Single cell transcriptomics of human nephritis reveal the cellular origin of kidney disease associated genes

Background Single cell RNA sequencing (scRNA-seq) have become a powerful tool in discovering a novel cell type and pinpointing cellular specific gene within tissues. However, in diseased kidneys, especially caused by glomerulonephritis, there have few study focusing on revealing gene expression changes at the cellular level. Methods To reveal cellular gene expression profiles of glomerulonephritis, we performed scRNA-seq of 2 human kidney transplantation donor samples, 4 human glomerulonephritis samples, 1 human malignant hypertension sample and 1 human chronic interstitial nephritis sample, all tissues were taken from biopsy. Results Upon disease occur, immune cells infiltrate which can be proved by our dataset. In the cluster of podocyte, two glomerulonephritis related genes named FXYD5 and CD74 were found, and interferon alpha/beta signalling pathway and antigen processing and presentation was enriched in podocyte of LN and IgAN comparing with other samples, thus inhibiting these signalling pathway may alleviate the symptom of these disease. found


Background
The kidney is a highly complex tissue with a broad range of specialized cell types organized into functionally distinct compartments. Previous bulk RNA-seq cannot detect heterogeneity of renal cells, thus specific renal cells functioning abnormal may be covered up by the majority of cell types(1).
Glomerulonephritis is a major reason leading to renal failure. The clinical manifestation of glomerulonephritis is a heterogeneous group of diseases, which were diagnosed by biopsy. However specific pathogenesis underlying histologic findings seems to be covered (2).
As one of most common renal diseases, IgA nephropathy is characterized by deposition of IgA immune complexes in glomerulus. The mesangial IgA is exclusively of the IgA1 subclass, which is mostly in a dimeric form that is composed of two IgA1 monomers and J chain(3).
Genome-wide association studies(GWAS) have found IgAN contributing genes including HLA-DRB1, -DQA1 and DQB1, which mediate the MHC class II response. In addition, TAP1, TAP2, PSM8 and PSM9 involved in antigen generation are also IgAN associated genes (4). However, in addition to primary MN (caused by auto antibody), there have other forms of MN in which anti-PLA2R antibodies can not be detected (7). The mechanisms underlying the common clinical manifestations also need to be clarified.
Lupus nephritis(LN) is a common and serious complication of systemic lupus erythematosus (SLE).To date, renal biopsy remains to be the gold standard for the severity of LN as current laboratory markers for LN lack sensitivity and specificity for renal disease activity and damage in lupus nephritis (8). Thus, novel biomarkers should be found to act as surrogate indicators instead of invasive method. However whether these functionally disease-related genes originating from specific cell type remain unknown. So this study wants to find whether the disease associated genes originate from specific cell type .

Clinical Sample
The patient described in this study consented under Ethics committee review of renji hospital affiliated to Shanghai Jiao Tong University School of Medicine.

Tissue Processing and Single-Cell Dissociation
The renal biopsy was preserved in cold PBS, then minced into small pieces with a razor blade and incubated at 37°C in freshly prepared dissociation buffer containing enzymes from Multi Tissue Dissociation Kit

Single-cell RNA sequencing and library construction
According to manufacturer's(10X Genomics) protocol, single cell suspensions were used to generate single-cell libraries with the Chromium Single Cell Gene Expression system using 3′ Library& Gel Bead Kit v2 (10X Genomics) and paired-end sequencing was performed on a HiSeq.

Bioinformatic Analysis
According to the instructions of SATIJA LAB(https://satijalab.org/seurat/), integrated sample was performed by using R toolkit, then calculation of highvariance genes, dimensional reduction, graph-based clustering, and the identification of cluster markers was done.

Overall cell clusters and marker genes of 21 cell clusters from integrated 8 samples.
After filtering the cells with <200 genes and >10% MT genes, the resulting 14932 cells, as we can see in figure   1A, can be divided into 20 cell clusters including 5

Sub cell clusters of NK, NKT, LTB high and CD4 T and characteristic of PT
As increased immune cells were observed in kidney biopsy of diseased patients comparing with that of donor ones and a special NK subtype expressing LTB exists, next we want to subdivide NK cell clusters to find whether there have some diseased associated subtypes of NK. As figure2A and 2B showed, there exists a subtype adjacent to NK/NKT cell cluster expressing IL7R, CD3D and CD3E instead of NKG7. In our data, besides LTB high cluster, the level of LTB was also higher in CD4 T of LN and CIN than that of other samples( Figure 2D).
Next we subdivide the PT cells. Interestingly, an inflammatory subtype named PT6 was found in our dataset( Figure 2H). Figure 2F show the top genes, defined as high fold change, of PT6.And we can find wnt-β catenin target genes(MMP7 AND MYC) was also activated in PT6( Figure 2H). Comparing with diseased samples, the number of PT1, PT4 and PT7 cell clusters was increased, while the number of other PT cell clusters was decreased in donor samples(Figure2G). Figure 2I show the cell cycle status of each cell clusters. B and NK cell seems to be at status of cell division comparing with other cell types.

GWAS genes and target genes of promising biologics in the specific cell clusters
The dotplot in Figure 3A shows that GWAS genes focus on clusters of glomerulus and immune cells. GWAS genes expressed across different samples were showed in   As more immune cells infiltrate the diseased sample.
Next, we want to find diseased associated NKT, and LTB high T cell cluster was found in our data, this cell cluster seems to enrich in IgAN, the up regulated genes in this cluster of IgAN comparing with that of donor was enriched in B cell activation, this suggests LTB high cell may have a role in B cell activation. While the data from humphreyslab show LTB level was higher in lymphocyte of diabetes than that of control. Their data also show LTB was expressed only in T and B cell cluster (18)(19)(20).
As an inducer of the inflammatory response system, LTB was also involved in normal development of lymphoid tissue (21). Mast cell, which has a profibrogenic role (22,23), expressing TPSB2 and TPSAB1 was easy to find in IgAN (24)

Conclusion
Single cell RNA-seq is a powerful tool for mining kidney disease associated genes and the activated signaling pathways the genes involved in at cellular level, and such datasets of nephritis were provided by this paper to some extent.

Competing interests：
The authors declare that they have no competing interests.

Availability of data and material
The datasets generated during the current study are available for uploading if required.

Consent for publication
Not applicable

Ethics approval and consent to participate
The patient described in this study consented under