Spatially Resolved Transcriptomic Analysis of Acute Kidney Injury in a Female Murine Model

Abstract

Background: Single-cell sequencing technologies have advanced our understanding of kidney biology and disease, but the loss of spatial information in these datasets hinders our interpretation of intercellular communication networks and regional gene expression patterns. New spatial transcriptomic sequencing platforms make it possible to measure the topography of gene expression at genome depth.

Methods: We optimized and validated a female bilateral ischemia-reperfusion injury model. Using the 10× Genomics Visium Spatial Gene Expression solution, we generated spatial maps of gene expression across the injury and repair time course, and applied two open-source computational tools, Giotto and SPOTlight, to increase resolution and measure cell-cell interaction dynamics.

Results: An ischemia time of 34 minutes in a female murine model resulted in comparable injury to 22 minutes for males. We report a total of 16,856 unique genes mapped across our injury and repair time course. Giotto, a computational toolbox for spatial data analysis, enabled increased resolution mapping of genes and cell types. Using a seeded nonnegative matrix regression (SPOTlight) to deconvolute the dynamic landscape of cell-cell interactions, we found that injured proximal tubule cells were characterized by increasing macrophage and lymphocyte interactions even 6 weeks after injury, potentially reflecting the AKI to CKD transition.

Conclusions: In this transcriptomic atlas, we defined region-specific and injury-induced loss of differentiation markers and their re-expression during repair, as well as region-specific injury and repair transcriptional responses. Lastly, we created an interactive data visualization application for the scientific community to explore these results (http://humphreyslab.com/SingleCell/).

Keywords: AKI; spatial; transcriptomics.

Graphical abstract

Figure 1.

Establishing kidney injury in a female IRI model. (A) Schematic representation of Bi-IRI and timeline for tissue collection in male (blue circles) and female (red squares) 8- to 10-week-old C57BL6/J mice. (B) BUN levels at 4 hours, 12 hours, 2 days, and 6 weeks postinjury in both males and females (n=4–6 for each sex and condition; *P<0.05). (C) Creatinine levels of male and female control and 12-hour Bi-IRI mice (n=5–6 for each sex and time point; ***P<0.001). (D) Representative traces of female GFR measurements and GFR quantification in male and female control and 12-hour Bi-IRI mice (n=4–6 for each sex and condition; ****P<0.0001). (E) Kidney expression changes of Kim-1 (Havcr1) and Ngal (Lcn2) detected by quantitative real-time PCR along the female Bi-IRI time course (n=3–6 per time point; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 2.

Assessing spatial library quality in Seurat. (A) SrT representations of regional marker expression in female sham for cortex (Lrp2), medulla (Aqp2), and glomeruli (Nphs1) using Seurat. (B) Widefield immunofluorescent image of cortex (lotus tetragonolobus lectin [LTL], cyan), medulla (Aquaporin-2 [AQP2], purple), and glomeruli (nephrin, red) in female sham. Objective 20×, scale bar 500 µm. (C) UMAPs of cell type clustering in male (blue) and female (pink) sham kidneys, showing the similarity of present cell types in Seurat. Cell types include proximal tubule segments 1–2 (PTs12), proximal tubule segment 3 (PTs3), distal convoluted tubules (DCT), intercalated cells (IC), podocytes (Pod), fibroblasts (Fib), thick ascending limb (TAL), principal cells (PC), urothelium (Uro), and adipocytes (Adipo). (D) DEGs (Cyp7b1, male enriched; Cyp4a14, female enriched; Slco1a6, female enriched in PTs3) in male and female sham kidneys. (E) Similarly expressed genes in PTs3 (Slc22a7) and PC (Aqp2) in male and female sham kidneys.

Figure 3.

Resolving spatial relationships of cell types and gene expression with Giotto. (A) Leiden clustering UMAP and visualization for PAGE enrichment of female sham. (B) Spatial plots of increased resolution for major cell types in female sham using PAGE enrichment for podocytes (Pod), proximal tubule segments 1–2 (PTs1–2), connecting tubule (CNT), thick ascending limb (TAL), and thin limb (TL). (C) Spatial expression of DEGs from PTs3 (Aadat), TAL (Lcn2), fibroblasts (Cfh), and collecting duct (CD) (Cryab) along the female injury time course.

Figure 4.

Revealing changes in cell type interactions along the Bi-IRI time course with SPOTlight. (A) Representation of isolated Visium regions for cortex, outer medulla (OM), and papilla, after SPOTlight deconvolution with snRNA-seq in a female sham. Each spot is divided into a scatterpie of positional cell types (no immune cells) found at each specific slide coordinate. Cell types are connecting tubule (CNT), thick ascending limb of loop of Henle in cortex (CTAL1), distal convoluted tubule (DCT), descending and ascending thin limp of loop of Henle (DTL.ATL), endothelial cells (EC1–2), fibroblasts (Fib), intercalated cells (ICA, ICB), macula densa (MD), thick ascending limb of loop of Henle in medulla (MTAL), injured proximal tubule cells (InjPT), failed repair proximal tubule cells (FR-PTC), principal cells (PC1–2), pericytes (Per), podocytes (Pod), proximal tubule segments 1–3 (PTS1–3), and urothelium (Uro). (B) Spatial cell type interaction graphs for cell types of interest to injury generated for each time point (sham, 4 hours, 12 hours, 2 days, 6 weeks) after SPOTlight deconvolution. Edges between cell types represent the proportion of spots in which colocalization is detected and each node size corresponds to the number of connections to each cell type. Additional cell types are T cells and macrophages (Mø). (C) Remaining tissue covered spots expressing macrophage markers 6 weeks postinjury. (D) Representative immunofluorescent images of macrophage marker, F4/80 (green), and InjPT marker, Kim-1 (red), across the injury time course (sham, 12 hours, and 6 weeks). Kim-1 positive tubules at 6 weeks (white arrows) have increased F4/80 positive signal compared with sham and 12-hour time points, consistent with Figure 4B. Objective 20×, scale bar 50 µm.