Blockade of the natriuretic peptide clearance receptor attenuates proteinuria in a mouse model of focal segmental glomerulosclerosis

Abstract

Glomerular podocytes play a key role in proteinuric diseases. Accumulating evidence suggests that cGMP signaling has podocyte protective effects. The major source of cGMP generation in podocytes is natriuretic peptides. The natriuretic peptide clearance receptor (NPRC) binds and degrades natriuretic peptides. As a result, NPRC inhibits natriuretic peptide-induced cGMP generation. To enhance cGMP generation in podocytes, we blocked natriuretic peptide clearance using the specific NPRC ligand ANP(4-23). We then studied the effects of NPRC blockade in both cultured podocytes and in a mouse transgenic (TG) model of focal segmental glomerulosclerosis (FSGS) created in our laboratory. In this model, a single dose of the podocyte toxin puromycin aminonucleoside (PAN) causes robust albuminuria in TG mice, but only mild disease in non-TG animals. We found that natriuretic peptides protected cultured podocytes from PAN-induced apoptosis, and that ANP(4-23) enhanced natriuretic peptide-induced cGMP generation in vivo. PAN-induced heavy proteinuria in vehicle-treated TG mice, and this increase in albuminuria was reduced by treatment with ANP(4-23). Treatment with ANP(4-23) also reduced the number of mice with glomerular injury and enhanced urinary cGMP excretion, but these differences were not statistically significant. Systolic BP was similar in vehicle and ANP(4-23)-treated mice. These data suggest that: 1. Pharmacologic blockade of NPRC may be useful for treating glomerular diseases such as FSGS, and 2. Treatment outcomes might be improved by optimizing NPRC blockade to inhibit natriuretic peptide clearance more effectively.

Keywords: cell signaling; focal segmental glomerulosclerosis; glomerular podocyte; natriuretic peptides.

FIGURE 1

Effect of natriuretic peptides on podocyte cGMP generation and apoptosis. (A) Both ANP (1 µM) and CNP (1 µM) potently stimulated cGMP generation. In contrast, SNAP (100 µM)‐induced little cGMP generation. (b) PAN significantly enhanced apoptosis and this increase in apoptosis was inhibited by ANP and CNP. (c & d) Podocyte express mRNA for NPRC, neprilysin, PDE5, and PDE9. NPRC and neprilysin were highly expressed in cultured podocytes at both the mRNA and protein level. (e) Podocytes were treated with the indicated doses of ANP in the presence or absence of the NPRC ligand ANP(4‐23) (1 µM), the neprilysin inhibitor LBQ657 (10 µM), the PDE5 inhibitor tadalafil (5 µM) or the PDE9 inhibitor PF‐04449613 (5 µM). Pharmacologic blockade of NPRC was the most effective strategy to potentiate natriuretic peptide‐induced cGMP generation. Four to six tissue culture wells were studied in each group. ƒp < 0.01 or ^‡ p < 0.001 versus vehicle by ANOVA followed by Sidak's multiple comparisons post‐test, *p < 0.05 versus PDE5 by Kruskal–Wallis test followed by Dunn's multiple comparisons test, ^§ p < 0.01 versus PAN or ^†† p < 0.001 versus basal by an ANOVA followed by Sidak's multiple comparisons post‐test, **p < 0.01 versus LBQ657 or ^† p < 0.001 versus tadalafil, or PF4449613 by ANOVA followed by Sidak's multiple comparisons post‐test

FIGURE 2

Effect of ANP(4‐23) on urinary cGMP excretion in non‐TG wild type mice. (a) Mice were given a single, IP injection of 10 nmol/kg ANP(4‐23) or vehicle at the beginning of period 1 (11AM). Urine was then collected during periods 1, 2, and 3 at the indicated times. (b) Urinary cGMP excretion was significantly increased in period 2, and the increase in urinary cGMP excretion was sustained in period 3. (c) Urine output increased significantly during period 2 and returned to baseline in period 3. Four non‐TG mice were studied in each group. *p < 0.05 versus vehicle by ANOVA followed by Dunnett's multiple comparisons post‐test. Data in (c) were divided by time (h) to correct for the different durations of the urine collections. Note that the y‐axis of (a) is logarithmic

FIGURE 3

Effect of ANP(4‐23) on kidney disease in TG mice. (a) PAN‐induced heavy proteinuria at day 14 in TG mice treated with vehicle compared to baseline albuminuria. The increase in albuminuria at day 14 was significantly reduced by treatment with ANP(4‐23). (b–f) Treatment with ANP(4‐23) reduced the number of mice with glomerular injury but this difference was not statistically significant. (g) Systolic BP was similar in TG mice treated with vehicle and ANP(4‐23). Ten to 13 TG mice were studied in each group. **p = 0.0002 versus baseline or ^† p = 0.003 versus vehicle‐treated TG mice at day 14 by ANOVA followed by Sidak's multiple comparisons post‐test. Kidney sections were stained with Masson's trichrome stain

FIGURE 4

Effect of sex on the experimental outcomes. (a) Albuminuria was significantly increased in female mice on day 14 and this increase in albuminuria was significantly reduced by treatment with ANP(4‐23). By a two‐way ANOVA, there was a significant effect of treatment with ANP(4‐23) on albuminuria but no significant effect of male or female sex on the experimental outcome. (b and c) There was no significant difference in glomerulosclerosis in male mice compared to female mice in both the vehicle and ANP(4‐23)‐treated groups. In addition, there was no significant difference in glomerulosclerosis by sex between mice treated with ANP(4‐23) versus mice treated with vehicle (compare panels b & c). *p ≤ 0.0126 versus vehicle‐treated female mice or **p = 0.0062 versus basal levels in female mice by two‐way ANOVA followed by Sidak's multiple comparisons post‐test

FIGURE 5

Effect of ANP(4‐23) on glomerular podocytes and kidney fibrosis. (a & b) Expression of nephrin was significantly reduced in the vehicle‐treated TG mice compared to non‐TG mice treated with PAN. ANP(4‐23) significantly inhibited this decrease in nephrin expression. (a & c) Expression of α‐SMA was similarly increased in both groups of TG mice compared to non‐TG controls treated with PAN. (d) A similar nonsignificant increase in fibrosis was observed in the TG mice compared to the non‐TG mice by Sirius red staining in kidney cortex. (e) There were no significant differences in podocytes per glomerular profile in the TG and non‐TG groups. Nine mice were studied in each group for the immunoblotting studies. **p < 0.01 versus non‐TG mice by an ANOVA followed by Sidak's multiple comparisons post‐test; ^† p < 0.001 versus non‐TG mice by a Kruskal–Wallis test followed by Dunn's multiple comparison post‐test

FIGURE 6

Effect of ANP(4‐23) on urinary cGMP excretion in TG and non‐TG mice. There were no significant differences in urinary cGMP excretion at baseline. Treatment with PAN increased urinary cGMP excretion, and this increase in urinary cGMP excretion from baseline was statistically significant in TG mice treated with either vehicle or ANP(4‐23). There was no significant difference in urinary cGMP excretion between TG mice treated with PAN and vehicle compared to TG mice treated with PAN and ANP(4‐23). Seven to 10 mice were studied in each group. ^† p< 0.01 or **p < 0.001 versus baseline in TG mice by an ANOVA followed by Sidak's multiple comparisons post‐test