Multiomics profiling of mouse polycystic kidney disease progression at a single-cell resolution

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to kidney failure. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single-nucleus multimodal atlas of an orthologous mouse PKD model at early, mid, and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalog differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single-nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

Keywords: PKD1; mouse model; multiomics; polycystic kidney disease; single cell analysis.

Fig. 1.

Single-nucleus multiomics profiling for mouse PKD kidneys. (A) Overview of experimental strategy. Single-nucleus multiomics atlas was generated from PKD model mouse kidneys and littermate control kidneys along a time course (three time points; postnatal day 66 (P66), 100 (P100), or 130 (P130) after Pkd1 deletion (n = 2 to 3 pairs for each time point). (B) Representative immunofluorescence images of lotus tetragonolobus lectin (LTL, green), uromodulin (UMOD, red), and lectin Dolichos biflorus agglutinin (DBA, white) in the cortex (Left) or corticomedullary junction (Right) in the PKD kidneys at P66 (n = 2), P100 (n = 3), or P130 (n = 3), or control kidneys (n = 3). Scale bar indicates 100 µm. (C) UMAP plot of the integrated single-nucleus multiomics dataset with weighted nearest neighbor (wnn) clustering. Clusters were annotated by lineage marker gene expression. PTS1/S2/S3, proximal tubule S1/S2/S3 segments; FRPTC_PEC, failed-repair proximal tubular cells and parietal epithelial cells; DTL1/DTL2/ATL, descending thin limb 1/2 and ascending thin limb of Henle’s loop; TAL, thick ascending limb of Henle’s loop; DCT, distal convoluted tubule; CNT, connecting tubule; PC1/2, principle cells 1/2; URO, uroepithelial cells; ICA, Type A intercalated cells; ICB, Type B intercalated cells; PODO, podocytes; ENDO, endothelial cells; FIB, fibroblasts; Myel, myeoloid cells; FAT, adipocyte.

Fig. 2.

Heterogeneity of collecting duct principal cells in mouse PKD. (A) Subclustering of distal nephron clusters (CNT, PC1, PC2, and URO) on the UMAP plot, colored by unsupervised clustering (Left) or genotype (Right). cPC, cortical principal cells; mPC, medullary principal cells. (B) Dot plot showing expressions of the genes enriched in each of the subtypes. The diameter of the dot corresponds to the proportion of cells expressing the indicated gene and the density of the dot corresponds to average expression. (C) Representative immunofluorescence images of CALB1 (green) and AQP2 (red) in the PKD kidneys at P130 (n = 3). Arrowheads indicate AQP2+ cyst lining. Arrows mark AQP2+/CALB1+ cyst lining. Scale bar indicates 50 µm. (D) Distal nephron subtypes in the human advanced ADPKD data are label-transferred from those in the mouse PKD data, and the frequencies of predicted mouse subtypes in each human subtype are shown on the heatmap. (E) Heatmap showing enrichment of gene expressions of the hallmark gene sets among cPC2 and mPC1 clusters at each time point. The pathways associated with DNA damage response are in blue characters and those associated with metabolic regulation are in red characters. (F) Volcano plot showing differentially expressed genes in mPC1 of PKD mice compared to that of control at P66 (Upper) or P130 (Middle). The x-axis represents the log fold change, and the y-axis represents the P values. Dot plot (Lower) showing representative differentially expressed genes among control and PKD mPC1 at P66 and P130. The diameter of the dot corresponds to the proportion of cells expressing the indicated gene and the density of the dot corresponds to average expression. (G) Representative immunofluorescence images of DBA (green) and TMSB4X (red) in the PKD kidneys at P130 (n = 3). An arrow indicates a glomerulus. Scale bar indicates 50 µm.

Fig. 3.

