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Literature Interpretation | Spatial Transcriptomic Sequencing Analysis of Acute Kidney Injury in Mice

Introduction:

Acute Kidney Injury (AKI): Refers to a sudden decline in renal function that occurs within 1–7 days and persists for more than 24 hours, characterized by an increase in serum creatinine (SCr) of at least 0.5 mg/dL. It manifests as azotemia, disturbances in fluid, electrolyte, and acid–base balance, accompanied by oliguria (<400 mL/24 h or 17 mL/h) or anuria (<100 mL/24 h), along with systemic symptoms.

Single-nucleus RNA sequencing (snRNA-seq) has revealed cell states and gene expression patterns induced by AKI injury, but it fails to elucidate intercellular communication networks or regional gene expression patterns. To address this gap, researchers employed spatial transcriptomics.

Single-cell sequencing technologies have transformed how researchers study renal cell biology and disease. Recently, single-nucleus RNA sequencing (snRNA-seq) has unveiled cell states and gene expression patterns induced by acute kidney injury (AKI). This study characterizes the injured proximal tubule cell states, which can be further categorized into severe injury and failed repair.

Due to tissue dissociation, single-cell RNA sequencing (scRNA-seq) cannot capture spatial information, leaving the physiological context of macrophage and leukocyte recruitment to injured cells after kidney injury poorly understood. For this reason, researchers employed spatial transcriptomics to map transcriptional changes during the repair process in acute kidney injury (AKI). The vast majority of mouse AKI studies have utilized male mice, as they are more susceptible to bilateral ischemia-reperfusion injury (IRI); however, research on AKI in females remains relatively limited.

The researchers optimized the timing at which C57BL/6J female and male mice develop equivalent injury, employed 10x Genomics’ Visium spatial transcriptomics, and leveraged the analytical tools SPOTlight and Giotto to enhance resolution. They improved the visualization of epithelial cells and distinct cell types associated with injury. The generation of this spatial transcriptomic atlas represents the first step toward reconstructing the spatial organization of gene expression in female AKI and provides a reference for multi-omics and spatial transcriptomic studies of AKI.

Experimental Procedure

Eight- to ten-week-old female C57BL/6J mice were used, with ad libitum access to food and water. IRI was induced, and BUN and creatinine levels were measured, along with glomerular filtration rate (GFR). PCR, immunohistochemistry, and immunofluorescence were also performed.

Experimental Results

Reconstruction of the IRI Model in Female C57Bl6/J Mice

Researchers induced renal injury in 8- to 10-week-old male and female mice and found that, following bilateral renal pedicle clamping for 34 minutes, the extent of injury in females was comparable to that observed in males after 22 minutes. Tissue samples were collected at three time points: the acute early phase (4 hours and 12 hours), the acute phase (2 days), and the late phase (6 weeks) (Figure 1A).

At each time point, researchers collected plasma samples to measure blood urea nitrogen and assessed plasma creatinine and glomerular filtration rate (GFR) at 12 hours post-injury (Figure 1A). BUN levels were comparable between males (n = 4–6 per time point) and females (n = 4–6 per time point) throughout the study period, except for a statistically significant decrease in female BUN levels on postoperative day 2 (P = 0.028) (Figure 1B).

At 12 hours post-injury, plasma creatinine levels were also significantly elevated in both males and females (Figure 1C). The researchers further confirmed that, at the 12-hour time point, a 22-minute clamping procedure in males and a 34-minute clamping procedure in females resulted in comparable declines in renal function (Figure 1D). Additionally, the researchers employed quantitative real-time PCR to assess the transcriptional induction of established injury biomarkers (Figure 1E).

As expected, at the acute early (12-hour) and acute (2-day) time points, Havcr1 (kidney injury molecule‑1 [Kim‑1]) and Lcn2 (neutrophil gelatinase‑associated lipocalin [Ngal]) were correspondingly upregulated; all injury biomarkers returned to pre‑injury expression levels by 6 weeks post‑injury.

Figure 1: Establishment of a renal injury model in female IRI mice

(A) Schematic diagram of the Bi-IRI and timeline for tissue collection from 8–10-week-old C57BL/6J male (blue circles) and female (red squares) mice.

(B) BUN levels in males and females at 4 hours, 12 hours, 2 days, and 6 weeks post-injury.

(D) Representative traces of female GFR measurements and GFR quantification in male and female control mice, as well as in 12-hour Bi-IRI mice.

(E) Real-time fluorescent quantitative PCR was used to assess the temporal changes in renal expression of Kim-1 (Havcr1) and Ngal (Lcn2) during female Bi-IRI.

Pathological examination

The researchers used Seurat to visualize marker gene expression and observed the proximal tubule (Lrp2) and the collecting duct (Aqp2) (Figure 2A). However, the spatial resolution of the Visium platform is relatively low (Figure 2A). To highlight the contrast in resolution, the specificity of these markers to individual tubules, and the resolution criteria that the researchers ultimately aim to achieve in SrT, they validated the expression patterns of these cell types by immunofluorescence (Figure 2B).

Accordingly, to validate the detection of sex-specific differences in gene expression and cell-type similarities, the researchers compared SrT samples from male sham-operated kidneys with those from female sham-operated kidneys, identifying comparable major renal cell types (Figure 2C).

Figure 2

(A) SrT characterization of marker expression in the cortical (Lrp2), medullary (Aqp2), and glomerular (Nphs1) regions using Seurat.

(B) Wide-field immunofluorescence images of the female renal cortex, medulla, and glomeruli. In the male sham kidney (blue) and the female sham kidney, the proportion of UMAP clusters corresponding to specific cell types is 500 µm.

Using open-source toolkits to analyze cell types and the spatial expression patterns of genes.

By employing Giotto to enhance computational resolution, researchers can identify patterns of gene expression changes during injury and repair.

Figure 3: Using Giotto to analyze the spatial relationships between cell types and gene expression.

(A) Leiden clustering, UMAP, and visualization of female sham-operated PAGE enrichment.

(B) Spatial maps showing increased resolution of major cell types in female sham-operated animals, generated using PAGE to enrich podocytes (Pod), proximal tubule segments 1–2 (PTs1–2), connecting tubules (CNT), thick ascending limb (TAL), and thin descending limb (TL).

(C) Spatial expression of DEGs from PTs3 (Aadat), TAL (Lcn2), fibroblasts (Cfh), and collecting ducts (Cryab) during the female injury process.

Researchers used SPOTlight to reveal changes in cell-type interactions during the Bi-IRI process.

Figure 4

(A) In female sham-operated animals, the expression profiles of isolated Visium regions in the cortex, outer medulla (OM), and papilla after SPOTlight deconvolution using snRNA-seq.

(B) Spatial interaction map of injury‑sensitive cell types at each time point (sham surgery, 4 hours, 12 hours, 2 days, 6 weeks) following SPOTlight deconvolution.

(D) Representative immunofluorescence images of the macrophage marker F4/80 (green) and the InjPT marker Kim-1 (red) at different time points after injury (sham surgery, 12 hours, and 6 weeks).

Discussion

SCRNA-seq and snRNA-seq technologies have enabled researchers to characterize renal cell types and states under both healthy and disease conditions; however, these studies have lacked spatial information. By comparing a female Bi-IRI model with an equivalent injury in male mice, the researchers generated a spatial transcriptomic atlas of kidney injury and, using the open-source toolboxes Giotto and SPOTlight, successfully analyzed data at higher resolution.

Reference: doi.org/10.1681/ASN.2021081150

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