Abstract: FR-PO123
TGFβR1 Activation in Tubular Epithelial Cell Recruits Leukocytes That Deliver Lethal Hit in a Model of AKI
Session Information
- AKI: Mechanisms - II
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Lassen, Emelie, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Yu, Liping, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Daehn, Ilse S., Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background
Activation of the TGFβ signaling pathway plays an important role in both acute kidney injury (AKI) and chronic kidney disease pathogenesis. We have previously shown that ligand-independent activation of TGFβR1 in proximal tubules (doxycycline inducible transgenic Pax8Tgfbr1) results in rapid epithelial cell injury and death, as well as immune cell infiltration. This study aims to determine the drivers and mechanism of epithelial cell injury in this novel model of TGFβR1 mediated AKI.
Methods
To determine the mechanisms involved in AKI observed in transgenic Pax8Tgfbr1 mice, we isolated and immortalized proximal tubular epithelial cells (mPTECs) from Pax8Tgfbr1 mice with hTERT and treated with doxycycline. Characterization was done by IF for proximal tubule markers (AQP1, APN) and by RT-PCR. Splenocytes were isolated by sieving spleens from single transgenic mice of the same background (FVB). Cell viability was assessed by MTT, cell death by Annexin V/PI (FACS) and by cleaved caspase 3 (IF). Chemokines and cytokines released by mPTECs were determined by Luminex.
Results
We confirmed activation of TGFβR1 and the canonical TGFβ pathway in doxycycline treated mPTECs. To our surprise, TGFβR1 activation in these cells caused mild cell damage that did not reflect the dramatic phenotype observed in the mouse. This suggested to us that additional mechanisms could be at play in driving the severe tubular injury phenotype. We examined the secretome of mPTECs -/+ doxycycline to determine whether these cells could induce infiltration of proinflammatory cells that could potentiate injury. TGFβR1 activation induced the release of GM-CSF, LIF and MCP-1, suggesting recruitment and activation of infiltrate. Interestingly, an observed increase in VEGF by activated mPTECs could be involved in promoting vascular permeability. We next performed co-cultures with mixed allogenic leukocytes, and after 24hrs cell commitment to death was initiated by cleaved caspase 3, resulting in significant cell death of the mPTECs.
Conclusion
TGFβR1 in mPTECs promotes the recruitment of leukocytes by the secretion of key chemokines and facilitating vascular permeability. Activated leukocytes subsequently contribute to an injurious interplay triggering cell death pathways in mPTECs.