Meprin β Regulates Protein Kinase A Mediated TGF‐β Signaling in Ischemia Reperfusion‐Induced Kidney Injury

Abstract

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and is associated with high morbidity and mortality rates. 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 processing the catalytic subunit of protein kinase A (PKA‐C) and several mediators of inflammation. The protein kinase A (PKA) signaling pathway plays an important role in renal fibrosis, promoting the production of transforming growth factor‐β (TGF‐β) and/or TGF‐β‐dependent molecules. The TGF‐β signaling is involved in wound healing and tissue repair of injured kidneys. Activation of Smad2 and Smad3 is the major downstream event of the TGF‐β signaling pathway. The objective of the current study was to determine how meprin β expression impacts PKA C‐mediated TGF‐β signaling in IR‐induced kidney injury. To this end, we used surgical procedures for to achieve unilateral IR with contralateral nephrectomy in wild‐type (WT) and meprin β knockout (βKO) mice. Meprin βKO mice are deficient in the meprin B (β‐β) and the heterodimeric isoform of meprin A (α‐β). The mice were sacrificed at 96 h post‐IR and kidney tissues obtained for analysis. Real‐time PCR, and immunohistochemical staining, were used to evaluate the expression levels of PKA C and TGF‐β. Statistical analysis utilized 2‐way ANOVA. Real‐time PCR data showed significant increases in mRNA levels of PKA C only in βKO kidneys after IR (p< 0.05). In contrast, the mRNA levels for TGF‐β were higher in both WT and βKO at 96 h post‐IR (p< 0.01). Interestingly, the baseline mRNA levels for TGF‐β were higher in meprin βKO mice (p< 0.001). Immunohistochemical staining for PKA C and TGF‐β was quantified using Image J software. The data showed significant increases in the levels of PKA C proteins in select kidney tubules of both genotypes (p≤ 0.001) after IR. Similarly, staining intensity for TGF‐β were higher in select kidney tubules of both WT (p=0.01) and meprin β deficient mice at 96 h IR (p=0.001). Immunofluorescence counterstaining of kidney tissues with anti‐meprin β, anti‐PKA C and anti‐TGF‐β antibodies showed a positive correlation between meprin β expression and tubular PKA C and TGF‐β levels in kidneys subjected to IR. These findings suggest that meprin β regulates PKA C‐mediated TGF‐β signaling pathway in IR‐induced kidney injury and provides new insights on the mechanisms underlying meprin β modulation of the pathophysiology of IR‐induced renal injury.