Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair

Abstract

Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 ( FANCD2 ) and RAD51 recombinase ( RAD51 ) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies.

Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids

“…Residual Rh123 uptake in the presence of OCTi inhibitors suggests the presence of alternate basolateral uptake pathways for this molecule. Nonetheless, our results show that organoids express OCT2, consistent with previous reports that organoids were able to recapitulate kidney injury induced by cisplatin, a substrate of OCT2 (Morizane et al, 2015;Digby et al, 2020;Gupta et al, 2022).…”

“…Bulk RNA-seq analysis in our recent study suggests that kidney organoids mature during 7 weeks of differentiation in culture (Gupta et al, 2022). To evaluate the functional maturation of tubular transport, we compared the Rh123 transport activities in kidney organoids longitudinally during this period of differentiation, and specifically on days 21 (d21), 35 (d35), and 49 (d49).…”

Production of kidney organoids arranged around single ureteric bud trees, and containing endogenous blood vessels, solely from embryonic stem cells

“…Human kidney organoids made from induced pluripotent stem (iPS) cells have been used to model human development 46 , and to model some human kidney pathologies from patient-derived iPS cells 47 – 49 and from cells engineered to have genetic defects 48 . They have also been used to investigate critical pathways in renal repair 50 , and to model pathways of drug-induced nephrotoxicity 51 and to screen for and report nephrotoxicity fluorescently 52 . It is important to note, though, that there are differences between human and mouse development 53 and caution should be used in extrapolating from one to the other.…”

Potential Strategies for Kidney Regeneration With Stem Cells: An Overview

“…Tanigawa et al established organoids created from NPHS1 missense mutations, where the podocyte foot process showed impaired slit diaphragm formation ( Tanigawa et al, 2018 ). Furthermore, Gupta et al identified the novel intrinsic repair mechanism using kidney organoids ( Gupta et al, 2022 ). By using single-nuclear RNA sequencing analysis of kidney organoids after cisplatin exposure, they identified the transient up-regulation of Fanconi anemia complementation group D2 ( FANCD2 ) and RAD51 recombinase ( RAD51 ) during intrinsic repair, and the down-regulation of these genes in incomplete repair.…”