Deficiency of Hedgehog Interacting Protein (Hhip) in Endothelial Cells Prevents Kidney Ischemia-Reperfusion-Mediated Renal Tubular Cell Senescence

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Background: Acute kidney injury (AKI) caused by renal ischemia/reperfusion (R I/R) injury is a major clinical issue lacking viable therapies, and with incompletely delineated underlying mechanisms. Given the important roles of hedgehog interacting protein (Hhip) in glomerular endothelial cells (ECs), we asked whether Hhip knockout (KO) in ECs prevents R I/R-induced kidney injury in vivo and in vitro. Methods: Both male HhipEC KO and control Hhipfl/fl mice at 10-weeks of age were subjected to 45 minutes of unilateral R I/R or sham surgery along with contralateral nephrectomy. The time course of AKI was followed at Days 1, 3 and 7. We also studied naïve renal proximal tubular cells (IRPTCs) exposed to the conditioned media harvested from mouse endothelial cell (mECs) with or without Hhip (siRNA) ± cisplatin (20µM) treatment. Results: Compared to the respective sham controls, R I/R mice (HhipEC KO and Hhipfl/fl) lost body weight starting from Day 1 to Day 7. In line with the AKI time course, Hhipfl/fl-R I/R mice had higher mortality with significant kidney apoptosis documented by IVISense™ Annexin-V 750 fluorescent imaging probe; increased urinary albumin-to-creatinine ratio; and displayed renal dysmorphology (PAS, Masson’s trichrome and Sirius red) with enhanced Cystatin C- and kidney injury molecule-1 immunoexpression in their kidneys. Notably, renal tubular cell senescence (SA-β-galactosidase, p21, p16, MCP-1 staining) was significantly increased in Hhipfl/fl-R I/R mice. In contrast, those changes were significantly ameliorated in the kidneys of HhipEC KO -R I/R mice. In vitro, naïve IRPTCs exposed to the conditioned media harvested from mECs treated with cisplatin exhibited higher cellular senescent activity with enlargment of nuclear size with both mono- or multi-nuclei and increased cytoskeleton destabilization as evidenced by dysmorphic patterns of α-tubulin and F-actin staining. Those changes were largely prevented in naïve IRPTCs exposed to the conditioned media from Hhip KO mECs treated with cisplatin. Conclusion: Our data suggest that Hhip KO in ECs prevented or ameliorated AKI-induced renal tubular injury. This potential protection may be mediated, at least in part, by the inhibition of Hhip-mediated tubular cell senescence.

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