Abstract: TH-PO234
Klotho Reduces Necroptosis by Inhibiting Oxidative Stress Involved in Renal Ischemic-Reperfusion Injury
Session Information
- AKI Basic: Cell Death and Biomarkers
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Acute Kidney Injury
- 001 AKI: Basic
Authors
- Qian, Yingying, Renji Hospital, Shanghai, China
- Gu, Leyi, Renji Hospital, Shanghai, China
- Ni, Zhaohui, Renji Hospital, Shanghai, China
Background
Klotho is mainly expressed in kidney, but its functional relevance with AKI remains largely unclear. Although necroptosis has been suggested as a hallmark of the pathological progress of renal IRI, it is currently unknown what triggers this death mode during I/R induced AKI and whether the protective actions of Klotho have any relationship to necroptosis.
Methods
Male BALB/c mice were grouped as AKI and sham group. AKI model was generated by bilateral renal pedicles clamping. For the therapeutical investigation, recombined Klotho protein or nec-1 was intraperitoneal applied at 30 min or 0 min of reperfusion respectively. Besides, TCMK-1 cell was suffered to hypoxia/reoxygenation or H2O2 insult for pre-determined time. Recombinant Klotho protein or NAC was pre-applied for 1 h. Klotho levels in serum and urine were determined using ELISA. Expression of Klotho, RIP1, RIP3, Il-1 ß, 3-nitrotyrosine and SOD2 were assessed by Western Blot or/and Real-Time PCR. TUNEL staining and immunohistochemistry for Kim-1 and Klotho were performed. The release of LDH was measured to assess the membrane integrity. Urinary 8-OHdG, renal MDA levels and SOD activity were determined to detect oxidative injury.
Results
Klotho levels were decreased in serum and kidney, but increased in urine post-IRI, accompanied by enhancement of oxidative stress and necroptosis. In contrast, Klotho administration ameliorated AKI. Klotho application reduced the expression of RIP1, RIP3, and Il-1 ß, and the number of TUNEL positive cells. In TCMK-1 cell, Klotho downregulated the expression of RIP1 and RIP3, and attenuated the release of LDH induced by H/R or H2O2 insult by dose dependent. These functional effects of Klotho on necroptosis can also be duplicated by NAC application. These indicate a critical role of ROS in triggering necroptosis in tubular epithelial cell, which can be partly abolished by Klotho. In support of this, Klotho exerted an anti-oxidant role evidenced by reducing the levels of u-8-OHdG, renal MDA and 3-Nitrotyrosine, and upregulating SOD2 expression and total SOD activity in AKI mice. Meanwhile, Klotho also abolished the generation of ROS and increased the expression of SOD2 in TCMK-1 cell when exposed to H/R injury.
Conclusion
Klotho protects tubular epithelial cell from I/R-induced necroptosis, which may be attributable to its inhibition of oxidative stress.