Sodium depletion enhances renal expression of (pro)renin receptor via cyclic GMP-protein kinase G signaling pathway

Abstract

(Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-κB p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-κB p65 to nuclear factor-κB elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-κB p65, and c-Jun to PRR promotor.

Figure 1

Effects of low sodium on the expression of PRR in rat kidney. (A) PRR mRNA, n = 8; (B) PRR protein, n = 8; (C) PRR immunohistochemical staining. NS: normal sodium; LS: low sodium; IgG control: IgG control negative staining; PT: proximal tubules; DT: distal tubules; CD: collecting ducts. *p<0.01; **p<0.001.

Figure 2

Effects of cGMP stimulation and guanylyl cyclase inhibition on PRR expression in PTCs. NS: normal sodium; LS: low sodium; 8-bromo-cGMP: 8-bromocGMP; ODQ: guanylyl cyclase inhibitor; Mean is the average of three independent experiments. *p<0.01; **p<0.001.

Figure 3

Effects of low sodium and PKG inhibition on PRR expression, relative PP2A activity and protein phosphorylation in PTCs. NS: normal sodium; LS: low sodium; PKGi: PKG inhibitor; Mean is the average of three independent experiments. *p<0.05; **p<0.01; †p<0.001.

Figure 4

Effects of low sodium on CREB1 binding to CRE elements in PRR promoter in PTCs. (A) Illustrated diagram of positions of CRE elements and ChIP primer pairs in predicted PRR promoter. Δ Fold: relative enrichment; (B)-(D): ChIP; (E)-(G): EMSA. NS: normal sodium; LS: low sodium; 1: Negative control; 2 & 4: NS; 3 & 5: LS; 4 & 5: CREB-1 antibody competitive assay. Mean is the average of three independent experiments. *p<0.05; **p<0.01.

Figure 5

Effects of low sodium on NF-κB p65 binding to NF-κB elements in PRR promoter in PTCs. (A) Illustrated diagram of positions of NF-κB elements and ChIP primer pairs in predicted PRR promoter. Δ Fold: relative enrichment; (B), (D) and (F): ChIP; (C), (E) and (G): EMSA. NS: normal sodium; LS: low sodium; 1: Negative control; 2 & 4: NS; 3 & 5: LS; 4 & 5: NF-κB p65 antibody competitive assay. Mean is the average of three independent experiments. *p<0.001.

Figure 6

Effects of low sodium on c-Jun binding to activator protein 1 (AP-1) elements in PRR promoter in PTCs. (A) Illustrated diagram of positions of AP-1 elements and ChIP primer pairs in predicted PRR promoter. Δ Fold: relative enrichment; (B)-(E): ChIP; (F)-(I): EMSA. NS: normal sodium; LS: low sodium; 1: Negative control; 2 & 4: NS; 3 & 5: LS; 4 & 5: c-Jun antibody competitive assay. Mean is the average of three independent experiments. *p<0.001.