Meprin β meidates Apoptosis/Survival Through The IL‐6‐Mediated JAK/STAT Signaling Pathway In Ischemia/Reperfusion‐Induced Kidney Injury

Abstract

Meprin metalloproteinases have been implicated in the pathophysiology of IR‐induced kidney injury. Existing in vitro data show that meprins could modulate interleukin‐6 (IL‐6)‐mediated inflammation via proteolytic processing of IL‐6 and its receptor. However, it is not known whether meprin β cleaves IL‐6 in vivo, and how inactivation of IL‐6 modulates downstream mediators of the IL‐6 signaling pathway in kidney tissue. The goal of the current study was to determine how meprin β expression affects IL‐6 and its correlation to kidney injury and cellular apoptosis/survival in mice kidneys subjected to IR. We used the unilateral IR as a model of renal inflammation in wild‐type (WT) and meprin β knockout (βKO) male mice, with the contralateral kidneys serving as controls. The mice were sacrificed at 24 h post‐IR, and kidney tissue processed for evaluation by RT‐PCR, western blot, and immunohistochemistry (IHC). IHC staining for kidney injury molecule 1 (KIM‐1) was used to assess kidney injury. Statistical analysis utilized 2‐way ANOVA. Real‐time PCR data showed a significant increase (P ≤ 0.0001) in mRNA levels for IL‐6, Caspase3, and BCL‐2 in both WT and βKO mice subjected to IR when compared to counterpart control kidneys. Western blot data showed that protein levels for IL‐6, P‐STAT3β, Caspase3, and BCL‐2 significantly increased in βKO kidneys (P ≤ 0.0001) but not in WT counterparts at 24 h post‐IR, suggesting meprin β‐mediated decreases in IL‐6, Caspase3 and BCL‐2. Interestingly, immunohistochemical staining of kidney sections for IL‐6, P‐JAK2 and P‐STAT3, Caspase3 and BCL‐2 showed significant increases only in select kidney tubules for both genotypes. In the renal corpuscles, P‐JAK2 levels significantly increased only in the βKO (P ≤ 0.0001) and not in WT kidneys at 24h post‐IR. In contrast, BCL‐2 expression in the renal corpuscle was significantly higher in WT kidneys subjected to IR compared to control kidneys (P ≤ 0.0001) but not in βKO mice. To assess the correlations in localization of IL‐6 expression and the kidney injury and cellular apoptosis in kidney tubules, we used immunofluorescence counterstaining of IL‐6 with either kidney injury biomarker, KIM‐1, or cellular apoptosis biomarker, Caspase 3. Our data showed that IL‐6 expression was positively associated with both KIM‐1 and Caspase 3, and thus kidney injury and cellular apoptosis in several tubules in both genotypes subjected to IR. Data from immunofluorescence counterstaining of kidney tissues also showed that the levels of IL‐6, Caspase3 and BCL‐2 were higher in meprin β‐expressing proximal tubules (PTs), at 24 h post‐IR when compared to the distal kidney tubules (DTs), which lack meprins. High levels of IL‐6, Caspase3 and BCL‐2 were also detected at 24 h post‐IR in the lumen of PTs and DTs from WT and βKO kidneys, suggesting increased release into filtrate and subsequently into urine. However, P‐JAK2 and P‐STAT3 expression were high in PTs of both genotypes with accumulation in the lumen of PTs in βKO kidneys only at 24 h post‐IR. In conclusion, data showed that Meprin β expression modulates IR‐induced kidney injury and cellular apoptosis/survival in part through IL6‐ mediated JAK/STAT signaling.