Heterogeneity of failed-repair proximal tubular cells in mouse PKD. (A) Subclustering of FRPTC_PEC cluster on the UMAP plot. (B) Dot plot showing expressions of the genes enriched in each of the subtypes. The diameter of the dot corresponds to the proportion of cells expressing the indicated gene and the density of the dot corresponds to average expression. (C) UMAP plot showing a gene expression level of Vcam1 (Left) and coverage plot showing accessible regions around Vcam1 promoter in each subtype is also shown (Right). The color scale represents a normalized log-fold-change (LFC, Left). The scale bar indicates 2 kbp (Right). (D) UMAP plot displaying Hnf4a (Left) or Cdh6 (Right) expression. The color scale represents LFC. (E) Representative immunofluorescence images of LTL (green) and VCAM1 (red) in the PKD or control kidneys at P66 (n = 2). Scale bar indicates 50 µm. (F) Representative immunofluorescence images of LTL (green) and VCAM1 (red) in the mildly or severely cystic regions in PKD kidneys at P100 or P130 (n = 3). The arrow indicates VCAM1+ PTC. Arrowheads mark cystic epithelial lining losing both VCAM1 and LTL staining. Scale bar indicates 50 µm. (G) Dot plot showing expressions of the differentially expressed genes among different time points in FR-PTC1 (Left) or FR-PTC2 (Right). The diameter of the dot corresponds to the proportion of cells expressing the indicated gene and the density of the dot corresponds to average expression. Spp1 expression is up-regulated at P130 in both subtypes (red). (H) Representative immunofluorescence images of VCAM1 (green), SPP1 (red), and LTL (white) in the PKD kidneys at P130 (n = 3). Scale bar indicates 50 µm.

Fig. 4.

Differentially activated molecular signaling pathways among PT subtypes in PKD. (A) The proximal tubular cell subtypes of human advanced ADPKD data are label-transferred from FR-PTC subtypes in mouse PKD data, and the frequencies of predicted mouse subtypes in each human subtype are shown on the heatmap. (B) Frequency of each PT subtype among whole PTC for each time point in PKD or control data. (C) Heatmap showing relative transcription factor binding motif enrichment among PT subtypes in snATAC-seq. Most enriched transcription binding motifs in each subtype are shown. (D) Violin plots displaying relative motif enrichment (chromVAR score) among PTC for TEAD3 (MA0808.1) (E) Violin plots displaying relative gene set enrichment among PTC for Hippo pathway genes (REACTOME-SIGNALING-BY-HIPPO).

Fig. 5.

GPRC5A as a shared cyst lining cell marker for mouse and human PKD. (A and B) UMAP plot showing a gene expression level of Gprc5a in the whole dataset (A) or distal nephron subclustering (B). The color scale represents a normalized log-fold-change (LFC). (C) Representative immunofluorescence images of LTL (green), GPRC5A (red), and DBA (white) in the PKD kidneys at P130 (n = 3). Arrowheads indicate GPRC5A+DBA+ cyst lining. Scale bar indicates 50 µm. (D) UMAP plot showing a gene expression level of Gprc5a in the FR-PTC subclustering. The color scale represents a normalized LFC. (E and F) Representative immunofluorescence images of VCAM1 (green), GPRC5A (red), and LTL (white) in the PKD kidneys at P130 (n = 3). The arrows indicate VCAM1+ atrophic tubules. Arrowheads indicate cyst lining with GPRC5A mutually exclusive with LTL (E) or colocalized with VCAM1 (F). Scale bar indicates 50 µm. (G) Areas of GPRC5A+ cysts and the adjacent cysts lacking GPRC5A signals in the PKD mouse kidneys at P130. The quantification was performed in five 200× images including GPRC5A+ cysts, taken from each of n = 3 PKD kidneys. The box-and-whisker plots depict the median, quartiles, and range. Wilcoxon rank sum test. (H and I) Representative immunofluorescence images of LTL (green), GPRC5A (red), and DBA (white) in the Pkd1^RC/RC mouse cystic kidneys at 11 mo of age (n = 3) (G) or Six2-Cre; Pkd1^F/F cystic kidneys at P7 (n = 2) (H). Scale bar indicates 50 µm (H), 100 µm (I, Left), or 10 µm (I, Right). (J) Cis-coaccessibility network (CCAN, gray arcs) of a conserved cis-regulatory region (CRE) 5’ distal to Gprc5a promoter in the mouse PKD FR-PTC (lower) or human ADPKD (upper) among accessible regions (red boxes) is shown. (K) Coverage plot showing accessibility of conserved CRE 5’ distal to Gprc5a gene among PT subtypes. The conserved CRE has several TEAD family binding motifs both in humans and mice. (L) Quantitative PCR for GPRC5A expression in primary human PTC with siRNA knockdown of LATS1 and LATS2 (n = 3). Bar graphs represent the mean and error bars are the s.d. Student’s t test (Upper). LATS1/2 knockdown inhibits Hippo pathway, activating TEAD and subsequently up-regulating GPRC5A expression (Lower). Schematic was created with BioRender.