Plasminogen Deficiency and Amiloride Mitigate Angiotensin II-Induced Hypertension in Type 1 Diabetic Mice Suggesting Effects Through the Epithelial Sodium Channel

Abstract

Background Diabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro. Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus. Methods and Results Male plasminogen knockout (plasminogen-deficient [Plg^-/-]) and wild-type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30-60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild-type, diabetic ANGII-treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24-hour urine albumin and plasminogen excretion. Diabetic Plg^-/- mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild-type, diabetic wild-type, and Plg^-/- control mice, ANGII did not change blood pressure in diabetic Plg^-/- mice. Compared with ANGII infusion alone, wild-type ANGII-infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild-type mice evoked larger amiloride-sensitive current than urine from Plg^-/- mice with or without diabetes mellitus. Full-length γ-ENaC and α-ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa γ-ENaC cleavage product was increased in diabetic versus nondiabetic mice. Conclusions Plasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.

Keywords: albuminuria; aldosterone; protease; renin; sodium channels.

Figure 1. Effect of streptozotocin (STZ) treatment on plasma glucose and urinary excretion of albumin and plasminogen in wild‐type (WT) and plasminogen deficient (Plg^−/−) mice.

A, Diabetic wildtypes (WT STZ, n=10) and Plg^−/− (KO STZ, n=7) mice displayed a progressive increase in plasma glucose over 4 weeks, whereas nondiabetic controls (WT VEH, n=14; KO VEH, n=4) continued to be at the same level as the baseline. B, The 24‐four hour urine albumin excretion was measured 3 weeks after streptozotocin treatment. Urine albumin was significantly elevated by streptozotocin treatment compared with vehicle in both genotypes (WT STZ, n=8, and KO STZ, n=5; WT VEH, n=10, and KO VEH, n=6). Albuminuria was significantly higher in KO VEH compared with WT VEH. **P<0.01 ****P<0.0001, Mann–Whitney. C, Immunoblotting demonstrated plasminogen in plasma and 24‐hour urine from diabetic WT (WT STZ) mice as a band migrating at ≈90 to 100 kDa, which was absent in urine from Plg^−/− (KO) mice. In the bottom blot, the proteins migrated a little slower at the right margin, which caused the apparent different molecular weight.

Figure 2. Effect of streptozotocin‐induced diabetes mellitus (STZ) and angiotensin II (ANGII) infusion on arterial blood pressure in wild‐type (WT) and plasminogen‐deficient (Plg^−/−) mice.

A,Traces show mean arterial blood pressure at baseline (day [D] and night [N] 1–2) and in response to ANGII‐infused intravenously for 8 days in diabetic WT (WT STZ, n=7) and knockout mice (KO STZ, n=6) and control WT (WT VEH, n=12) and knockout mice (KO VEH, n=8). B, ANGII increased mean arterial blood pressure significantly in WT vehicle, WT streptozotocin, and knockout vehicle. The response was abolished in knockout streptozotocin, and mean arterial blood pressure was significantly lower in knockout streptozotocin compared with WT streptozotocin and knockout vehicle. Baseline corresponded to mean of days 1 to 2 and ANGII to mean of days 6 to 9 (when the response in mean arterial blood pressure to ANGII reached a plateau) in (A). *P<0.05, **P<0.01 (compared between groups), ***P<0.001 (compared within or between groups), ****P<0.0001. D1=day 1 (6 am to 6 pm), N1=night 1, etc.

Figure 3. Effect of amiloride infusion on blood pressure in angiotensin II (ANGII)–infused wild‐type (WT) mice with diabetes mellitus (STZ).

A, Traces show mean arterial blood pressure in diabetic WT mice at baseline (day [D] and night [N] 1–2), in response to ANGII infused intravenously for 7 days (D and N 3–9) with (WT STZ amiloride, n=8) or without (WT STZ controls, n=7) amiloride (Amil) for 4 days (D and N 6–9). Mean arterial blood pressure was significantly lower in WT streptozotocin Amil from N7 when evaluated by repeated‐measures analysis of variance. B, ANGII increased mean arterial blood pressure significantly and similarly in both groups before Amil intervention. Following Amil treatment, mean arterial blood pressure decreased and was no longer different from baseline. Baseline corresponded to days 1 to 2, ANGII to days 4 to 5, and with Amil (ANGII w Amil) or controls without (ANGII w/o Amil) corresponded to days 8 to 9. **P<0.01, ***P<0.001, ****P<0.0001. D1=day 1 (6 am to 6 pm), N1=night 1, etc.

Figure 4. Effect of proteolytic activity in urine from diabetic and plasminogen‐deficient (Plg^−/−) mice on inward current in M1 cells and abundance of γ‐epithelial sodium channel (ENaC) and α‐ENaC subunits in kidney tissue.

A, Patch‐clamp current trace of M1 cell before (gray line) and after (black line) addition of 24‐hour urine aliquot from a diabetic wild‐type (WT) mouse. B, Urine from aging WT vehicle mice (n=5 in all groups) significantly stimulated inward current. Pretreatment with amiloride (Amil, 2 μmol/L) or α₂‐antiplasmin (AP, 1 μmol/L) abolished inward current evoked by urine. The response with urine from knockout vehicle mice was impaired compared with WT mice. ****P<0.0001. C, 24‐hour urine from WT streptozotocin mice stimulated significantly Amil‐sensitive inward current relative to knockout streptozotocin (n=5 in all groups). Urine samples from streptozotocin animals were dilute as a result of significant 5 to 6 times greater diuresis (Table S1). **P<0.01. (D‐H) Immunoblot analysis of kidney homogenates from the vehicle (VEH) and diabetic (STZ) treated wild‐type (WT) and Plg‐/‐ (KO) mice for ϒ‐ENaC (D) and α‐ENaC (H). SIze markers are shown in kilo Daltons (kDa). Concentration‐response with an increasing amount of homogenate protein shows a linear relation with densitometry for γ‐ENaC (D) and α‐ENaC (H). The predicted sizes of full‐length glycosylated proteins are 85‐90 kDa. E shows a representative blot for γ‐ENaC and F‐G shows the densitometric evaluation of full length protein migrating at 80‐85 kDa and proteolytic product at 65‐70 kDa. ∗ P≤0.05, n=10 (WT‐STZ), n=8 (KO VEH), n= 6 (KO‐STZ).

Figure 5. Effect of diabetes mellitus and plasminogen deletion on plasma renin, angiotensin II (ANGII), and aldosterone concentrations and kidney PRR (prorenin receptor) abundance in ANGII‐infused mice.

A, Renin, (B) ANGII, and (C) aldosterone levels were measured in plasma drawn from resting conscious mice after 8 days of ANGII infusion in diabetic wild‐type (WT STZ) and knockout mice (KO STZ) and control WT (WT VEH) and knockout mice (KO VEH). D, Immunoblot; E, densitometry analysis of the PRR protein abundance in kidney tissue homogenate. There were no differences between groups.