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When 23 samples were divided into 2 groups by the median value of expression i. These results indicate that VISTA could be a biomarker related to glomerulonephritis and its fibrotic outcome.

Tubulointerstitial fibrosis after glomerular injury determines patient outcomes, but its immunological link and relevant sentinel molecules are not completely understood.

The present results may provide clues, and VISTA was identified as a sentinel molecule particularly expressed in kidney macrophages. These changes provoked IL-9 production in surrounding parenchymal tissue cells and aggravated interstitial fibrosis. VISTA is an immunoregulatory and inhibitory checkpoint expressed in cells of the myeloid and lymphoid lineages The expression can be inducible in inflammatory or cancerous conditions 12 — 15 , but an interesting aspect is its high expression in kidney-resident macrophages When the expression level was compared among tissue-resident macrophages, the kidney-resident subset had the highest expression, followed by peritoneal, splenic, and bone marrow macrophages These results indicate that the tissue environment may determine VISTA expression in macrophages, and the unique pattern of kidney-resident macrophages implies their homeostatic role under normal conditions or during kidney inflammation.

Our previous work identified kidney-resident macrophages as playing a repair role after ischemic tubular damage, and VISTA was determined to be a molecule relevant to this repair role These changes may be dependent on tissue status or specific to diseases such as severe glomerulonephritis.

In addition to direct damage by immune cells, the responses of parenchymal tissue cells, including glomeruli, tubules, and other stromal cells, are important for determining kidney outcomes Parenchymal tissue cell—derived cytokines can be inflammatory or antiinflammatory, and all of these cytokines participate in tubulointerstitial fibrosis as a repair or sometimes ongoing process 40 , IL-9, as a Th2-type cytokine, has been related to immune tolerance and tissue fibrosis 42 , IL-9 can be produced in kidneys after injury 44 , but the subsequent pathophysiology by IL-9 is less understood than that in other organs, such as the lung and liver.

The all-or-none status of IL-9 in transgenic and knockout mice results in unexpectedly reduced and increased inflammatory responses, respectively 45 , 46 , indicating that proper or specific window levels may be needed to maintain homeostasis of the kidney or repair after injury.

VISTA can tune the activity of T cells in a contact manner, but there is a gap in understanding human kidneys. However, gene expression levels were different such that human monocyte-like MNPs R2 or monocytes in the mouse kidney had the highest expression levels among immune cell subsets, higher than those of R1-like MNPs R1 in the mouse kidney.

The expression level within kidneys was associated with the fibrotic outcomes of ANCA vasculitis, a severe form of glomerulonephritis. These results will help us understand the difference between mice and humans and could improve intervention strategies related to VISTA or kidney immune cells.

A clear understanding of the pathogenesis of glomerulonephritis will provide valuable insight into disease progression and options for intervention. VISTA, as an immunoregulatory sentinel molecule, may be highlighted in future biomarker research and therapy development. All mice used in the experiments were 8 weeks of age and male. Animals were housed under specific pathogen—free conditions at the facility of Seoul National University College of Medicine. As a metabolic inhibitor, 10 ng of oligomycin Sigma Aldrich or etomoxir Sigma Aldrich was administered via subcapsular injection at day 7 after NTN induction.

The mice were sacrificed at the indicated time points after reperfusion. The antibodies used for flow cytometry are listed in Supplemental Table 1. Paraffin-embedded tissue sections were deparaffinized and rehydrated with xylene and ethanol. After retrieval of antigens, sections were blocked in 0. The contact analysis between macrophages and infiltrated T cells was performed using the Imaris program version 9. Surfaces and spots were created for T cells and macrophages, and the number of spots was calculated within the indicated distance from the surface.

The antibodies used for immunofluorescence are listed in Supplemental Table 1. The steps before blocking were the same as those described in the immunofluorescence section. Blocking reagent was added for 1 hour at room temperature. All morphometric parameters were determined using a microscope coupled to a computerized morphometry system Qwin3, Leica. The primers used are listed in Supplemental Table 2.

After digesting kidneys with 0. Briefly, the cell suspensions were diluted in nuclease-free water to achieve a target count of 10, RNA transcripts were uniquely barcoded and reverse-transcribed within droplets. The cDNA molecules were pooled and subjected to an end-repair process, the addition of a single A base, and ligation of the adapters.

