Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade

Abstract

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.

Figure 1. Significant reduction of albuminuria by combination therapy: Final kidney parameters in diabetic eNOS knockout mice treated with riociguat (3 mg/kg/d), telmisartan (1 mg/kg/d), both (3 mg/kg/d and 1 mg/kg/d) or vehicle, and non-diabetic eNOS knockout mice treated with vehicle, respectively.

Urinary albumin-creatinine ratio (A), percentage reduction in urinary albumin excretion per day compared with diabetic controls (B) and cystatin C in Plasma (C). Values are given as means ± SEM. For comparisons, student's t test and Mann-Whitney u test, respectively, were used. ^* p<0.05 vs. diabetic vehicle; ^# p<0.05 vs. non-diabetic vehicle.

Figure 2. Final plasma parameters in treatment groups (riociguat, telmisartan, combination of both), diabetic and non-diabetic eNOS knockout mice.

Values are given as means ± SEM. For comparisons, student's t test and Mann-Whitney u test, respectively, were used. ^* p<0.05 vs. diabetic vehicle. Abbreviations used: MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-alpha.

Figure 3. Significant reduction of MDA- positive renal tissue by combination therapy: Immunhistochemical detection of MDA in non-diabetic (A) and diabetic (B) untreated eNOS knockout mice.

Percent volume of MDA-positive area in renal cortex (F). Values are given as means ± SEM. For comparisons, student's t test was used. ^** p<0.01; ^***p<0.001 vs. diabetic vehicle and ^#p<0.05; ^##p<0.01 vs. non-diabetic vehicle. Abbreviations used: MDA, malondialdehyde.