Renal Single-Cell RNA Sequencing and Digital Cytometry in Dogs with X-Linked Hereditary Nephropathy

Abstract

Chronic kidney disease (CKD) significantly affects canine health, but the precise cellular mechanisms of this condition remain elusive. In this study, we used single-cell RNA sequencing (scRNA-seq) to profile renal cellular gene expression in a canine model of X-linked hereditary nephropathy (XLHN). Dogs with this condition exhibit juvenile-onset CKD similar to that seen in human Alport syndrome. Post-mortem renal cortical tissues from an affected male dog and a heterozygous female dog were processed to obtain single-cell suspensions. In total, we recovered up to 13,190 cells and identified 11 cell types, including major kidney cells and immune cells. Differential gene expression analysis comparing the affected male and heterozygous female dogs identified cell-type specific pathways that differed in a subpopulation of proximal tubule cells. These pathways included the integrin signaling pathway and the pathway for inflammation mediated by chemokine and cytokine signaling. Additionally, using machine learning-empowered digital cytometry, we deconvolved bulk mRNA-seq data from a previous canine study, revealing changes in cell type proportions across CKD stages. These results underline the utility of single-cell methodologies and digital cytometry in veterinary nephrology.

Keywords: Alport syndrome; RNA-seq; X-linked hereditary nephropathy; digital cytometry; dog; kidney; scRNA-seq.

Figure 1

Representative renal cortical histopathology from (A,C) an affected male dog and (B,D) a heterozygous female dog with end-stage XLHN. (A,B) Hematoxylin and eosin (H&E) stain; (C,D) Masson’s trichrome stain; magnification, 10×; scale bar: 100 µm.

Figure 2

Single-cell transcriptome atlas of renal cells. (A) Single-cell RNA-sequencing workflow. (B) Uniform manifold approximation and projection (UMAP) visualization of 24 cell types in the integrated dataset of affected male dog and heterozygous female dog with X-linked hereditary nephropathy (XLHN). This plot displays a comprehensive comparison of all renal cells using UMAP analysis to illustrate the distribution of various cell types and their subpopulations in both the affected male (left) and the heterozygous female (right). Each color represents a different cell type, and annotations indicate their spatial distribution and density. (C) Comparative expression levels of integrin genes ITGA6, ITGAV, and ITGA2 in the podocytes of the affected male (red) and heterozygous (carrier) female (teal). The violin plots illustrate the distribution and median expression levels of each gene. Each dot represents an individual podocyte within the group. (D) Enrichment of cytochrome P450 family 4 subfamily A member 11 (CYP4A11) in a subgroup of proximal tubule cells (proximal tubule cell-2) on the UMAP plots of the affected male (affected) and heterozygous female (carrier).

Figure 3

Deconvolved bulk mRNA-seq data from six male dogs with X-linked hereditary nephropathy (XLHN) and two age-matched same-sex littermates. The bar charts show how cell type proportion changed during disease progression. The asterisks denote cell types that differed in proportion between affected and control dogs at three clinical time points (p < 0.001).