Intrarenal Metabolite (α‐Ketoglutarate) Signaling Pathway Is Activated by Hnf1α‐Mediated Transcriptional Regulation of OAT10

Abstract

We recently discovered an unusual pathway that couples alterations in intermediate metabolism to salt retention. We found that thiazide diuretics or genetic loss of NCC phosphorylation (SPAK KO) or hypokalemia with intravascular volume contraction activates the synthesis and secretion of α‐ketoglutarate (αKG) from the proximal tubule (PT). αKG is then delivered in the tubular fluid to the distal nephron where it activates its cognate G‐protein coupled receptor, OxGR1, and this stimulates a salt reabsorption network (Pendrin/NDCBE) as a part of a homeostatic response that limits urinary sodium and potassium loss. Hypokalemia appears to stimulate αKG synthesis, whereas volume contraction stimulates αKG transport through angiotensin II (AngII). In the present study, we explore the mechanism by which αKG secretion is activated.Gene profiling studies identified OAT10 (Slc22a13) as an upregulated gene in each of the hypokalemic volume contraction models. As assessed by quantitative immunofluorescence confocal microscopy, OAT10 localized to the PT, and was increased at the apical membrane in SPAK null and thiazide treated WT mice relative to WT control mice. In LLC‐PK1 cells (porcine PT model), we found AngII (100nM) was sufficient to increase OAT10 transcript abundance, and protein localization at the apical membrane. siRNA mediated knockdown of OAT10 resulted in a proportional decrease in αKG secretion. Taken together, the results indicate that OAT10 is regulated to stimulate αKG secretion in response to hypokalemic volume contraction.Activation of OAT10 gene expression is paralleled by up‐regulation of 7 other PT genes that transport amino‐acid substrates for αKG synthesis. In silico promoter analysis (1500bp from start codon) identified Hnf1α as a candidate transcriptional regulator of all these transport genes. Treating LLC‐PK1 cells with AngII decreased phosphorylated Hnf1α and increased its nuclear localization. Luciferase assays using the porcine and mouse Slc22a13 promoter verified that Hnf1α mediates AngII‐stimulated transcription of OAT10 gene.Collectively these data support OAT10 as a mediator of regulated PT αKG secretion and demonstrate that AngII stimulates Slc22a13 transcription via Hnf1α.Support or Funding InformationSupport was provided to PAW from NIH (DK63049, DK54231, and DK093501) and to PRG from the National Kidney Foundation of Maryland.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.