Introduction:
Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are the major cause of end-stage kidney disease in children. Genomic variants account for only a minority (20%) of CAKUT, suggesting a pathogenic role for environmental and epigenetic factors. Yet, epigenetic control of human kidney development is largely undefined. Genetic variants of Lysine-specific Methyltransferase 2D (KMT2D), a histone methyltransferase that promotes gene transcription in nonrenal tissues via its catalytic SET domain and its non-catalytic Coiled Coil (CC) domain, are common in Kabuki Syndrome (KS) with CAKUT. Here, we investigate KMT2D function in both mouse and human embryonic kidney and the impact of a human CC domain genetic variant on CAKUT.
Methods:
Genotype versus phenotype was determined in published KS patients via meta-analysis. KMT2D-deficient human kidney organoids were generated (Takasato et al., 2015, Kumar et al., 2019) from CRISPR-edited induced pluripotent stem cells (iPSCs) targeting the SET domain (KS-SET), and from urine-derived iPSCs of a KS patient with a CC domain variant (KS-CC) and CAKUT. ALPHAFOLD, an AI tool, was used to model mutated KMT2D-CC protein structure. Kmt2d function in distinct renal cell lineages was studied in mice with Cre recombinase-mediated Kmt2d deficiency.
Results:
Meta-analysis of reported KS patients revealed that mutations in the CC domain are associated with a 2.5-fold greater risk of CAKUT compared to mutations in other KMT2TD domains (P≤0.01). ALPHAFOLD analysis revealed altered KMT2D tertiary protein structure of the CC and SET domains in KS patients with CC domain variants and CAKUT (n=2 unique patients). KMT2D protein, identified by in situ immunofluorescence, was detected in iPSC-derived intermediate mesoderm (IM), and in SIX2+ nephron progenitor cells and podocytes, but not in PBX1+ renal stroma in control iPSC-derived human kidney organoids. Human KS-CC and KS-SET kidney organoids displayed a dysplastic phenotype, with either complete absence of nephrons or a decreased number of nephron-like structures (n=5). qPCR of both KS-CC and KS-SET human IM-RNA demonstrated upregulation of IM markers and downregulation in SIX2 (n=3, P≤0.0001), while qPCR of mature KS-CC and KS-SET kidney organoid RNA showed downregulation of nephron segment differentiation markers and upregulation in stromal markers MEIS1 and PBX1 (n=3, P≤0.001). Cre-mediated heterozygous Kmt2d deficiency in mouse Osr1+ IM, which gives rise to both nephrogenic and stromal precursor cells, caused severe kidney hypoplasia with surface hemorrhages, loss of Six2+ nephrogenic and Pbx1+ stromal cells (n=3, P≤0.01), as well as unilateral (n=2) or bilateral (n=1) hydronephrosis. Cre-generated Kmt2d deficiency, targeted to mouse Six2+ nephrogenic cells, resulted in renal hypoplasia, loss of Six2+ precursor cells and decreased nephron number (n=4, P≤0.001).
Conclusions:
KMT2D is required for both mouse and human kidney development. Both KMT2D catalytic and non-catalytic domains function within nephrogenic cells in a cell autonomous fashion to control nephrogenesis. Human kidney KMT2D-mutant organoids provide a basis for determining underlying molecular mechanisms that control epigenetic regulation in the human embryonic kidney. Early versions of these results here were presented at the Canadian Society of Nephrology Annual Meeting.
I have no potential conflict of interest to disclose.
I did not use generative AI and AI-assisted technologies in the writing process.