Regulation of Renal Hemodynamics and Function by RGS2

Abstract

Regulator of G protein signaling 2 (RGS2) controls G protein coupled receptor (GPCR) signaling by acting as a GTPase-activating protein for heterotrimeric G proteins. Certain Rgs2 gene mutations have been linked to human hypertension. Renal RGS2 deficiency is sufficient to cause hypertension in mice; however, the pathological mechanisms are unknown. Here we determined how the loss of RGS2 affects renal function. We examined renal hemodynamics and tubular function by monitoring renal blood flow (RBF), glomerular filtration rate (GFR), epithelial sodium channel (ENaC) expression and localization, and pressure natriuresis in wild type (WT) and RGS2 null (RGS2-/-) mice. Pressure natriuresis was determined by stepwise increases in renal perfusion pressure (RPP) and blood flow, or by systemic blockade of nitric oxide synthase with L-NG-Nitroarginine methyl ester (L-NAME). Baseline GFR was markedly decreased in RGS2-/- mice compared to WT controls (5.0 ± 0.8 vs. 2.5 ± 0.1 μl/min/g body weight, p<0.01). RBF was reduced (35.4 ± 3.6 vs. 29.1 ± 2.1 μl/min/g body weight, p=0.08) while renal vascular resistance (RVR; 2.1 ± 0.2 vs. 3.0 ± 0.2 mmHg/μl/min/g body weight, p<0.01) was elevated in RGS2-/- compared to WT mice. RGS2 deficiency caused decreased sensitivity and magnitude of changes in RVR and RBF after a step increase in RPP. The acute pressure-natriuresis curve was shifted rightward in RGS2-/- relative to WT mice. Sodium excretion rate following increased RPP by L-NAME was markedly decreased in RGS2-/- mice and accompanied by increased translocation of ENaC to the luminal wall. We conclude that RGS2 deficiency impairs renal function and autoregulation by increasing renal vascular resistance and reducing renal blood flow. These changes impair renal sodium handling by favoring sodium retention. The findings provide a new line of evidence for renal dysfunction as a primary cause of hypertension.

Fig 1. Baseline glomerular filtration rate (A, B), mean arterial pressure (MAP; C), renal blood flow (RBF; D), renal vascular resistance (RVR; E), and filtration fraction (F) of wild type (RGS2+/+), RGS2 heterozygous (RGS2+/-), and RGS2 knockout (RGS2-/-) mice.

Values are mean ± SE. *^,**P < 0.05, 0.01 vs. RGS2+/+; ^# P < 0.05 vs. RGS2+/-. BW, body weight.

Fig 2. Structural analysis of renal vasculature in wild type (RGS2+/+) and RGS2 knockout (RGS2-/-) mice.

Sirius red staining for tissue fibrosis in RGS2+/+ (A and B) and RGS2-/- (C and D) kidneys. Arrows indicate renal microvessels and arrowheads indicate glomeruli. E, Quantification of perivascular fibrosis in RGS2+/+ and RGS2-/- kidneys. RGS2-/- renal microvessels showed a 11-fold increase in perivascular fibrosis relative to RGS2+/+ vessels. F, 24-hr proteinuria was similar between RGS2+/+ (n = 7) and RGS2-/- (n = 5) mice. **P < 0.01 vs. RGS2+/+.

Fig 3. Effects of increasing renal perfusion pressure (MAP) on renal blood flow (RBF, A), renal vascular resistance (RVR, B), and renal conductance in wild type (RGS2+/+, n = 7) and RGS2 knockout (RGS2-/-, n = 8) mice.

Increased MAP caused a greater change in RVR and conductance of RGS2+/+ relative to RGS2-/- mice. D, autoregulation index indicating no difference in steady-state autoregulation efficiency between RGS2+/+ and RGS2-/- mice. Values are mean ± SE. **P < 0.01 vs. RGS2+/+ baseline RVR and conductance; n.s., not significant vs. RGS2+/+. BW, body weight.

Fig 4. Time course of renal hemodynamic response to a step increase in perfusion pressure.

Fig A-D show responses of mean arterial pressure (MAP, A), renal blood flow (RBF, B), renal vascular resistance (RVR, C), and renal conductance (D), 10 seconds before and 60 seconds after occluding celiac and superior mesenteric arteries of isoflurane-anesthetized mice. E, a trendline drawn through the first 10 seconds after the beginning of increase in RVR following a step increase in MAP to determine the speed of myogenic response. Each point is the average from several animals (RGS2+/+; n = 6 and RGS2-/-; n = 7) at the time points in each group. The average slope of each trendline shown in the linear equations in E is equal to the mean of slopes from trendlines of all animals (used in panel 3F) in each group. F, scatter plot showing the correlation between baseline MAP vs. the slope of the rapid phase of RVR response to a step increase in renal perfusion pressure of wild type (RGS2+/+) and RGS2 knockout (RGS2-/-) mice. Each point location represents the intersection of the RVR slope and baseline MAP of individual mice in each group. Values are mean ± SE. BW, body weight.

Fig 5. Effect of increasing renal perfusion pressure (MAP) on urine flow rate (A, UV), sodium excretion rate (UV_Na+, B), potassium excretion rate (C), and fractional sodium excretion (D, FENa) in wild type (RGS2+/+, n = 12) and RGS2 knockout (RGS2-/-, n = 8) mice.

Values are mean ± S.E. ^# P < 0.05 vs. RGS2+/+ baseline MAP; **P < 0.01 vs. RGS2+/+ UV and UV _Na+ at equivalent MAP of ~100 mmHg. BW, body weight.

Fig 6. Effects of systemic administration of the non-selective eNOS inhibitor, L-NAME, on renal perfusion pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), glomerular filtration rate (GFR), and sodium (UV_Na+) and potassium (U_K+V) excretion rate in wild type (RGS2+/+, n = 4) and RGS2 knockout (RGS2-/-, n = 5) mice.

Values are mean ± SE. ^{#, ##} P < 0.05, 0.01 vs. corresponding baseline; *^,**P < 0.05, 0.01 vs. RGS2+/+ mice. BW, body weight.

Fig 7. Analysis of renal tubular expression and distribution of sodium/proton exchanger-3 (NHE-3) and epithelial sodium channel (α-ENaC).

A and D show representative images of expression and distribution of NHE-3 in wild type (RGS2+/+) and RGS2 knockout (RGS2-/-) renal tubules. B and E show representative images of α-ENaC expression and distribution. C and F are expanded inserts in B and E, respectively, showing a marked increase in luminal border localization of α-ENaC in RGS2-/- relative to wild type tubules. G, Frequency distribution of α-ENaC punctate size in the luminal border of renal tubules. H, average size of luminal α-ENaC granules. Values are mean ± SE. *P < 0.05 RGS2+/+ vs. RGS2-/- mice.