Enhanced Expression of Janus Kinase–Signal Transducer and Activator of Transcription Pathway Members in Human Diabetic Nephropathy

OBJECTIVE—Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies

A Molecular Signature of Proteinuria in Glomerulonephritis

Proteinuria is the most important predictor of outcome in glomerulonephritis and experimental data suggest that the tubular cell response to proteinuria is an important determinant of progressive fibrosis in the kidney. However, it is unclear whether proteinuria is a marker of disease severity or has a direct effect on tubular cells in the kidneys of patients with glomerulonephritis. Accordingly we studied an in vitro model of proteinuria, and identified 231 “albumin-regulated genes” differentially expressed by primary human kidney tubular epithelial cells exposed to albumin. We translated these findings to human disease by studying mRNA levels of these genes in the tubulo-interstitial compartment of kidney biopsies from patients with IgA nephropathy using microarrays. Biopsies from patients with IgAN (n = 25) could be distinguished from those of control subjects (n = 6) based solely upon the expression of these 231 “albumin-regulated genes.” The expression of an 11-transcript subset related to the degree of proteinuria, and this 11-mRNA subset was also sufficient to distinguish biopsies of subjects with IgAN from control biopsies. We tested if these findings could be extrapolated to other proteinuric diseases beyond IgAN and found that all forms of primary glomerulonephritis (n = 33) can be distinguished from controls (n = 21) based solely on the expression levels of these 11 genes derived from our in vitro proteinuria model. Pathway analysis suggests common regulatory elements shared by these 11 transcripts. In conclusion, we have identified an albumin-regulated 11-gene signature shared between all forms of primary glomerulonephritis. Our findings support the hypothesis that albuminuria may directly promote injury in the tubulo-interstitial compartment of the kidney in patients with glomerulonephritis

Safety of procuring research tissue during a clinically indicated kidney biopsy from patients with lupus: data from the Accelerating Medicines Partnership RA/SLE Network

Objectives In lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis.Methods 475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines.Results 34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved.Conclusions Procurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required

Comparative Transcriptional Profiling of 3 Murine Models of SLE Nephritis Reveals Both Unique and Shared Regulatory Networks

<div><p>Objective</p><p>To define shared and unique features of SLE nephritis in mouse models of proliferative and glomerulosclerotic renal disease.</p><p>Methods</p><p>Perfused kidneys from NZB/W F1, NZW/BXSB and NZM2410 mice were harvested before and after nephritis onset. Affymetrix based gene expression profiles of kidney RNA were analyzed using Genomatix Pathway Systems and Ingenuity Pathway Analysis software. Gene expression patterns were confirmed using real-time PCR.</p><p>Results</p><p>955, 1168 and 755 genes were regulated in the kidneys of nephritic NZB/W F1, NZM2410 and NZW/BXSB mice respectively. 263 genes were regulated concordantly in all three strains reflecting immune cell infiltration, endothelial cell activation, complement activation, cytokine signaling, tissue remodeling and hypoxia. STAT3 was the top associated transcription factor, having a binding site in the gene promoter of 60/263 regulated genes. The two strains with proliferative nephritis shared a macrophage/DC infiltration and activation signature. NZB/W and NZM2410 mice shared a mitochondrial dysfunction signature. Dominant T cell and plasma cell signatures in NZB/W mice reflected lymphoid aggregates; this was the only strain with regulatory T cell infiltrates. NZW/BXSB mice manifested tubular regeneration and NZM2410 mice had the most metabolic stress and manifested loss of nephrin, indicating podocyte loss.</p><p>Conclusions</p><p>These findings identify shared inflammatory mechanisms of SLE nephritis that can be therapeutically targeted. Nevertheless, the heterogeneity of effector mechanisms suggests that individualized therapy might need to be based on biopsy findings. Some common mechanisms are shared with non-immune–mediated renal diseases, suggesting that strategies to prevent tissue hypoxia and remodeling may be useful in SLE nephritis.</p></div

A. One way cluster analysis of genes with significantly altered expression in the PCR validation set (See Table S3). Gene expression was scaled to the mean of pre-nephritic controls for each strain. Significantly up or downregulated (>2 fold) genes by SAM with q value <0.05 are shown (corresponding to 137 genes). B. Summary of the unique and shared pathogenic pathways identified in the kidneys of the three mouse strains.

