Combining Neprilysin Inhibitor With AT2R Agonist Is Superior to Combination With AT1R Blocker in Providing Reno-Protection in Obese Rats

Abstract

Clinical use of the combination therapy of the neprilysin inhibitor sacubitril and angiotensin II type 1 receptor blocker valsartan is known to be associated with albuminuria. Albuminuria is both a risk factor for and an indicator of kidney injury. Earlier work from our laboratory reported that the agonist of angiotensin II type 2 receptor Compound 21 (C21) prevents proteinuria, albuminuria, and is reno-protective in obese Zucker rats fed high salt diet (HSD). Thus, we hypothesized that sacubitril/C21 combination provides superior reno-protection compared to sacubitril/valsartan. Male obese Zucker rats 10-11 weeks old were treated daily with vehicle, sacubitril + C21, or sacubitril + valsartan while fed HSD for 16 days. HSD-feeding caused kidney dysfunction, evident by significant increases in urinary protein, osteopontin, and cystatin C. HSD-feeding lowered plasma cystatin C and creatinine concentrations suggestive of hyperfiltration, which was not affected by either treatment. Unlike sacubitril/valsartan, sacubitril/C21 treatment significantly decreases proteinuria, albuminuria, the expression of nephrin, and kidney weight, independent of hyperfiltration, compared with HSD alone. Moreover, sacubitril/valsartan therapy increased plasma renin and did not prevent HSD-induced increases in renal angiotensin II, while sacubitril/C21 completely prevented these changes. Together, this study suggests that sacubitril/C21 afforded superior reno-protection compared to sacubitril/valsartan therapy in high salt-fed obese Zucker rats.

Keywords: angiotensin II type 2 receptor agonist; angiotensin receptor blocker; neprilysin inhibition; obesity; proteinuria; reno-protection.

FIGURE 1

(A) kidney weight, (B) urinary protein normalized to 24 h urine volume, (C) urinary albumin normalized to 24 h urine volume in obese Zucker rats on NSD or HSD and treated with vehicle, SAC/C21, or SAC/VAL. Data shown as mean ± SEM, analyzed by one-way ANOVA, followed by Fisher’s LSD test. Results are considered significant at *p < 0.05, with a 95% confidence interval; n = 8. NSD—normal salt diet fed + vehicle treated obese control, HSD—high salt diet fed + vehicle treated obese control, SAC/C21—high salt diet fed obese rat treated with sacubitril + C21, SAC/VAL—high salt diet fed obese rat treated with sacubitril + valsartan.

FIGURE 2

Analysis of renal injury via markers and indicators of glomerular and tubular damage (A) urinary osteopontin normalized to 24 h urine volume, (B) urinary cystatin C normalized to 24 h urine volume, (C) urinary creatinine, (D) plasma cystatin C, (E) plasma creatinine, (F) eGFR, (G) nephrin expression in kidney cortex, (H) podocin expression in kidney cortex, and (I) megalin expression in kidney cortex from obese Zucker rats on NSD or HSD and treated with vehicle, SAC/C21, or SAC/VAL. Data shown as mean ± SEM, analyzed by one-way ANOVA, followed by Fisher’s LSD test. Results are considered significant at *p < 0.05, **p < 0.01, ****p < 0.0001 with a 95% confidence interval; n = 6–8 for urinary osteopontin, plasma creatinine, and eGFR and n = 8 for urinary and plasma cystatin C, urinary creatinine, nephrin, podocin, and megalin expression. NSD—normal salt diet fed + vehicle treated obese control, HSD—high salt diet fed + vehicle treated obese control, SAC/C21—high salt diet fed obese rat treated with sacubitril + C21, SAC/VAL—high salt diet fed obese rat treated with sacubitril + valsartan.

FIGURE 3

(A) plasma ANP, (B) Neprilysin activity in kidney cortex, (C) Neprilysin expression in kidney cortex, (D) NPR-C expression in kidney cortex, (E) NPR-C expression in white adipose tissue, (F) plasma bradykinin, (G) ACE2 expression in kidney cortex, and (H) ACE2 activity in kidney cortex from obese Zucker rats on NSD or HSD and treated with vehicle, SAC/C21, or SAC/VAL. Data shown as mean ± SEM, analyzed by one-way ANOVA, followed by Fisher’s LSD test. Results are considered significant at *p < 0.05, with a 95% confidence interval; n = 6–8 for neprilysin activity and renal NPR-C and ACE2 expression and n = 8 for plasma ANP and bradykinin, expression of renal neprilysin and adipose NPR-C, and renal ACE2 activity. NSD—normal salt diet fed + vehicle treated obese control, HSD—high salt diet fed + vehicle treated obese control, SAC/C21—high salt diet fed obese rat treated with sacubitril + C21, SAC/VAL—high salt diet fed obese rat treated with sacubitril + valsartan.

FIGURE 4

Angiotensin peptides, receptors and downstream signaling markers in obese Zucker rats on NSD or HSD and treated with vehicle, SAC/C21, or SAC/VAL. (A) cortical Ang II, (B) renin activity in kidney cortex, (C) plasma renin, (D) AT₁R expression in kidney cortex, (E) AT₂R expression in kidney cortex from obese Zucker rats on NSD or HSD and treated with vehicle, SAC/C21, or SAC/VAL. Data shown as mean ± SEM, analyzed by one-way ANOVA, followed by Fisher’s LSD test. Results are considered significant at *p < 0.05, with a 95% confidence interval; n = 6–8 for Ang II, plasma renin and AT₂R expression and n = 8 renin activity and AT₁R expression. NSD—normal salt diet fed + vehicle treated obese control, HSD—high salt diet fed + vehicle treated obese control, SAC/C21—high salt diet fed obese rat treated with sacubitril + C21, SAC/VAL—high salt diet fed obese rat treated with sacubitril + valsartan.