Abstract: SA-PO314

Screening Fibroblasts for Novel Therapeutic Targets in CKD

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

Authors

  • Logan, Ian, newcastle hospitals, Newcastle----, United Kingdom
  • Sheerin, Neil S., Newcastle University, Newcastle upon Tyne, United Kingdom
  • Shuttleworth, Victoria Gemma, Newcastle University, UK, Newcastle Upon Tyne, United Kingdom
Background

The most significant problem in nephrology is the progression of Chronic Kidney Disease (CKD) to End Stage Renal Disease (ESRD), which requires either dialysis or transplantation to sustain life. Despite an our knowledge that ESRD is caused by accumulation of fibrotic scar tissue leading to organ failure, no treatments are available to treat fibrosis. Targeting disease-causing renal fibroblasts may hold the key to new drugs to treat CKD.

Methods

Drug screening: Human renal fibroblasts were isolated under ethical approval from the normal poles of 3 nephrectomy specimens and expanded by outgrowth. They were cultured in the presence of TGF B-1 and 181 drugs targetting human epigenetic enzymes prior to WST-1 proliferation assays.
Flow cytometry: Cell cycle profiles were evaluated by propidium iodide staining fixed cells, whereas apoptosis was analysed using Annexin V staining in live cells.
Animal studies: Black 6 mice underwent unilateral ureteric obstruction (UUO) by ligation, under ethical approval and licencing, then treated with Aurora kinase inhibitors from day zero. Animals were sacrificed on day 10. Immunohistochemistry for alpha-SMA was performed on kidney tissue and analysed by automated microscopy. Values were subject to T-test statistical analysis.

Results

1. Drug screening revealed that 15 Aurora kinase inhibitors were effective at inhibiting proliferation of primary renal fibroblasts to more than 85%. Counterscreening showed these had no such impact on primary tubular cells.

2. Flow cytometry demonstrated that fibroblast cell cycle profiles were relatively unchanged upon Aurora kinase inhibitor treatment, but we observed a large enrichment in apoptosis to > 85% upon drug treatment.

3. We observed a 75% reduction in alpha-SMA staining upon treatment with Aurora kinase A inhibitor in injured kidneys compared to vehicle-treated animals.

Conclusion

1. Primary renal fibroblasts can be propagated and used as a model of CKD in target identification studies.

2. Targeting Aurora kinases with small molecule inhibitors causes selective apoptosis in fibroblasts but not tubular cells.

3. UUO mice demonstrate reduced fibroblast accumulation, by alpha-SMA expression, upon Aurora kinase A inhibition.

Overall, our data suggest that Aurora kinase inhibitors may have therapeutic utility in slowing progression of CKD to ESRD.