Advancing Diabetic Kidney Disease Research through Innovative Kidney Tubuloid Model

Abstract

Introduction: Diabetic kidney disease (DKD) is a severe complication of diabetes that affects the kidney glomerulus as well as tubules due to hyperglycemia induced oxidative stress. Traditional animal models and cell lines fail to fully replicate DKD's genetic and cellular complexity, prompting a shift to advanced models like kidney organoids. Kidney tubuloids, a three-dimensional model mimicking native tubules, offer a physiologically relevant platform to study renal physiology, disease mechanisms, and drug responses, aiding in the development of personalized therapies. Herein, we aim to develop an in vitro 3D tubuloid model from control and diabetic murine origins to elucidate the cellular and molecular mechanisms underlying DKD. Methods: Mice were allocated into 2 groups FVB-NJ control group (C, n=3) and a non-obese type 2 diabetic MKR group (D, n=3). At 45 weeks of age, mice of both groups were euthanized, and kidneys isolated for further studying. The first murine kidney was used for adult stem cells (ASCs) isolation, that were cultured in 3D Matrigel to promote tubuloid formation. On the other hand, the second kidney was used for molecular and functional analysis. Results: Metabolic parameters showed no differences in body weight and kidney weight/body weight ratio between the C and D groups. However, blood glucose levels and HBA1C were significantly elevated in the D group. Kidney function tests shows elevation of BUN, UACR and proteinuria in the D group compared to the C group. H&E staining revealed significant glomerular hypertrophy in the D group, while PAS and MT staining demonstrated increased glycogen and collagen deposition, respectively, in the glomeruli as well as tubules. NOX1 and NOX4 expression were slightly elevated in the D group in comparison to C. Examining the established tubuloid model, tubuloids from both groups were uniform in size upon passaging and evolved from cystic to tubular structures, with higher organoid-forming efficiency observed in the D group. In addition, the formed organoids lacked podocyte markers nephrin and podocin, however, they expressed the tubular epithelial marker PAX8, proximal convoluted tubule marker SLC3A, loop of Henle marker UMOD, distal convoluted tubule marker SCNN1G, but not the collecting duct marker AQP2. No significant changes were found in KIM1 expression. Moreover, our established tubuloids were found to express NOX4 but not NOX1. Conclusion: In conclusion, the successful generation of kidney tubuloids in our study present to be a promising model system for studying renal physiology, disease mechanisms, and therapeutic interventions. The established tubuloid model closely resembles tubular cells under physiological conditions. However, further molecular testing is required to evaluate the impact of in vivo DKD on established in vitro tubuloids.