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Kidney Week

Abstract: FR-PO966

RIPK3-MLKL Signaling Drives Mitochondrial Dysfunction and Kidney Fibrosis During CKD

Session Information

  • CKD: Pathobiology - I
    November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Srivastava, Anjali, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
  • Tomar, Bhawna, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
  • Mulay, Shrikant R., CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
Background

TGFβ is believed to be a major driver of kidney fibrosis involved in all forms of chronic kidney diseases (CKD). Despite several reports, anti-TGFβ therapies have consistently failed to reduce kidney fibrosis in CKD patients indicating the need for novel therapeutic strategies. Here, we aim to investigate the molecular mechanisms of kidney fibrosis during CKD.

Methods

We performed a series of in-vitro and in-vivo experiments using murine fibroblasts cell lines and an oxalate-induced CKD mouse model, respectively. We used biochemical analysis, RNA sequencing, histology, immunohistochemistry, immunoblotting, etc. techniques to understand the extent of injury, inflammation, and fibrosis in the kidney.

Results

First we analyzed increased expressions of necroptosis signaling molecules (RIPK1, RIPK3, MLKL) and profibrotic markers in both humans and oxalate-induced mice fibrotic kidneys subjected to RNA seq.
Next, using CRISPR/Cas9 we generated stable Ripk3 and Mlkl-deficient murine fibroblasts. Further, we suppressed the expression of RIPK3 and MLKL specifically in kidneys by renal intraparenchymal injections of lentivirus containing CRISPR gRNAs for either RIPK3 or MLKL along with Cas9 protein preceding oxalate-induced CKD. Knockdown of either RIPK3 or MLKL exerted overall renoprotective effects and rmTGFβ-induced fibrotic response.
Moreover, CKD mice were administered either TGFβ signaling inhibitor SIS3 or RIPK1 inhibitor necrostatin-1s, RIPK3 inhibitor dabrafenib with or without SIS3. Similarly, fibroblasts were treated with inhibitors before rmTGFβ exposure. Inhibition of RIPK3 or MLKL also prevented overall fibrotic response.
Interestingly, rmTGFβ exposed RIPK3-MLKL deficient fibroblasts did not induce mitochondrial ROS. Declined mitochondrial genes expression and impaired oxidative phosphorylation was observed in CKD mice kidneys subjected to RNA seq. Also, we observed RIPK3-MLKL translocation on mitochondria isolated from oxalate-induced CKD mice kidneys and rmTGFβ exposed fibroblasts, confirming that mitochondrial RIPK3 and MLKL translocation induce mitochondrial dysfunction.

Conclusion

We show that TGFβ induces translocation of RIPK3-MLKL to mitochondria, where it induces ROS production subsequently leading to Smad2/3 phosphorylation and ECM production. Together, RIPK1, RIPK3, MLKL, and Smad2/3 are molecular targets to inhibit kidney fibrosis during CKD.

Funding

  • Government Support – Non-U.S.