Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 May 19;13(1):92.
doi: 10.1186/s13578-023-01041-3.

Integrated single-nucleus sequencing and spatial architecture analysis identified distinct injured-proximal tubular types in calculi rats

Zhu Wang  1 , Qiong Deng  1 , Yanli Gu  2 , Min Li  3 , Yeda Chen  1 , Jieyan Wang  1 , Ying Zhang  1 , Jianwen Zhang  1 , Qiyi Hu  1 , Shenping Zhang  1 , Wei Chen  4 , Zhenhua Chen  4 , Jiaying Li  4 , Xisheng Wang  5   6 , Hui Liang  7   8
Affiliations

Integrated single-nucleus sequencing and spatial architecture analysis identified distinct injured-proximal tubular types in calculi rats

Zhu Wang et al. Cell Biosci. .

Abstract

Background: Urolithiasis with high prevalence and recurrence rate, has impacts on kidney injury in patients, becomes a socioeconomic and healthcare problem in worldwide. However, the biology of kidney with crystal formation and proximal tubular injury remains essentially unclear. The present study aims to evaluate the cell biology and immune-communications in urolithiasis mediated kidney injury, to provide new insights in the kidney stone treatment and prevention.

Results: We identified 3 distinct injured-proximal tubular cell types based on the differentially expression injury markers (Havcr1 and lcn2) and functional solute carriers (slc34a3, slc22a8, slc38a3 and slc7a13), and characterized 4 main immune cell types in kidney and one undefined cell population, where F13a1+/high/CD163+/high monocyte & macrophage and Sirpa/Fcgr1a/Fcgr2a+/high granulocyte were the most enriched. We performed intercellular crosstalk analysis based on the snRNA-seq data and explored the potential immunomodulation of calculi stone formation, and founded that the interaction between ligand Gas6 and its receptors (Gas6-Axl, Gas6-Mertk) was specifically observed in the injured-PT1 cells, but not injured-PT2 and -PT3 cells. The interaction of Ptn-Plxnb2 was only observed between the injured-PT3 cells and its receptor enriched cells.

Conclusions: Present study comprehensively characterized the gene expression profile in the calculi rat kidney at single nucleus level, identified novel marker genes for all cell types of rat kidney, and determined 3 distinct sub-population of injured-PT clusters, as well as intercellular communication between injured-PTs and immune cells. Our collection of data provides a reliable resource and reference for studies on renal cell biology and kidney disease.

Keywords: Kidney stone; Proximal tubular; Single cell sequencing; Spatial transcriptomics; Urolithiasis.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interest exists.

