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

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Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and associated with high morbidity and mortality rate. The underlying mechanisms involved in IR-induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically process several mediators of cell signaling pathways mediators involved in apoptosis and extracellular matrix (ECM) metabolism such as osteopontin (OPN). The objective of the current study was to determine how meprin β expression affects OPN and downstream mediators of the OPN-signaling pathway. Surgical procedures were used to induce unilateral IR with contralateral nephrectomy in wild-type (WT) and meprin β knockout (βKO) mice which are deficient in the meprin B (β-β) and the heteromeric isoform of meprin A (α-β). The mice were sacrificed at 24 h 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 NGAL level were elevated significantly in WT (P=0.0001) and βKO (P=0.01) at 24 h post-IR compare to their control counterparts. However, the fold change was higher in WT when compared to βKO mice indicating that meprin β enhances kidney injury. OPN mRNA increased in both genotypes at 24 h post-IR. In contrast, OPN protein levels only increased in WT kidneys. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules. To determine whether meprins 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 distal tubules (DTs), which do not express meprins. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti-apoptotic molecules 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 24 h post-IR. Immunohistochemical staining for caspase-3, Bcl-2 and NFκB also showed high staining levels in tubules of WT kidney compared to βKO at 24 h post-IR. These findings suggest that meprin β expression has a direct impact on the levels of OPN in the kidney and affects apoptotic genes regulated by the OPN signaling pathway.