(665.6) The activation status of integrin β1 determines the ACE-2 expression and functions in renal epithelial and cancer cells

Poster Board Number: A304

Md Saimon Mia (North Dakota State University), Hum Shrestha (North Dakota State University), Riya Palamuttam (Vanderbilt University Medical Center), Bony Kumar (Yale School of Medicine), Sijo Mathew (North Dakota State University)

Background: Integrins are heterodimeric cell surface receptors that participate in intracellular signaling and regulate multiple cellular functions. These membrane receptors are also crucial for the organ development, structural maintenance, and disease pathologies of the kidney. Integrin β1 is the major integrin subunit present in the kidney. Alteration of integrin β1 expression is a characteristic of many kidney diseases such as kidney cancer and chronic kidney disease. The purpose of this study is to find the importance of integrin β1 in the expression and function of angiotensin-converting enzyme 2 (ACE-2) in the kidney. We hypothesized that the expression and function of ACE-2 depend on the activation status of integrin β1 in the renal epithelial and cancer cells.

Method: Kidney epithelial-specific integrin β1 KO mice were generated by crossing between ITGBfl/fl and γGT-Cre mice. PCR-based analysis of genomic DNA was used to confirm the gene deletion. Expression of integrin β1 in the kidney cortex was monitored by qRT-PCR.  Immunoblotting and qRT-PCR were performed to observe protein expression and mRNA level of ACE-2 in mouse kidneys. The pharmacological compound, BTT 3033, was used to inhibit integrin β1 in HK-2 and Caki-1 cells. Integrin β1 activation status was monitored by immunocytochemistry using a conformation-specific antibody. SARS-CoV-2 spike protein was used to find out the functional importance of the integrin β1-ACE-2 complex. Statistical significance of the observed data was carried out using a two-tail t-test.

Results: Deletion of integrin β1 from the renal epithelial cells reduced expression of ACE-2 in the mice kidneys (n=3; plt;0.05). Immunoblotting and qRT-PCR showed, expression of integrin β1 (n=3; plt;0.05) and ACE-2 (n=3; plt;0.05) were higher in renal cancer cells than renal epithelial cells indicating a positive correlation in their expression patterns.  With the treatment of BTT 3033, there was a significant decrease in the activation status of integrin β1 in kidney epithelial (HK-) and cancer (Caki-1) cells (n=30; plt;0.05). ACE-2 mRNA levels decreased in HK-2 and Caki-1 cells when treated with an integrin β1 antagonist, BTT 3033 (n=3; plt;0.05). Treatment of HK-2 and Caki-1 with BTT 3033 also decreased the internalization of the SARS-CoV-2 spike protein.

Conclusion: Results from this study demonstrate the importance of matrix proteins receptor integrin β1 in maintaining the ACE-2 expression and function in the kidney. This study will have implications in various chronic diseases where significant changes in the composition of matrix proteins and integrin expressions are observed. Further studies are needed to understand the role of altered matrix composition in ACE-2 mediated physiological functions.

American Heart Association Scientist Development Grant (16SDG29740001) and DaCCoTA CTR pilot and feasibility grant supported by NIGMS U54GM128729