Investigating the Role of NOX5 in Diabetic Kidney Disease with Kidney Organoids

Background: Diabetic kidney disease (DKD) is the leading cause of kidney failure, accounting for 44% of dialysis needs. DKD is a progressive disease of the tubulointerstitial and glomerular compartments, causing distinctly different histologic lesions. In the peritubular space, activated pericytes transdifferentiate into mobile myofibroblasts leading to hallmarks of DKD, includingperitubular capillary rarefaction and interstitial fibrosis. In glomeruli, an early podocyte injury is characterized by foot process effacement, loss of slit diaphragm proteins, and oxidative-stress related vacuolization. In humans, NOX5 enhances reactive oxygen species and is upregulated in diabetic and non-diabetic podocyte injury. Unfortunately, NOX5 is absent in rodents, greatly limiting mechanistic studies to artificially-derived models. Given interspecies differences, and the requirement of tubulointerstitial and glomerular compartments, here we propose a human in vitro DKD model using pluripotent stem cells (PSCs)-derived from kidney organoids. Methods: Kidney organoids were differentiated from H9 human embryonic PSCs using the Morizane protocol. Differentiation day 42 organoids were subject to a published diabetic milieu (DM; Glucose 75mM, IL-6 1 ng/mL, TNF-α 1 ng/mL) vs vehicle control, and analyzed biweekly. Wholemount immunostaining (PODXL, LTL, CDH1, CD31), cryosection confocal microscopy (NPHS1, NPHS2, Phalloidin, αSMA, COL1A1, NOX5), and transmission electron microscopy (TEM) assessed for tubulointerstitial and glomerular injury. Results: Acute histologic changes indicative of a proteinuric glomerular injury was suggested by progressive podocyte actin cytoskeletal dysmorphia and the significant loss of NPHS2 over 2-4 weeks of exposure to a DM. In a similar timeframe, upregulated NOX5 in podocytes by confocal microscopy was associated with podocyte vacuolization and foot process effacement by TEM. Chronic changes indicative of kidney fibrosis included the development of αSMA+ myofibroblasts and COL1A1+ interstitial fibrosis after 6 weeks’ exposure. Conclusion: Kidney organoids recapitulate the multicompartmental histopathological changes of DKD and may be a useful model to investigate human-specific pathomechanisms, such as NOX5-mediated oxidative stress.