Figures

Fig. 1
Fig. 1
A single-cell transcriptomic analysis of rats with calculi. A Study design and workflow of rat kidney sample processing for snRNA-seq and spatial architecture analysis. B Histochemical validation and von Kossa staining of the calcium crystals in the urolithiasis model via HE staining; the green arrows indicate the crystals. Original magnification, 10 × 40. C Uniform manifold approximation and projection (UMAP) of snRNA-seq cells recovered from the kidneys of both normal rats and rats with calculi, as well as proximal tubular and BI subsets
Fig. 2
Fig. 2
snRNA-seq revealed the distinct cell types in the kidneys of rats with calculi A Bar plots of the proportion of cell types by origin and total cell number. B Distribution and relative expression of established marker genes (violin plots) for identifying the cell populations in rat kidneys. C Dot plot of top five cluster-specific genes of each cell type in the kidneys of rats. D Spatial feature plots of cell types identified in the kidney of calculi rats
Fig. 3
Fig. 3
Single-cell RNA sequencing identified distinct proximal tubular populations in the kidneys of rats. A Heatmap of novel marker genes of subpopulations in proximal tubular. B, C UMAP snRNA-seq and spatial feature plots of marker genes expressed by proximal tubular subpopulations. D Violin plots of selected markers expression in distinct injured-proximal tubular cells. The representative immunohistochemistry images were adopted from the Human Protein Atlas (https://www.proteinatlas.org/). E Left: Spp1 and Havcr1 expression feature plots generated via ST platform; Right: Violin plots of Spp1 and Havcr1 in the kidney of calculi rats
Fig. 4
Fig. 4
Profiling of distinct genes in the proximal tubular cells of rats with calculi. A Heatmap of functional genes and injury markers expressed in the proximal tubular cells. B Spot plot of functional genes and injury markers expressed in the proximal tubular cells. C Representative images of immunohistochemistry staining of injury markers (Havcr1 and Spp1) in the kidney of calculi rats. D Expression profiles of distinct markers in the proximal tubular cells of rats with calculi by UMAP and column plots. The gene expression level in different groups and sub-populations were showed in different plots with error bars. *, P < 0.05, **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001; ns, non-significant
Fig. 5
Fig. 5
Biological significance of the DEGs in proximal tubular. A Bar plot of the top 20 KEEG signaling pathways enriched in the proximal tubular of rats with calculi. B Bubble plot of the top 20 KEEG signaling pathways enriched in the proximal tubular of rats with calculi. C KEGG pathway annotation in the proximal tubular of calculi rats. D The most significantly upregulated signaling pathways in the proximal tubular cells identified by GSEA.
Fig. 6
Fig. 6
snRNA-seq revealed the immune landscape of kidneys of rats with calculi. A Heatmap of novel markers expressed in the BI cell sub-clusters. B Monocyte & Macrophage and Granulocyte populations were identified through the expression of Mrc1, CD163, CD63 and Sirpa. C An undefined cell population in the BI cluster was identified with high expression of Lrrk2 and Dok2. The representative immunohistochemistry images were adopted from the Human Protein Atlas (https://www.proteinatlas.org/)
Fig. 7
Fig. 7
Profiles of gene expression in the BI cluster in the kidneys of rats with calculi. A Scatter plot of DEGs of the BI cluster in the kidneys of rats with calculi versus normal control. B Heatmap of distinct markers expression in the kidneys of rats with calculi versus normal control. C Dot plot of distinct novel marker expression in the BI cluster in the kidneys of rats with calculi versus normal control. D Column plot and UMAP analysis of Fth1. E Column plot and UMAP analysis of Spp1. F Column plot and UMAP analysis of Mgp. *, P < 0.05, **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001; ns, non-significant
Fig. 8
Fig. 8
Novel interactions between injured-proximal tubular and immune cells. A Schematic of ligand–receptor interaction in injured-PT subpopulations at the leading edge and in immune cells. B and C Bar plots of significant ligand–receptor (L-R) pairs (P < 0.05) when PTs expressed ligands and immune cells expressed receptors matched to the cell types in snRNA-seq data. D Left: heatmap of snRNA-seq average log fold change (logFC) of NicheNet top predicted ligands expressed by injured-PT cells that modulated immune cell types. Middle: heatmap of significant L-R pairs between injured-PT subpopulations and immune cell types in snRNA-seq. Bottom: heatmap of snRNA-seq average logFC of ligand-matched receptors expressed by immune cell types. E and F Bar plots of significant L-R pairs (P < 0.05) when monocytes and macrophages and granulocytes expressed ligands and injured-PTs expressed receptors matched to cell types in snRNA-seq data. G Left: heatmap of snRNA-seq average log fold change (logFC) of NicheNet top predicted ligands expressed by monocyte and macrophages and granulocytes that modulated injured-PT cells. Middle: heatmap of significant ligand–receptor pairs between immune cell types and injured-PT subpopulations pair in snRNA-seq. Bottom: heatmap of snRNA-seq average logFC of ligand-matched receptors expressed by injured-PT subpopulations
See this image and copyright information in PMC

References

    1. Hsiao CY, Chen TH, Lee YC, Wang MC. Ureteral stone with hydronephrosis and urolithiasis alone are risk factors for acute kidney injury in patients with urinary tract infection. Sci Rep. 2021;11(1):23333. doi: 10.1038/s41598-021-02647-8. - DOI - PMC - PubMed
    1. Alexander RT, Hemmelgarn BR, Wiebe N, Bello A, Morgan C, Samuel S, et al. Kidney stones and kidney function loss: a cohort study. BMJ. 2012;345:e5287. doi: 10.1136/bmj.e5287. - DOI - PMC - PubMed
    1. Kittanamongkolchai W, Vaughan LE, Enders FT, Dhondup T, Mehta RA, Krambeck AE, et al. The changing incidence and presentation of urinary stones over 3 decades. Mayo Clin Proc. 2018;93(3):291–9. doi: 10.1016/j.mayocp.2017.11.018. - DOI - PMC - PubMed
    1. Zeng G, Mai Z, Xia S, Wang Z, Zhang K, Wang L, et al. Prevalence of kidney stones in China: an ultrasonography based cross-sectional study. BJU Int. 2017;120(1):109–16. doi: 10.1111/bju.13828. - DOI - PubMed
    1. Uribarri J, Oh MS, Carroll HJ. The first kidney stone. Ann Intern Med. 1989;111(12):1006–9. doi: 10.7326/0003-4819-111-12-1006. - DOI - PubMed