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Modelling APRT deficiency in kidney cells in vitro

Hildur Rún Helgudóttir, Svandís Davíðsdóttir, Runólfur Pálsson, Viðar Örn Eðvarðsson, Þórarinn Guðjónsson

Introduction: Adenine phosphoribosyltransferase deficiency (APRTd) 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. This study aims to create a cell culture model to investigate DHA crystal-induced kidney injury for potential clinical intervention targets.
Methods: Three kidney cell lines MDCK, HK-2 and HEK293 were used in the study. All cell lines were treated with DHA in concentrations found in the urine of untreated humans with APRTd, both in monolayers and 3D/transwell assays. Further, siRNA against APRT was utilized to knock down the gene in HEK293 and HK-2 cells. Read-out assay included cell viability, RT-PCR, western blot and immunostaining.
Results: APRT gene expression was significantly reduced in HEK293 and HK-2 cell lines after successful knockdown. All cell lines showed reduced viability upon exposure to DHA. HEK293 cells exhibited increased expression of proinflammatory markers IL-1β and IL-8 with DHA treatment, indicating an inflammatory response. Additionally, HEK293 cells showed increased expression of the CD44 marker which is believed to be important for crystal binding to renal epithelial cells upon exposure to higher DHA concentrations.
Conclusion: We have established a cell culture model that captures APRT deficiency in kidney cell lines. Preliminary data suggest that DHA treatment of these cell lines in vitro induces an inflammatory response and reduces cellular viability. This cell culture model provides insight into the inflammatory response and potential disease-specific targets for clinical intervention.


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