Contribution of the basolateral isoform of the Na-K-2Cl- cotransporter (NKCC1/BSC2) to renin secretion

Abstract

Acute administration of loop diuretics like furosemide leads to a stimulation of renin secretion, an effect thought to result from inhibition of Na-K-2Cl cotransporter (NKCC2)-mediated salt transport at the luminal surface of the macula densa (MD). However, loop diuretics also inhibit NKCC1, the second isoform of the Na-K-2Cl cotransporter, with similar potency. In the present study, we examined the influence of furosemide on renin secretion in NKCC1-deficient mice to distinguish between effects of the loop diuretic involving NKCC2 and, by implication, the MD pathway, and effects that might occur via inhibition of NKCC1. Baseline plasma renin concentration (PRC) was 1,212 +/- 211 in NKCC1+/+ (n = 13) and 3,851 +/- 579 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice (n = 14; P = 0.00024). Acute administration of furosemide (50 mg/kg i.p.) increased PRC significantly to 9,324 +/- 1,018 ng ANG I.ml(-1).h(-1) in NKCC1+/+ (n = 13; P < 0.0001 compared with basal) and to 14,188 +/- 2,274 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice [n = 14; P = 0.0002 compared with basal; P = 0.034 compared with wild-type (WT) plus furosemide]. Renin mRNA expression was about threefold higher in NKCC1-/- compared with WT mice. There was considerable recruitment of granular cells to upstream regions of afferent arterioles in NKCC1-/- mice. Patch-clamp studies in single juxtaglomerular granular (JG) cells from WT mice showed an approximately 10% increase in membrane capacitance during incubation with furosemide (10(-4) M), indicating a direct effect of the loop diuretic on renin secretion. No effect of furosemide on membrane capacitance was observed in JG cells from NKCC1-deficient mice. Furosemide (10(-3) M) significantly stimulated renin release from primary cultures of JG cells from WT mice, whereas no response was observed in NKCC1-/- mice. Our data suggest that a functional NKCC1 suppresses basal renin release, at least in part, through a direct effect on JG cells.

Fig. 1

PRC in NKCC1++ (left) and NKCC1−/− mice (right) mice under basal conditions and after acute administration of furosemide (50 mg/kg). Lines connect data from individual animals. Bold symbols and connecting lines represent means.

Fig. 2

Upper: Visualization of renin granules after acid maceration of microdissected renal arterial vessel trees from a NKCC1+/+ (left) and a NKCC1−/− mouse. High contrast regions at the end of the arterioles represent clusters of renin granules (glomeruli are sheared off in the microdissection process). Lower: Count of negative, JG positive and JG positive afferent arterioles with upstream recruitment.

Fig. 3

Basal renin mRNA from kidneys of NKCC1+/+ and −/− mice (n=5 each) determined by real time RT-PCR. Values are given after normalization with β-actin or 18S rRNA, respectively.

Fig. 4

Fractional renin release in preparations of isolated JG cells in primary culture. Basal renin release is compared to the effect of forskolin (10⁻⁵ M) and to that of increasing concentrations of furosemide for JG cells from wild type (upper) and NKCC1-deficient mice (lower). * p<.05 vs. control.

Fig. 5

A: Original recording of membrane capacitance in a single mouse juxtaglomerular cell from NKCC1+/+ (left) and NKCC1−/− mice (right). The pipette contained control internal solution and the cell was bathed in control external solution supplemented with furosemide (10⁻⁴M) at the time indicated by the arrow (see method section). B: Mean relative change of C_m for JG cells from NKCC1+/+ (left) and NKCC1−/− mice (right). C: Whole-cell currents were measured in response to 9 pulses from −150 mV to +90 mV in 30 mV steps for 200 ms from a holding potential of −30 mV. These pulses were applied before and after superfusion with furosemide (10⁻⁴M). Mean steady-state I-V curves from 5 independent experiments before (circle) and after (square) 20 minutes of capacitance measurements are given. * p<.05 vs. control.

Fig. 6

RT-PCR on RNA prepared from single isolated juxtaglomerular granular cells. cDNA from the renal medulla was used as positive control.