The Cell Ranger pipeline version 3. As a quality control of pooled scRNA-sequencing data of normal human kidneys 29 — 32 , the same quality control as in the NTN model was used.

R version 4. Sections were then stained with periodic acid—Schiff before microscopy analysis Leica Microsystems. The areas of fibrosis and positive staining were evaluated using ImageJ software version 1. For a competitive assay, 2. Composite renal risk e. Normal human kidney tissue was obtained from patients who underwent radical nephrectomy due to urogenital malignancy, but did not have hydronephrosis or any infectious diseases.

All analyses and calculations were performed using GraphPad Prism version 8. Survival curves were drawn using the Kaplan-Meier method.

A P value of less than 0. All patients provided written, informed consent for the donation and use of their specimens. DY and SSH analyzed bioinformatics data. All authors approved the final manuscript. Co—first authorship order was determined based on contribution to results generation. Conflict of interest: The authors have declared that no conflict of interest exists. This is an open access article published under the terms of the Creative Commons Attribution 4.

Reference information: J Clin Invest. National Center for Biotechnology Information , U. Journal List J Clin Invest v. J Clin Invest. Published online Jan 4. Find articles by Chang Wook Jeong. Find articles by Cheol Kwak. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Min-Gang Kim: moc. Phone: Received May 7; Accepted Nov 5. This work is licensed under the Creative Commons Attribution 4. This article has been cited by other articles in PMC.

Associated Data Supplementary Materials Supplemental data. Abstract Severe glomerular injury ultimately leads to tubulointerstitial fibrosis that determines patient outcome, but the immunological molecules connecting these processes remain undetermined. Keywords: Nephrology. Keywords: Fibrosis, Macrophages, T cells. Introduction Glomerulonephritis is the most common form of glomerular injury, and if severe, it can lead to tubular dysfunction and leave irreversible fibrotic matrix in the interstitium 1 , 2.

Open in a separate window. Figure 1. Figure 2. Figure 3. Immunometabolic changes in T cells. Interaction between macrophages and T cells. Figure 4. Contact between macrophages and T cells.

Figure 5. Figure 6. Figure 7. Figure 8. VISTA expression in human glomerulonephritis. Figure 9. Translation of results to human glomerulonephritis. Discussion Tubulointerstitial fibrosis after glomerular injury determines patient outcomes, but its immunological link and relevant sentinel molecules are not completely understood. Methods Mice. Flow cytometry. Real-time qPCR.

Single-cell RNA sequencing. Kidney histology. Adoptive transfer of T cells. Human samples. Study approval. Supplementary Material Supplemental data: Click here to view. Supplemental data set 1: Click here to view. Version Changes Version 1. Version 2. Footnotes Conflict of interest: The authors have declared that no conflict of interest exists. References 1. Strutz F, Muller GA. Interstitial pathomechanisms underlying progressive tubulointerstitial damage. Kidney Blood Press Res.

Jinde K, et al. Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis. Am J Kidney Dis. Iseki K, et al. Proteinuria and the risk of developing end-stage renal disease. Kidney Int. Tubular toxicity of proteinuria. Nat Rev Nephrol. Chen SJ, et al. Crosstalk between tubular epithelial cells and glomerular endothelial cells in diabetic kidney disease. Cell Prolif. Schnaper HW. The tubulointerstitial pathophysiology of progressive kidney disease.

Adv Chronic Kidney Dis. Bleriot C, et al. Determinants of resident tissue macrophage identity and function. Szabo PA, et al. Location, location, location: tissue resident memory T cells in mice and humans. Sci Immunol. Park JG, et al. Immune cell composition in normal human kidneys. Sci Rep. Cao Q, et al. J Am Soc Nephrol. Gao J, et al. VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer.

Nat Med. Wang L, et al. J Exp Med. Flies DB, et al. Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype with predisposition to the development of autoimmunity.

Hoppe JM, Vielhauer V. Induction and analysis of nephrotoxic serum nephritis in mice. Methods Mol Biol. Desalegn G, Pabst O. Inflammation triggers immediate rather than progressive changes in monocyte differentiation in the small intestine. Nat Commun. For more information about Air Engineering, including an aerial tour of our service and parts facility, please visit our about us.

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