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Identifying Common Disease Pathways via Human Genetics
Summary: Richard Lifton uses genetic approaches to identify the genes and pathways that contribute to common human diseases, including cardiovascular, renal, and bone disease.
Dissecting Complex Biological Systems with Genetics: Identification of a Molecular Switch That Regulates Salt and Potassium Balance
Complex biological systems integrate diverse physiologic processes to produce global homeostasis. Genetic analysis can identify genes that perturb these integrating functions. One such system orchestrates net renal NaCl and K+ flux to achieve homeostasis of both intravascular volume and serum K+. Precise control of NaCl reabsorption is required for maintenance of normal blood pressure and tissue perfusion. Control of serum K+ levels, determined by renal secretion, is essential for normal neuromuscular function.
Pseudohypoaldosteronism type II (PHAII) is an autosomal-dominant disease featuring hypertension with hyperkalemia and renal tubular acidosis, despite otherwise normal renal function. By positional cloning, we identified mutations in two novel serine-threonine kinases, WNK1 and WNK4, as the cause of this disease. Large intronic deletions in WNK1 increase WNK1 expression. Missense mutations in WNK4 cluster within a highly conserved segment of the protein.
To gain insight into the function of these kinases, we have determined the tissue distribution of each. Both kinases are present in many organs. They are, however, highly restricted in cell type, expressed almost exclusively in epithelia involved in chloride flux. These epithelia include the pancreatic ducts, bile ducts, colonic crypts, sweat ducts, and lung. In the kidney, these kinases are expressed in the distal nephron, the portion of the kidney that regulates net NaCl and K+ balance. This distribution and the phenotype resulting from their mutation strongly suggest that WNK kinases will be involved in regulation of chloride flux, and suggest a number of targets for their action.
By expression studies in Xenopus oocytes, we have identified four such targets. These include NKCC1 and CFEX, which mediate entry and exit of chloride across the basolateral and apical surfaces in extrarenal epithelia. In the kidney, wild-type WNK4 inhibits the NaCl cotransporter NCCT and the K+ channel ROMK, which are respectively involved in net NaCl reabsorption and K+ secretion. WNK4 thus inhibits a diverse group of flux mediators, including ion channels, cotransporters, and exchangers.
We have investigated the inhibition mechanism. WNK4 inhibits each of these flux pathways by inducing their clearance from the cell surface. The mechanisms, however, are distinct. WNK4 inhibition of NCCT requires the kinase domain of WNK4, whereas this domain is not required for WNK4's inhibition of ROMK. Also, WNK4-induced clearance of ROMK occurs via clathrin-dependent endocytosis, whereas clearance of NCCT occurs independently of this pathway. Finally, the PHAII-causing missense mutations in WNK4 have striking and divergent effects on NCCT and ROMK. While WNK4 harboring these mutations loses inhibition of NCCT, inhibition of ROMK is dramatically increased. These findings can thus explain the pathophysiology of PHAII: The loss of NCCT inhibition resulting from WNK mutation results in increased NaCl reabsorption, which results in increased plasma volume and cardiac output, leading to hypertension. The increased inhibition of ROMK impairs K+ secretion, resulting in hyperkalemia.
These findings establish WNK4 as a molecular switch and can explain a classic paradox in integrated renal physiology: Aldosterone secretion occurs in response to two distinct stimuli—angiotensin II in the setting of intravascular volume depletion and K+ in the setting of hyperkalemia. How does the kidney "know" how to respond appropriately to aldosterone in these different physiologic states? The genetic data suggest that the WNK signaling pathway is the integrator that allows independent modulation of the NaCl-retaining and K+ secretion pathways.
These observations have potentially important implications for the regulation of blood pressure. There have long been data that K+ supplementation lowers blood pressure; however, the mechanism has been unknown. The WNK4 switch provides a potential mechanism, as setting the switch to promote increased K+ secretion results in obligatory reduction in NaCl reabsorption, thereby lowering blood pressure.
Hypertension, Hypercholesterolemia, and Hypomagnesemia: A New Disease Caused by Mitochondrial Mutation, with Implications for the Metabolic Syndrome
Hypertension, dyslipidemia, and insulin resistance each contribute to risk of atherosclerosis, myocardial infarction, and stroke. These traits are concordant in individual patients far more often than expected by chance alone; this combination is referred to as the metabolic syndrome. Other traits, including obesity and hypomagnesemia, are common components of this syndrome. The underlying factors that account for the concordance of these phenotypes have been unknown. Convergent lines of evidence have suggested a major role for mitochondrial dysfunction in insulin resistance and diabetes, raising the question of whether other components of the metabolic syndrome might also result from mitochondrial abnormalities.
We have studied 142 blood relatives of a single kindred with a high prevalence of hypomagnesemia, hypertension, and hypercholesterolemia. Analysis of the distribution of each of these traits in the kindred revealed a striking increase of each on the maternal lineage, with a high rate of transmission from affected mothers to offspring and markedly reduced transmission from affected fathers to offspring. Segregation with the maternal lineage was highly significant for hypomagnesemia (p = 10–11) and both blood pressure (p = 10–4) and hypercholesterolemia (p = 10–4) after adjustment for age, sex, and body mass index. Since we inherit virtually all of our mitochondria from our mothers, this distribution strongly implicates a mitochondrial mutation as the cause of this syndrome.
Sequencing the mitochondrial genome revealed a mutation never previously seen that alters the uridine immediately 5' to the anticodon of the mitochondrial isoleucine tRNA. This is perhaps the most highly conserved base in the biological world, present in virtually all tRNAs owing to the critical role of the amino group of this uridine in stabilizing the anticodon loop by hydrogen bonding with the phosphate backbone of the third base of the anticodon. Biochemical analysis of tRNAs in which this base is mutated confirms markedly reduced ribosome binding.
Muscle biopsy of an affected subject revealed typical features of mitochondrial abnormalities on both light and electron microscopy, including ragged red fibers and abnormal mitochondrial cristae. Further evidence came from measurement of TCA cycle flux and ATP synthetic rate by in vivo NMR (nuclear magnetic resonance) spectroscopy of skeletal muscle, performed by Kitt Petersen and Gerald Shulman (HHMI, Yale University). This revealed normal TCA flux but substantially reduced ATP synthesis.
These findings for the first time establish a causal relationship between mitochondrial mutation and hypertension, hypercholesterolemia, and hypomagnesemia. Together with prior work implicating loss of mitochondrial function in insulin resistance, these findings indicate that abnormal mitochondrial function can account for all components of the metabolic syndrome, and suggest that these diverse manifestations occur as pleiotropic consequences of mitochondrial dysfunction. These findings suggest that age-related loss of mitochondrial function can contribute to common forms of hypertension and hypercholesterolemia and may provide a unifying explanation for the metabolic syndrome.
Last updated: November 17, 2008
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