Kidney Week

Abstract: SA-PO855

Targeted Disruption of CD40 in Human Proximal Tubular Epithelial Cells Significantly Reduces Pro-Inflammatory and Pro-Fibrotic Signaling

Session Information

• Molecular Mechanisms of CKD - III
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

• Zhang, Shungang, University of Toledo, Toledo, Ohio, United States

Shungang Zhang,

• Hinds, Terry, University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States

Terry Hinds,

• Khalaf, Fatimah Kareem, University of Toledo, Toledo, Ohio, United States

Fatimah Kareem Khalaf,

• Lad, Apurva, University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States

Apurva Lad,

• Kleinhenz, Andrew, University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States

Andrew Kleinhenz,

• Malhotra, Deepak K., The University of Toledo, Toledo, Ohio, United States

Deepak K. Malhotra,

• Kennedy, David J., The University of Toledo Health Science Campus, Toledo, Ohio, United States

David J. Kennedy,

• Haller, Steven T., University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States

Steven T. Haller,

Background

We have demonstrated that levels of the TNF-α superfamily member CD40 predict progression of renal dysfunction in patients with ischemic and chronic kidney disease, and that CD40’s soluble ligand (sCD40L) is significantly elevated in these settings. In the two-kidney one clip (2K1C) model of renal ischemia, CD40 expression is significantly elevated in the ischemic renal proximal tubule epithelium, and CD40 knockout (KO) rats show significantly reduced tubular interstitial fibrosis in the ischemic kidney and improved renal function verses controls. To test the hypothesis that activation of CD40 in the proximal tubule epithelium induces a significant pro-inflammatory and pro-fibrotic response contributing to ischemic renal injury we performed the following study.

Methods

Renal ischemia was induced via 2K1C in both Dahl-S wild type and Dahl-S CD40 KO male rats and kidneys were assessed for evidence of inflammation and fibrosis after 4 weeks. CRISPR/Cas9 was used for the creation of a CD40 KO human proximal tubular cell line (HK2/CD40KO) and confirmed by immunoblotting and gene expression. Parent HK2 cells and HK2/CD40KO cells were treated with sCD40L (100 ng/ml) or TNF-α (10 ng/ml) for 24h.

Results

Renal expression of the pro-fibrotic marker plasminogen activator inhibitor-1 (PAI-1) was elevated in Dahl-S rats after 2K1C renal ischemia and this was significantly attenuated in kidneys from Dahl-S CD40KO animals (p<0.05). In HK2 proximal tubular cells, sCD40L induced a significant increase in cytokine gene expression [CXCL2, CXCL5, IL23A, colony stimulating factor-1, and lymphotoxin beta (all > 5 fold increase)] as assessed by quantitative PCR. Importantly, sCD40L treatment also induced a significant increase in monocyte chemoattractant protein-1 (>20 fold increase) and PAI-1 (>2 fold increase) gene compared to TNF-α treatment. The responses to both sCD40L and TNF-α were substantially attenuated in the HK2/CD40KO cells (p<0.05).

Conclusion

Activation of CD40 in the proximal tubule epithelium induces a significant pro-inflammatory and pro-fibrotic response, and represents an attractive therapeutic target for treatment of ischemic renal disease.