Differential regulation of Na+ transporters along nephron during ANG II-dependent hypertension: distal stimulation counteracted by proximal inhibition

Abstract

During angiotensin II (ANG II)-dependent hypertension, ANG II stimulates, while hypertension inhibits, Na(+) transporter activity to balance Na(+) output to input. This study tests the hypothesis that ANG II infusion activates Na(+) transporters in the distal nephron while inhibiting transporters along the proximal nephron. Male Sprague-Dawley rats were infused with ANG II (400 ng·kg(-1)·min(-1)) or vehicle for 2 wk. Kidneys were dissected (cortex vs. medulla) or fixed for immunohistochemistry (IHC). ANG II increased mean arterial pressure by 40 mmHg, urine Na(+) by 1.67-fold, and urine volume by 3-fold, evidence for hypertension and pressure natriuresis. Na(+) transporters' abundance and activation [assessed by phosphorylation (-P) or proteolytic cleavage] were measured by immunoblot. During ANG II infusion Na(+)/H(+) exchanger 3 (NHE3) abundance decreased in both cortex and medulla; Na-K-2Cl cotransporter 2 (NKCC2) decreased in medullary thick ascending loop of Henle (TALH) and increased, along with NKCC2-P, in cortical TALH; Na-Cl cotransporter (NCC) and NCC-P increased in the distal convoluted tubule; and epithelial Na(+) channel subunits and their cleaved forms were increased in both cortex and medulla. Like NKCC2, STE20/SPS1-related proline alanine-rich kinase (SPAK) and SPAK-P were decreased in medulla and increased in cortex. By IHC, during ANG II NHE3 remained localized to proximal tubule microvilli at lower abundance, and the differential regulation of NKCC2 and NKCC2-P in cortex versus medulla was evident. In summary, ANG II infusion increases Na(+) transporter abundance and activation from cortical TALH to medullary collecting duct while the hypertension drives a natriuresis response evident as decreased Na(+) transporter abundance and activation from proximal tubule through medullary TALH.

Keywords: ENaC; NCC; NHE3; NKCC2; SPAK; pressure natriuresis.

Fig. 1.

Chronic ANG II (400 ng·kg⁻¹·min⁻¹) infusion increases urinary angiotensinogen. A: representative immunoblots of angiotensinogen in urine of rats infused with either vehicle (control) or ANG II (n = 8 each) assayed in 0.02% of overnight urine volume before (baseline) and after 2 wk of ANG II infusion (final). B: relative abundance displayed as individual records with means ± SE. Density values normalized to mean density of baseline values from their respective groups. *P < 0.05, final ANG II vs. final control. #P < 0.05, final ANG II vs. baseline ANG II, assessed by two-way ANOVA analysis.

Fig. 2.

ANG II infusion decreases NHE3 abundance. A: immunoblots of total and phosphorylated NHE3 in renal cortex and medulla of rats infused with either vehicle (control) or ANG II (n = 8 each). Protein/lane in Table 1. B: relative abundance displayed as individual records with means ± SE. *P < 0.05. C: indirect immunofluorescence microscopy of NHE3 and villin in renal cortex. Sections were processed identically on same slide and imaged with the same settings. Antibody labeling specifics provided in Table 1. Bar, 20 μm.

Fig. 3.

ANG II infusion provokes differential regulation of NKCC2 in cortex vs. medulla. A: immunoblots of total NKCC2 and NKCC2-P in cortex and medulla of rats infused with either vehicle (control) or ANG II (n = 8 each). Protein per lane in Table 1. B: relative abundance displayed as individual records with means ± SE. *P < 0.05. C: indirect immunofluorescence microscopy of NKCC2 and NKCC2-P. Kidneys from 2 rats were fixed for each condition. Sections were processed identically on same slide and imaged with the same settings. Two sections were fully examined and the representative images were chosen with glomeruli for identification of the cortex. Antibody labeling specifics provided in Table 1. Bar, 20 μm. G, glomerulus.

Fig. 4.

ANG II infusion decreases sodium pump subunits' abundance in the medulla. Immunoblots of NKAα₁ (A) and NKAβ₁ (B) in renal cortex and medulla of rats infused with either vehicle (control) or ANG II (n = 8). Protein per lane in Table 1. Relative abundance is displayed as individual records with means ± SE. *P < 0.05.

Fig. 5.

Chronic ANG II (400 ng·kg⁻¹·min⁻¹) infusion increases NCC abundance and phosphorylation. A: immunoblots of NCC total, NCCpT53, NCCpS71, and NCCpS89 in renal cortex of rats infused with either vehicle (control) or ANG II (n = 8 each). Protein per lane in Table 1. B: relative abundance is displayed as individual records with means ± SE. Density values were normalized to mean density of control group. *P < 0.05. C: characterization of a new anti-NCC antibody produced in rabbits against NH₂-terminal amino acids 74–96 (PGEPRKVRPTLADLHSFLKQEG). Forty micrograms of homogenate from untreated rat cortex (contains NCC and NKCC), medulla (contains NKCC), and mouse whole kidney were resolved and probed with the new anti-NCC diluted 1:5,000. The antibody detects a band around 150 kDa in cortex and very little in medulla, indicating that it detects NCC and not NKCC2. The band at 100 kDa in the medulla is nonspecific since there should be no NCC in the medulla, and the band at 70 kDa in mouse is unknown. D: frozen slice of renal cortex was probed with the new anti-NCC antibody as well as an antibody directed to NCCpT58. The antibodies both detect apical NCC, with little nonspecific labeling. Antibody labeling specifics provided in Table 1.

Fig. 6.

ANG II infusion differentially regulates SPAK and SPAK-P abundance in cortex vs. medulla. A: immunoblots of SPAK and SPAK-P in renal cortex and medulla of rats infused with either vehicle (control) or ANG II (n = 8 each). Protein per lane in Table 1. B: relative abundance displayed as individual records with means ± SE. *P < 0.05. FL, full-length SPAK; KS, kidney-specific SPAK.

Fig. 7.

Chronic ANG II (400 ng·kg⁻¹·min⁻¹) infusion does not change the abundance or phosphorylation of regulatory kinase oxidative stress response-1 (OSR1) in cortex or medulla. A: immunoblots of total OSR1 and OSR1-P in renal cortex and medulla of rats infused with either vehicle (control) or ANG II (n = 8). OSR1-P is detected by the same antibody used for SPAK-P. Protein per lane in Table 1. B: relative abundance displayed as individual records with means ± SE. *P < 0.05. #, Tubulin recognized by anti-OSR1 antibody (33).

Fig. 8.

ANG II infusion increases proteolytic cleavage of the ENaC subunits in both cortex and medulla. Immunoblots of αENaC (A), βENaC (B), and γENaC (C) in renal cortex and medulla of rats infused with either vehicle (control) or ANG II (n = 8 each). Protein per lane in Table 1. Relative abundance displayed as individual records with means ± SE. *P < 0.05. FL, full-length; Cleaved, proteolytic cleavage product; #nonspecific band above FL-αENaC (40).

Fig. 9.

Na⁺ transporter regulation along the nephron during ANG II-dependent hypertension. ANG II infusion activates distal nephron Na⁺ transporters (highlighted in red) consistent with increased Na⁺ reabsorption, elevated ECFV, and higher blood pressure. Hypertension provokes pressure-natriuresis to normalize ECFV by decreasing NHE3, medullary NKCC, and regulatory kinase SPAK (highlighted in green). See text for abbreviations.