Genetic polymorphisms in genes regulating renal ion excretion and diuretic drug effects

Regulation of human salt and fluid homeostasis is of basic biological importance. Kidney functions allow surviving under a wide variety of conditions with high or low salt diets and with high or low intake of fluids, even over long periods of times. Ion and water excretion is regulated by a complex network which responds to the internal and environmental conditions. The response to hypovolemia could lead to retaining sodium and excreting more potassium, while in hyperkalemia the excretion of potassium may not be accompanied with differences in the sodium chloride excretion. This controlling network consists mainly of the with-no-lysine kinases (WNKs) and the mineralocorticoid receptor (aldosterone receptor, MR) and their downstream effectors. Genetic polymorphisms in ion transporters were shown to affect the ion-balance excretion with and without diuretic drugs, but no comprehensive study has been performed to study the effects of the polymorphisms in the regulating genes. 229 Healthy Caucasian volunteers from two clinical studies under different diuretic drugs and under sodium chloride restriction days were genotyped for 39 polymorphisms in 11 genes. Two genes arose as candidates for further investigation, the aldosterone receptor gene NR3C2 and the with-no-lysine 4 kinase gene WNK4. Twelve polymorphisms in the NR3C2 gene were selected for further analysis. The association between ion excretion and the minor A-allele of the intron 3 polymorphism (rs3857080) was further investigated in silico and in vitro. The WNK4 exon regions were sequenced for all healthy volunteers in both clinical studies by massive parallel sequencing (Next generation sequencing). The intron 3 polymorphism (rs3857080) in the NR3C2 gene was of high impact mostly on potassium excretion in both clinical studies. The effect of this polymorphism was seen after the sodium chloride restriction days and after the different used diuretics, but it was not seen after torsemide which may have antialdosteronergic effect. In silico and in vitro studies showed that the intron 3 polymorphism region has a possible binding site for the transcription factor LHX4, with higher affinity to bind when the double-strand DNA corresponded to the minor A-allele. The same region had an activating effect in reporter gene assays, but there was no consistent difference between the two different alleles. The WNK4 gene appeared to be not very polymorphic. The massive parallel sequencing of the whole exon regions of the WNK4 gene in both clinical studies revealed only five non-synonymous polymorphisms which were confirmed by Sanger-sequencing. Two novel polymorphisms in the kinase region of the WNK4 (S239W and V358A) were predicted to have deleterious effect on the protein function. Concerning the medical impact, carriers of the A-allele of rs3857080 may be more prone to hypokalemia from diuretic therapy and its devastating consequences than carriers of the major G-allele. The A-allele of the intron 3 polymorphism (rs3857080) may predict a sub-optimal outcome from diuretic therapy and heart diseases. More in vitro studies should follow, first to confirm the possible impact of the intron 3 polymorphism (rs3857080) in the NR3C2 gene, and second to confirm the consistency of the bioinformatics predictions on the WNK4 polymorphisms with the real effects.