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Abstract: TH-PO1061

β1 Integrin Activation and Signaling in Endothelium-Initiated Podocyte Injury

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Cara-Fuentes, Gabriel M., University of Michigan, Ann Arbor, Michigan, United States
  • Venkatareddy, Madhusudan M., University of Michigan, Ann Arbor, Michigan, United States
  • Verma, Rakesh, University of Michigan, Ann Arbor, Michigan, United States
  • Garg, Puneet, University of Michigan, Ann Arbor, Michigan, United States
Background

Integrins are heterodimeric transmembrane proteins that anchor cells to the extracellular matrix but also play a key role as bi-directional (outside-in and inside-out) signaling mediators. Our aim is to determine the activation state and role of podocyte β1 integrins in a transient model of podocyte injury.

Methods

8-10 week-old mice were injected intraperitoneally with LPS (10 µg/g) or PBS (control) and urine collected prior to and after injection. In another set of experiments, mice were injected with a β1 integrin “blocking” antibody (HMβ, 2.5 µg/g) 20 hours prior to LPS. Albuminuria was measured by ELISA. Mouse kidney tissue was processed for immunohistochemistry. Glomerular isolation was performed for western blotting and RT-PCR.

Results

LPS injected mice had significantly higher albuminuria than controls. In podocytes, β1 integrin activation, FAK and nephrin phosphorylation occurred 18 to 32 hours after LPS injection. Pre-treatment with HMβ1 antibody significantly reduced albuminuria 24 h following LPS, suggesting that activation of podocyte β1 integrin plays a role in albuminuria. Immunofluorescence confirmed binding of HMβ1 to glomerular endothelial cells and podocytes, suggesting a possible crosstalk between these cells. Since β1 integrin activation can occur due to outside-in and inside-out signaling, we investigated the chronology of events involving endothelial, glomerular basement membrane (GBM) and podocytes. By immunohistochemistry, western blotting and RT-PCR, we found that endothelial and GBM injury preceded β1 integrin activation, nephrin phosphorylation and foot process effacement, suggesting an outside-in β1 integrin activation.

Conclusion

LPS activates β1 integrin on podocytes and leads to FAK and nephrin phosphorylation in vivo. Targeting endothelia/podocyte β1 integrin reduces albuminuria. Changes in glomerular endothelial cells and GBM precede podocyte injury, suggesting that activation of podocyte β1 integrin may be triggered by an outside rather than inside signal in this model of podocyte injury.