Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System

Abstract

Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 ^FAN1+/-), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease.

Keywords: FAN1 gene; induced pluripotent stem cells; karyomegalic interstitial nephritis; kidney organoid.

Figure 1

FAN1 gene mutation in a patient with karyomegalic interstitial nephritis (KIN). (A) Hematoxylin and eosin (H&E) staining of kidney tissues of a patient with karyomegalic interstitial nephritis. Scale bar = 100 μm. White arrows in A point to karyomegaly. (B) Patient PBMC with KIN showing FAN1 gene mutation on deletion c.1985-1944delTTGGGTGGAT on 15q13.3. (C) Pedigree of a family showing individuals affected by karyomegalic interstitial nephritis resulting in the appearance of p.Gly663llefs*54.

Figure 2

Establishment of CRISPR/Cas9 ribonucleoproteins (RNP)-mediated FAN1 gene editing in WTC-11 hiPSCs. (A) Target site for guide RNA (gRNA) targeting FAN1. Target sites are indicated by a green color in exon 2 of the FAN1 gene. The downward-pointing arrowhead indicates the position of the canonical cut site and predicted specificity based on the number and distribution of homoeologous SNPs at the corresponding target site/PAM. (B) PAM sequences (5′-NGG-3′) in target site are indicated by red letters. The table shows no mismatched number with gRNA. The asterisk indicates on-target gRNA. (C) Indel sequences after transfecting WTC-11 hiPSCs with gRNA using the CRISPR/Cas9 RNP method. Indel sequences are indicated by red letters (a green color indicates a target site). (D) Read number of In-del frequency. Indel of 1bp (A ins) in the target sequence read of about 50%. (E) Morphology of FAN1-gene-edited WTC-11 iPSC (WTC-11^FAN1+/−). (F,G) Flow cytometry analysis and immunofluorescence image of cells expressing NANOG, SSEA-4, and TRA-1-81 in WTC-11^FAN1+/− hiPSCs. Scale bar = 50 μm. (H) Immunofluorescence staining of three germ layer markers. Ectoderm, mesoderm, and entoderm differentiation were detected using PAX4, SM22a, and FOX2A, respectively. Scale bar = 50 μm. (I) Expression analysis via RT-PCR of FAN1 and GAPDH in WTC-11 and WTC^FAN1+/− hiPSCs. All expression levels were normalized against GAPDH expression level. (J) Expression analysis via immunoblot of FAN1 and β-actin in WTC-11 and WTC^FAN1+/− hiPSCs. All expression levels were normalized against the β-actin expression level. Data are presented as mean ± standard error. *, p < 0.05 vs. WTC-11 iPSC.

Figure 3

Differentiation of kidney organoids from WTC-11, WTC-11^FAN1+/−, and KIN patient hiPSCs. (A) Schematic timeline of the hiPSC differentiation protocol. (B) Representative immunofluorescence images of podocalyxin (PODXL), lotus tetragonolobus lectin (LTL), and e-cadherin (ECAD) kidney organoids from WTC-11, WTC-11^FAN1+/−, and KIN patient, respectively. (C) Quantification of PODXL, LTL or ECAD-positive cells per organoid in each group. Scale bar = 50 or 100 μm.

Figure 4

Effect of mitomycin C (MMC) treatment in kidney organoids from WTC-11, WTC-11^FAN1+/−, and KIN patient hiPSCs. (A) Each kidney organoid was treated with 20 nM MMC. After 24 h or 48 h of incubation, each kidney organoid was stained with Ki67 antibody. Images with DAPI show nuclei positive for the Ki67 antibody. Scale bar = 50 or 100 μm. (B) Quantification of Ki67-positive cells in kidney organoids from WTC-11, WTC-11^FAN1+/−, and KIN patient hiPSCs. (C) Flow cytometry gating strategy illustrating a viable cell population being subgated to the level of podocalyxin+ (PODXL), lotus tetragonolobus lectin+ (LTL), or e-cadherin+ (ECAD) in kidney organoid cells from WTC-11, WTC-11^FAN1+/−, and the KIN patient hiPSCs, respectively, after treatment with MMC for 24 h or 48 h. (D–F) Quantification of percentage of viable cells in PODXL+, LTL+, or ECAD+ in cells from each kidney organoid. (G,H) Immunoblot analysis and its quantification of H2A.X in kidney organoids from WTC-11, WTC-11^FAN1+/−, and KIN patient hiPSCs after treatment with MMC for 24 h or 48 h. Data were normalized against the β-actin expression level. All data are presented as mean ± standard error. *, p < 0.05 vs. Nil group or 24 h MMC group.