Meprin Metalloprotease Deficiency Associated with Higher Mortality Rates and More Severe Diabetic Kidney Injury in Mice with STZ-Induced Type 1 Diabetes

Abstract

Meprins are membrane-bound and secreted metalloproteinases consisting of α and/or β subunits that are highly expressed in kidney epithelial cells and are differentially expressed in podocytes and leukocytes (macrophages and monocytes). Several studies have implicated meprins in the progression of diabetic nephropathy (DN) and fibrosis-associated kidney disease. However, the mechanisms by which meprins modulate DN are not understood. To delineate the role of meprins in DN, we subjected meprin αβ knockout (αβKO) mice and their wild-type (WT) counterparts to streptozotocin-induced type 1 diabetes. The 18-week survival rates were significantly lower for diabetic meprin αβKO mice when compared to those for their WT counterparts. There were significant decreases in mRNA and protein levels for both meprin α and β in diabetic WT kidneys. Furthermore, the blood urea nitrogen levels and urine albumin/creatinine ratios increased in diabetic meprin αβKO but not in diabetic WT mice, indicating that meprins may be protective against diabetic kidney injury. The brush border membrane levels of villin, a meprin target, significantly decreased in diabetic WT but not in diabetic meprin αβKO kidneys. In contrast, isoform-specific increases in cytosolic levels of the catalytic subunit of PKA, another meprin target, were demonstrated for both WT and meprin αβKO kidneys.

Figure 1

(a) Messenger RNA levels for meprin α and meprin β. Kidneys from WT mice injected with high-dose STZ were harvested at 12 weeks post-STZ injection and Trizol was used to extract RNA. Real-time PCR was used to amplify meprin α and meprin β mRNA. Data were normalized to β-actin and analyzed using the comparative threshold cycle method. The results are presented as fold expression ± SEM (n = 7) relative to nondiabetic (sodium citrate-injected) control mice. ^∗ p < 0.05, ^∗∗ p < 0.01. (b) Protein levels for meprin A and meprin B in the brush border membrane (BBM) of kidneys from wild-type (WT) mice with STZ-induced type 1 diabetes. Proteins were extracted from WT mice injected with low-dose STZ at 18 weeks post STZ injection, and Western blot analysis used to evaluate meprin protein levels in the BBM-enriched fraction. Diabetic kidneys had a significant decrease in the levels of both meprin A and meprin B proteins (p ≤ 0.05). However, the fold change in protein levels was greater for meprin B (53%) than for meprin A (18%). ^∗ p < 0.05, ^∗∗ p < 0.01.

Figure 2

Survival rates for mice with STZ-induced type 1 diabetes. Low-dose STZ was used to induce type 1 diabetes in 8-week-old male mice. The relative survival rates at 18 weeks post-STZ were normalized to those of nondiabetic control mice in each group. Meprin αβ double-KO mice had a significantly lower 18-week survival rate (40%) when compared to wild-type mice (100%) (p ≤ 0.02). ^∗ p ≤ 0.05.

Figure 3

Blood urea nitrogen (BUN) levels at 12 weeks post-STZ injection. Blood was collected by tail nicking and plasma BUN levels were determined by use of BUN slides that were read on a Vitros DT6011 analyzer (Ortho Clinical Diagnostics, Rochester, NY). BUN levels were significantly higher (p ≤ 0.05) in diabetic meprin αβ double-KO mice when compared to those in nondiabetic controls at 12 weeks post-STZ injection. This difference was not significant in the wild-type mice. ^∗ p ≤ 0.05.

Figure 4

Urine albumin/creatinine ratio. Urine samples were collected onto sterile petri dishes by bladder massage. Albumin levels were determined by ELISA using murine Albuwell ELISA kits (Exocell, Philadelphia, PA). Creatinine levels in the same samples were determined by use of ELISA (Diazyme Laboratories). The 8-week albumin/creatinine ratios were significantly higher in diabetic meprin αβKO mice when compared to those in their diabetic WT counterparts (p ≤ 0.05). ^∗ p ≤ 0.05, ^∗∗ p ≤ 0.01.

Figure 5

The kidney/body weight ratio at 18 weeks post-STZ injection. Kidneys were decapsulated and individually weighed. There was a significant renal hypertrophy in diabetic mice when compared to that in nondiabetic control mice in both genotypes. However, there was no significant difference between meprin αβKO mice and WT mice. ^∗ p ≤ 0.05.

Figure 6

(a) Representative immunoblot for the catalytic subunit of protein kinase A (PKA C) in cytosolic kidney protein fraction; (b) representative immunoblot for p-PKA C in cytosolic-enriched kidney proteins. Proteins were extracted from kidney tissue at 18 weeks post-STZ injection and fractionated into cytosolic- and BBM-enriched fractions using differential centrifugation. Western blot analysis using anti-PKA C and p-PKA C-specific antibodies coupled with optic densitometry were used to quantify the PKA C protein levels. The levels of the 42 kDa PKA C protein isoform (a) were significantly higher in kidneys from diabetic mice, but there were no significant changes in the levels of the 57 kDa isoform. The levels of the 42 kDa p-PKA C increased while the levels of the 57 p-KCA isoform decreased for both genotypes. ^∗ p ≤ 0.05, ^∗∗ p ≤ 0.01.

Figure 7

Localization of meprin B in the juxtamedullary region of the kidney (a), acid Schiff-stained kidney sections (b), and representative immunoblot for villin in BBM-enriched kidney protein fractions from wild-type and meprin αβKO mice at 18 weeks post-STZ (c). There was no noticeable redistribution of meprins from the BBMs to other cell compartments. Significant tubular dilation was evident in the kidneys from diabetic mice when compared to that from nondiabetic controls. There was more tubular dilation in STZ-treated mice when compared to that in nondiabetic vehicle-treated controls. The levels of villin were significantly lower in diabetic WT mice when compared to those in nondiabetic controls (p ≤ 0.05). This decrease was not observed in meprin αβKO mice. ^∗ p ≤ 0.05.