<p>A. One way cluster analysis of genes with significantly altered expression in the PCR validation set (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077489#pone.0077489.s003" target="_blank">Table S3</a>). Gene expression was scaled to the mean of pre-nephritic controls for each strain. Significantly up or downregulated (>2 fold) genes by SAM with q value <0.05 are shown (corresponding to 137 genes). B. Summary of the unique and shared pathogenic pathways identified in the kidneys of the three mouse strains.</p

Literature-based analysis of limited gene expression patterns using Genomatix Pathway System (GePS) software.

<p><b>A</b>. 103 shared genes were regulated in the same direction in the nephritic vs. prenephritic kidneys in NZW/BXSB and NZB/W mouse models. The picture shows the 72 that were co-cited in the same sentence of PubMed abstracts. <b>B</b>. 240 genes were regulated in the same direction in the nephritic vs. prenephritic kidneys in NZB/W and NZM2410 mouse models. The picture shows the 124 genes that were co-cited in PubMed abstracts in the same sentence. Orange represents the genes that are upregulated and green represents the genes that are downregulated in nephritic compared to prenephritic mice.</p

A. Glomerular and interstitial damage scores in pre-nephritic (Pre) and nephritic (N) mice (mean + SD) of NZB/W (B/W), NZW/BXSB (W/B) and NZM2410 NZM) strains (* p<0.001; ** p<0.01). The high tubulointerstitial score in the NZM2410 strain reflects severe tubular atrophy. B. Shared and unique gene expression profiles of each of the three strains. Parentheses indicate the number of genes with a human ortholog. C. 3D principal component analysis from the 14780 genes passing the cutoff value (see Materials and Methods) after normalization and batch correction of the arrays from the 3 mouse strains together.

<p>A. Glomerular and interstitial damage scores in pre-nephritic (Pre) and nephritic (N) mice (mean + SD) of NZB/W (B/W), NZW/BXSB (W/B) and NZM2410 NZM) strains (* p<0.001; ** p<0.01). The high tubulointerstitial score in the NZM2410 strain reflects severe tubular atrophy. B. Shared and unique gene expression profiles of each of the three strains. Parentheses indicate the number of genes with a human ortholog. C. 3D principal component analysis from the 14780 genes passing the cutoff value (see Materials and Methods) after normalization and batch correction of the arrays from the 3 mouse strains together.</p

Expression and significance of selected genes.

<p>The genes passing the defined filter criteria are highlighted in bold. np: genes not passing the Affymetrix negative controls cut-off. In italic are the genes not significantly regulated (q-value>0.05).</p

A–L. Flow cytometry analysis of proliferating cells. After gating for singlets (A), whole kidney cells from prenephritic (Pre - B, F) and nephritic (Neph - C, G, I, J) NZW/BXSB and nephritic NZB/W (D, H, K, L) mice were analyzed for BrDU incorporation. A BrDU⁺/F4/80⁻ population is seen only in nephritic NZW/BXSB mice (F–H). Proliferating CD11b⁺/F4/80⁺ macrophages are observed only in nephritic NZB/W mice (I–L). A non-BrDU treated control is shown in E. M–P. Immunohistochemistry of kidneys from nephritic NZW/BXSB (M, N), 8 week NZW/BXSB (O), and nephritic NZB/W (P) mice stained with antibodies to Ki67 (M) and PCNA (N–P). 40× magnification. Data are representative of 3 mice per stain.

<p>A–L. Flow cytometry analysis of proliferating cells. After gating for singlets (A), whole kidney cells from prenephritic (Pre - B, F) and nephritic (Neph - C, G, I, J) NZW/BXSB and nephritic NZB/W (D, H, K, L) mice were analyzed for BrDU incorporation. A BrDU⁺/F4/80⁻ population is seen only in nephritic NZW/BXSB mice (F–H). Proliferating CD11b⁺/F4/80⁺ macrophages are observed only in nephritic NZB/W mice (I–L). A non-BrDU treated control is shown in E. M–P. Immunohistochemistry of kidneys from nephritic NZW/BXSB (M, N), 8 week NZW/BXSB (O), and nephritic NZB/W (P) mice stained with antibodies to Ki67 (M) and PCNA (N–P). 40× magnification. Data are representative of 3 mice per stain.</p