salt reabsorption.
Common variants that contribute to cerebral hemorrhage.Cerebral hemorrhage from ruptured intracranial aneurysms affects 500,000 people annually, typically without prior warning. Half of these individuals die as a consequence, and half of the remainder have long-term neurologic deficits. New tools for preclinical diagnosis are consequently paramount. In collaboration with Murat Gunel's laboratory (Yale School of Medicine) and investigators in Europe and Japan, we completed the first genome-wide association study of intracranial aneurysm, studying 2,000 cases and 8,000 controls. The results identify three loci that pass genome-wide significance thresholds in Europe and which show significant replication in Japan. These loci include the same single-nucleotide polymorphisms (SNPs) on 9p adjacent to the CDKN2A/2B locus that have also shown significant association with myocardial infarction and abdominal aortic aneurysm as well as SNPs on 8q flanking Sox17, which is required for formation and maintenance of endothelial cells. These loci operate independent of known risk factors (age, gender, smoking, and hypertension) and confer a threefold range in disease risk. Further studies will be required to establish the mechanisms of their effects and to establish more comprehensively the genetic risk of this disease to enable preclinical identification of subjects at increased risk.
From rare Mendelian traits to the general population. In our work on rare Mendelian forms of high and low blood pressure, we have identified a number of rare recessive traits that cause low blood pressure. We hypothesized that the more frequent heterozygous state might have significant effects on blood pressure in the general population. To this end, Weizhen Ji and Jia Nee Foo sequenced SLC12A3 (encoding the thiazide-sensitive Na-Cl cotransporter), SLC12A1 (encoding the Na-K-Cl cotransporter NKCC2), and KCNJ1 (encoding the K+ channel ROMK), in 3,100 members of the Framingham Heart Study. Forty-nine subjects (1.6 percent of the population) had heterozygous mutations likely to cause loss of function (half were previously identified disease alleles and half were missense variants at positions conserved from invertebrates to humans). Mutation carriers had significantly lower blood pressure than other cohort members and their noncarrier siblings at all ages. At age 60, the average effect was ~9 mmHg, similar to the effect of a single antihypertensive drug, and the prevalence of hypertension was reduced by nearly two-thirds. While these mutations are collectively relatively common, they are individually rare, with allele frequencies all below 1/2,000. The relatively large effects of these rare mutations stand in contrast to the small effects of most common variants implicated in disease and suggest that large fractions of interindividual risk of disease for many common diseases will be attributable to the collective effects of rare variation.
Research on hypertension and intracranial aneurysm was also supported in part by grants from the National Institutes of Health.
As of August 10, 2010