Meprin β mediates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) causes acute kidney injury (AKI) and is associated with high morbidity and mortality rates. Meprins metalloproteases are expressed abundantly in the brush border membranes of proximal kidney tubules and implicated in the progression of AKI. However, the underlying mechanisms are not fully understood. Meprins are capable to proteolytically processing osteopontin (OPN), a highly phosphorylated multifunctional and matricellular glycophosphoprotein. OPN mediates cell signaling pathways involved in apoptosis and extracellular matrix metabolism. The objective of the present study was to determine the effect of meprin β expression on OPN and downstream mediators of the OPN-signaling pathway in IR-induced renal injury. Surgical procedures were used to induce unilateral IR in wild-type (WT) and meprin β knockout (βKO) mice. The mice were sacrificed at 24h post-IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real-time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase-3, Bcl-2, and NFκB. Statistical analysis utilized 2-way ANOVA. Our data show that NGAL levels were increased significantly in the kidney of WT (P=0.0001) and βKO (P=0.01) mice at 24h post-IR when compared to their control counterparts. OPN mRNA increased in both genotypes at 24h post-IR. Western blot analysis detected a full length OPN protein band at 66 kDa, with significant increases (p=0.001) in protein levels in WT kidney at 24h post-IR when compared to levels in control kidneys. This increase in OPN was not observed in meprin βKO mice subjected to IR. Additionally, we detected a cleaved OPN fragment at 32 kDa, for which there were no significant changes in either genotype at 24h IR post-IR. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules and in renal corpuscles. To determine whether meprin β expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in both proximal and distal kidney tubules. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is partly responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti-apoptotic protein in different diseases, therefore we evaluated its impact on caspase-3, Bcl-2, and NFκB. The mRNA levels for caspase-3, Bcl-2 and NFκB decreased in WT but increased in βKO mice at 24h post-IR. Western blot detected higher levels of caspase-3 and Bcl-2 only in WT kidneys. Immunohistochemical staining for caspase-3, Bcl-2 and NFκB also showed higher levels in tubules and renal corpuscle of WT kidneys when compared to βKO kidneys at 24h post-IR. Taken together, our data show that meprin β regulates OPN and down-stream mediators of OPN-signaling pathway in IR-induced AKI.