FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair

Abstract

Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.

Figure 1. Renal histology in individuals with karyomegalic interstitial nephritis (KIN)

Renal histology of individuals with FAN1 mutation exhibits the characteristic triad of nephronophthisis (NPHP), with cystic dilation of renal tubules (asterisks), interstitial infiltrations (encircled with a dotted line in a), and widespread fibrosis (blue-grey coloring in Trichrome-Masson staining in b). Karyomegaly is observed (white arrow heads in b and c) in tubules that have lost epithelial cells at their circumference (black arrow heads in b and c). The tubular basement membrane is thickened (double arrows in c) as well as attenuated (black arrow in c). a and b are from individual A4393-21, c is from individual A4466-21. Scale bars denote 100 μm.

Figure 2. Phenotypes of FAN1-mutant cells

(a) Western blot analysis with anti-FAN1 antibody raised against the N-terminal 90 amino acids of FAN1. Specificity of the antibody was confirmed by the abrogation of signal after transduction of BJ fibroblasts with shRNAs against FAN1 and in individuals with protein truncating mutations in FAN1 (see Table 1). (b) Examples of metaphases of the indicated cell lines after treatment with 50 nM mitomycin C (MMC). Arrows indicate radial chromosomes, arrowheads indicate chromatid breaks. (c–f) MMC and diepoxybutane (DEB) sensitivity of the indicated FAN1-mutant cell lines in comparison to FANCA mutant and wild type cell lines. Primary fibroblast (FIB) (c and d) or lymphoblastoid cell lines (LCL) (e and f) were treated in triplicate with increasing levels of indicated DNA ICL inducing agent. After 6 or 8 days, cell numbers were determined using a Coulter counter. Total cell numbers at each dose were divided by the number of cells in the initial untreated sample to arrive at percent survival. Error bars indicate standard deviations. (g) Cell cycle analysis of the indicated fibroblast cell lines after treatment with 100 nM MMC or 0.1 μg of DEB per ml of media. Untreated samples were analyzed in parallel.

Figure 3. Complementation of FAN1-mutant cells with FAN1 cDNAs, and epistasis analysis with genes implicated in ICL resistance

(a) Complementation of MMC sensitivity in fibroblasts of KIN individual A1170-22. Fibroblasts stably transduced with empty vector (vector control), vector expressing wild type FAN1 cDNA, or FAN1 cDNA with mutations incorporated (see Table 1) were exposed to different levels of MMC ranging from 0–100 nM. After 8 days, the cell number was determined using a Coulter counter. Total cell numbers at each dose were divided by the number of cells in the initial untreated sample to arrive at percent survival. Error bars indicate s.d. (b) Immunoblot showing expression levels of FAN1 alleles in A1170-22 fibroblasts used in the MMC sensitivity assay shown in panel a. Note that Trp707* and Arg679Thrfs*5 lack expression of FAN1 and Leu925Profs*25 results in a shortened product. (c) MMC sensitivity in A1170-22 fibroblasts transfected with the indicated siRNAs. (d) Immunoblot of expression levels of indicated proteins after siRNA-mediated depletion in the A1170-22 fibroblasts used in the experiment shown in panel c.

Figure 4. Phenotype of fan1 loss of function in zebrafish

(a–c) Knockdown of fan1 in zebrafish causes developmental abnormalities. A fan1D7 morpholino (MO) that targets the splice acceptor site of exon 7 (Supplementary Fig. 5) was injected into 1–4 cell stage embryos at the concentrations shown. Standard control MO (Ctrl) was injected at 0.2 mM. At 25 hpf (b–c), morphant embryos displayed shortened body axis, ventral body axis curvature, and massive cell death (dark grey tissue areas) throughout the embryo (see c), when compared to control MO (a). Scale bar in a–c is 750 μm for left panels and 150 μm for right panels. (d–e) Knockdown of fan1 in zebrafish induces widespread apoptosis. Upon fan1 knockdown, utilizing fan1D7 MO as in (a–c), widespread apoptosis was seen in the anterior body (d) and the tail (Supplementary Fig. 5) of 27 hpf zebrafish, as detected by an antibody against activated caspase-3 (CASP3) compared to negative control morpholino. Images are representative for 32 embryos evaluated for control and MO knockdown, respectively. (e) Knockdown of fan1 leads to increased staining with γH2AX antibody, indicating increased DDR. In knockdown embryos, 24 out of 27 embryos had elevated Caspase-3 staining, and 27 of 32 embryos stained with γH2AX had elevated expression of γH2AX. Scale bar is 250 μm. (See also Supplementary Fig. 5.) (f–h) Knockdown of fan1 on the background of p53 morphants reveals pronephric cysts. (f) p53 knockdown alone does not cause pronephric cysts in 72 hpf embryos exhibiting normal pronephric tubules (arrows). (g) However, 72 hpf zebrafish embryos co-injected with fan1D7 MO (0.1 mM) and p53 MO (0.2 mM) display pronephric kidney cysts (19±3%) (asterisks in g) and results in body axis curvature (not shown) (45±4%). (g–h) it causes body axis curvature in a significantly smaller fraction (11±2%; not shown). Scale bar is 50 μm.

Figure 5. Differential expression levels of FANCD2 vs. FAN1 in human tissue sources and increased DNA damage response in chronic kidney disease

Expression levels measured by quantitative real-time PCR were normalized against GAPDH expression. Error bars show SEM of triplicates. (a–b) Expression levels of FANCD2 vs. FAN1 in 25 different human tissue sources (of 48 total with differential expression). (a) FAN1 expression exceeded FANCD2 in parenchymatous organs including kidney, liver, neuronal tissue and female reproductive organs, whereas (b) FANCD2 expression exceeds FAN1 expression in lymphatic or bone marrow-derived sources, skin and testis. (c–h) DNA damage response is pronounced in chronic kidney disease. (c) Quantified percent of γ-H2AX staining of fawn-hooded hypertensive rat kidneys (between 2,000–3,000 cells scored per animal) with progressive kidney injury correlates with proteinuria (p<0.0054). (d) Example of γ-H2AX immunostaining in normal rat kidney. (e) Example of chronically damaged kidney in the fawn hooded hypertensive rat. (f–h) γ-H2AX staining of human kidney transplant biopsies: Transplant kidney, 4 months post-transplantation, no evidence for injury (f); transplant kidney, 16 yrs after transplantation, chronic damage (g); transplant kidney with chronic tissue damage, 10 years post-transplantation (h). Scale bars in d–h are 100 μm.