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Kidney Week

Abstract: FR-PO618

Tracking Molecular and Phenotypic Changes in Kidney Cells in 2D and 3D Cultures Following DHA Exposure: An Approach to Understand Cellular Changes in Adenine Phosphoribosyltransferase (APRT) Deficiency 

Session Information

Category: Genetic Diseases of the Kidneys

  • 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Helgudottir, Hildur Run, Faculty of Medicine, University of Iceland, Reykjavik, Iceland
  • Palsson, Runolfur, Faculty of Medicine, University of Iceland, Reykjavik, Iceland
  • Edvardsson, Vidar O., Faculty of Medicine, University of Iceland, Reykjavik, Iceland
  • Gudjonsson, Thorarinn, Faculty of Medicine, University of Iceland, Reykjavik, Iceland
Background

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in the generation and renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stones and crystal nephropathy characterized by inflammation and fibrosis. The aim of this study was to create a cell culture model to investigate DHA crystal-induced kidney injury in order to identify potential targets for clinical intervention.

Methods

Three kidney cell lines, MDCK, HK-2, and HEK293, were used in this study. All cell lines were treated with DHA at concentrations comparable to those found in the urine of untreated humans with APRT deficiency, both in monolayer and 3D assays. Furthermore, siRNA against APRT was used to knock down the gene in HEK293 and HK-2 cells. The readout assays included cell viability, RT-PCR, western blotting, and immunostaining.

Results

Following DHA exposure, decreased viability was observed in all cell lines. HEK293 cells exhibited increased expression of IL-8 following DHA treatment, indicating an inflammatory response. MDCK cells showed increased expression of N-cadherin when treated with high concentrations of DHA compared to the control, suggesting an epithelial–mesenchymal transition (EMT) response. Additionally, both HEK293 and MDCK cells showed increased CD44 expression, which is believed to be important for crystal binding to renal epithelial cells, upon exposure to higher DHA concentrations. When cultured in 3D condition with the addition of DHA, MDCK cells were still able to form polarized structures, and DHA accumulated within and around the structures. HEK293 cells form solid colonies and with the addition of DHA, the DHA accumulates inside the colonies. APRT expression was significantly reduced in HEK293 and HK-2 cells after successful knockdown, and HEK293 cells exhibited elongated protrusions.

Conclusion

We have established a cell culture model that captures kidney cell alterations observed in APRT deficiency. Data suggest that DHA treatment of these cell lines in vitro induces an inflammatory response, EMT, and reduces cellular viability. This cell culture model provides insights into the inflammatory response and potential disease-specific targets for clinical intervention.

Funding

  • Government Support – Non-U.S.