Defining the Role of Tubular MDM2 in the Transition from AKI to CKD in Kidney Organoids

Background: Chronic kidney disease (CKD) is a global health problem. Repeated acute kidney injury (AKI) promotes the onset of CKD by unclear mechanisms. Current treatments are largely supportive, with a lack of FDA-approved therapies potentially due to historically inadequate human models. Kidney organoids derived from human pluripotent stem cells (hPSCs) have demonstrated utility in modeling kidney injury and repair. By subjecting kidney organoids to repeated treatment with the nephrotoxicant, cisplatin, we modeled the transition from reversible AKI to irreversible CKD. To better understand the molecular mechanisms, unbiased single-nuclear RNA sequencing was performed in healthy, AKI, and CKD phases of organoids. Murine double minute 2 (MDM2), an inhibitor of p53-related cell cycle arrest and apoptosis, was the lead differentially expressed gene in repairing tubules. Here the association of MDM2 with tubular repair will be mechanistically probed, via modulation of MDM2 activity in an organoid model of AKI to CKD. Methods: Kidney organoids were differentiated from hPSCs into nephron-rich spheroids. From differentiation day 42 onwards, organoids were subject to repeated low-dose cisplatin (5μM) for 24 hours twice weekly for a total of 5 treatments, in the presence and absence of MDM2 inhibitor (Nutlin3 10μM). High-resolution confocal microscopy was used to determine the expression and localization of MDM2, γH2AX, and Ki67 in nephron epithelia of kidney organoids. Whole-mount immunostaining of nephron markers (PODXL+ podocytes, LTL+ proximal tubules, CDH1+ distal tubules) and for interstitial fibrosis (COL1A1) quantifiably assessed fortubular atrophy and interstitial fibrosis. Results: MDM2 was upregulated in DNA damaged proximal tubules (LTL+γH2AX+) after the initial cisplatin treatment, further increased during the tubular reparative phase (AKI), and reduced in the fibrotic phase (CKD). Following the third cisplatin injury in kidney organoids, inhibition of MDM2 resulted in tubular failed repair and early fibrosis characterized by reduced tubular Ki67+ proliferation, premature organoid atrophy, and peri-tubular COL1A1+ interstitial fibrosis. Conclusion: Kidney organoids model multicompartmental fibrotic phenotypes, permitting the identification of tubular MDM2 as mediating nephroprotective tubular repair during the transition from AKI to CKD.