Meprin β Activity Modulates IL‐6/JAK/STAT Signaling Pathway in Ischemia/Reperfusion Induced Acute Kidney Injury

Abstract

Meprin metalloproteases have been implicated in inflammation-induce kidney injury. Meprins proteolytically processes the pro-inflammatory cytokine, IL-6, causing IL-6 inactivation. However, it's not known how IL-6 inactivation by meprin β impact the inflammatory response in kidneys. The goal of this study was to determine whether meprin β expression modulates IL-6 signaling in ischemia/reperfusion (IR)-induced kidney injury. IL-6 trans-signaling is initiated when IL-6 binds to its soluble receptor (sIL-6r) and gp130 receptor, resulting in activation of Janus Kinase-Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway and activation of its mediators (e.g. suppressor of cytokine signaling 3, SOCS3) and downstream target genes (e.g. pro-apoptosis, anti-apoptosis and proliferation). We therefore sought to determine if meprin β expression impacts IL-6 levels and downstream mediators of the IL-6 trans-signaling mediated JAK-STAT pathway. 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 24h 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 two-way ANOVA. Our PCR data showed that meprin β deficiency associated with a significant upregulation of IL-6 mRNA (40%) in βKO mice at 24 h post-IR compared to WT. Similarly, Western blot data showed increased IL-6 protein levels. We then evaluated downstream mediators of the IL-6 signaling pathway and showed that WT kidneys which express meprin β had a 50% and 90% upregulation in the expression of the JAK/STAT negative regulator gene (SOCS3) and proliferating cell nuclear antigen (PCNA) gene, respectively when compared to βKO. However, meprin β expression did not affect the expression of the pro-apoptotic gene (BCL-2) or the anti-apoptotic gene (caspase3) at 24 h post-IR. Western Blot data showed increased levels of P-JAK2, P-STAT3, Caspase3, BCL2, SOCS3 and PCNA in βKO at 24 h post-IR compared to controls, but not in WT counterparts. Immunohistochemical analysis showed significant increases (P<0.001) in percentage of cells expressing IL-6, P-JAK2, P-STAT3, Caspase3 and BCL2 in some tubules in both genotypes subjected to IR when compared to their controls (OD levels >1.0). However, βKO kidneys had significantly higher expression level of IL-6, P-JAK2, P-STAT3, Caspase3 and BCL2 total proteins compared to WT 24h post-IR (P<0.001). Data from immunofluorescence counterstaining of kidney tissues showed that the levels of IL-6, P-STAT3, P-JAK-2, Caspase3 and BCL-2 were higher in meprin β-expressing proximal tubules (PT), at 24 h post-IR when compared to the distal kidney tubules (DT), which lack meprins. High level of IL-6, P-STAT3, P-JAK-2, Caspase3 and BCL-2 was also detected 24 post IR in the lumen of PT and DT from WT and βKO kidneys, suggesting increased release into filtrate and subsequently into urine. Taken together, our data demonstrates that meprin β expression modulates the IL-6/JAK/STAT signaling pathway in renal IR-induced inflammation.