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Abstract: FR-PO798

β2 Adrenergic Signaling Regulates a New OPN-LDLR-βPix-Rac1 Multimolecular Complex in Alport Syndrome

Session Information

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Yousefi, Keyvan, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Chahdi, Ahmed, University of Miami Miler School of Medicine, Miami, Florida, United States
  • John-Williams, Krista, University of Miami Miler School of Medicine, Miami, Florida, United States
  • Lambert, Guerline, University of Miami Miler School of Medicine, Miami, Florida, United States
  • Irion, Camila, University of Miami Miler School of Medicine, Miami, Florida, United States
  • Ramic, Melina, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Andrade, Nadja Suares, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Zeier, Zane, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Freundlich, Michael, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Shehadeh, Lina, University of Miami Miller School of Medicine, Miami, Florida, United States
Background

Alport syndrome is a rare hereditary renal disorder with no cure. Our group showed that Osteopontin (OPN) and low density lipoprotein receptor (LDLR) are highly expressed in the renal tubular epithelial cells (TECs) of the Col4a3-/- Alport mouse and play causative pathological roles. We showed that Alport kidneys had more epithelial to mesenchymal transition (EMT) and cholesterol accumulation in TECs, both phenotypes ameliorated by OPN deficiency. However, the molecular mechanisms involved are unknown.

Methods

Immunoprecipitation and cholesterol influx assays were used in human renal TEC line (HK2) with β2AR agonism/antagonism. Ksp-Cre mosaic analysis with double marker (MADM) mice were used to track EMT in Alport mice. CD44 was measured by immunostaining in Alport Kidneys, and by western blot in HK2 cells treated with OPN monoclonal antibody (mAb) to quantify EMT. PBMCs from Alport patients and healthy donors were used to generate iPSCs which were differentiated into KSP+ve TECs.

Results

In HK2 cells, OPN interacts with LDLR and the Rac1 guanine exchange factor βPix to form a multi-molecular complex controlled by β2AR signaling. Stimulating HK2 cells with adenylate cyclase activator forskolin or β2AR selective agonist salbutamol, decreased cholesterol influx by 15.4% and 17.4%, respectively-p<.0001. Conversely, treating HK2 cells with Rac1 inhibitor NSC23766, PKA inhibitor H89, or selective β2AR antagonist butoxamine significantly increased cholesterol influx -p<.05. OPN mAb blocked the Rac1 inhibitor effect, indicating the role of OPN in this complex. MADM mice showed that EMT in Alport kidneys is of tubular origin as was confirmed by excessive staining of interstitial CD44 (N=3 mice/group-p<.05). OPN mAb caused a 30% decrease in CD44 protein levels (N=3/group-p<.05). iPSC-TECs were functional in the cholesterol assay.

Conclusion

We conclude that elevated OPN in Alport TECs blocks cAMP production and reduces OPN binding to LDLR, thereby freeing LDLR to increase LDL cholesterol influx and drive EMT. OPN knockout or β2AR stimulation increases cAMP-mediated sequestration of LDLR by OPN-βPix complex that blocks LDLR function. Alport patient iPS-TECs may be a useful screening platform to identify β2AR stimulants regulating cholesterol influx.

Funding

  • Other NIH Support