CD248+ stromal cells are associated with progressive chronic kidney disease

Stromal fibroblasts are the primary cells of the kidney that produce fibrotic matrix. CD248 is a stromal marker expressed on fibroblasts and pericytes within the human kidney. Here, we tested whether CD248 expression in the kidney colocalizes with fibrosis and if it is associated with known determinants of chronic kidney disease (CKD). CD248 expression was located and quantified in situ by immunohistochemistry in kidney biopsies from 93 patients with IgA nephropathy and compared with 22 archived biopsies encompassing normal kidney tissue as control. In normal kidney tissue, CD248 was expressed by resident pericytes, stromal fibroblasts, and was upregulated in human CKD. The expression was linked to known determinants of renal progression. This relationship was maintained in a multivariate analysis with CD248 expression linked to renal survival. CD248 was expressed by a population of α-smooth muscle actin (SMA)+ myofibroblasts and α-SMA− stromal cells but not expressed on CD45+ leukocytes. Thus, CD248 defines a subset of stromal cells, including but not limited to some myofibroblasts, linked to albuminuria and tubulointerstitial damage during tissue remodeling in CKD

Identifying cell enriched miRNAs in kidney injury and repair

Small noncoding RNAs, miRNAs (miRNAs), are emerging as important modulators in the pathogenesis of kidney disease, with potential as biomarkers of kidney disease onset, progression, or therapeutic efficacy. Bulk tissue small RNA-sequencing (sRNA-Seq) and microarrays are widely used to identify dysregulated miRNA expression but are limited by the lack of precision regarding the cellular origin of the miRNA. In this study, we performed cell-specific sRNA-Seq on tubular cells, endothelial cells, PDGFR-β+ cells, and macrophages isolated from injured and repairing kidneys in the murine reversible unilateral ureteric obstruction model. We devised an unbiased bioinformatics pipeline to define the miRNA enrichment within these cell populations, constructing a miRNA catalog of injury and repair. Our analysis revealed that a significant proportion of cell-specific miRNAs in healthy animals were no longer specific following injury. We then applied this knowledge of the relative cell specificity of miRNAs to deconvolute bulk miRNA expression profiles in the renal cortex in murine models and human kidney disease. Finally, we used our data-driven approach to rationally select macrophage-enriched miR-16-5p and miR-18a-5p and demonstrate that they are promising urinary biomarkers of acute kidney injury in renal transplant recipients

In situ analysis of C-C chemokine mRNA in human glomerulonephritis

In situ analysis of C-C chemokine mRNA in human glomerulonephritis.BackgroundGlomerular and tubulointerstitial accumulations of macrophages and T cells are a prominent feature of immune inflammatory glomerulonephritis. The C-C family of chemokines are major mononuclear-cell chemoattractants and may be central to the recruitment of these cells.MethodsUsing in situ hybridization (ISH) we analyzed the expression of mRNA for the C-C chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α and β (MIP-1α, MIP-1β) and RANTES in renal biopsy material from twenty patients with glomerulonephritis.ResultsIn overt inflammatory glomerulonephritides, chemokine transcripts were differentially expressed by glomerular and tubulointerstitial leukocyte infiltrates, glomerular parietal and proximal tubular epithelial cells and endothelial cells. There was little expression in minimal change nephropathy and normal tissue. Expression of individual chemokines correlated with intrarenal T cell and macrophage infiltrates. Combined immunohistochemistry and ISH demonstrated that 56.9% of cells expressing MCP-1 mRNA were CD68+ve (monocytes/macrophages) and 53% of infiltrating CD68 +ve cells were MCP-1 mRNA positive.ConclusionsThese studies indicate that the in situ production of C-C chemokines by resident and infiltrating cells may play a crucial role in regulating macrophage and T-cell recruitment in glomerulonephritis

Mechanisms of ANCA-Mediated Leukocyte-Endothelial Cell Interactions In Vivo

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-α, IL-1β, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-α led to the greatest recruitment of inflammatory cells, and IL-1β led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor γ chain−/− mice. This study provides direct in vivo evidence of enhanced leukocyte–endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcγ receptors and β2 integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds

Antineutrophil Cytoplasmic Antibodies Induce Reactive Oxygen-Dependent Dysregulation of Primed Neutrophil Apoptosis and Clearance by Macrophages

This study assessed whether anti-neutrophil cytoplasmic antibodies (ANCAs) interfere with the safe deletion of neutrophils by apoptosis and phagocytic clearance. Tumor necrosis factor (TNF)-primed neutrophils were incubated with normal IgG (N IgG) or ANCA IgG for up to 36 hours. Compared with N IgG, ANCAs accelerated constitutive apoptosis of TNF-α primed neutrophils, as assessed by morphology and confirmed by DNA laddering pattern on gel electrophoresis, and accelerated progression to secondary necrosis. The accelerated apoptosis induced by ANCA was dependent on reactive oxygen species generation, as primed neutrophils from patients with chronic granulomatous disease failed to show an effect of ANCAs on apoptosis. However, there was no change in the rate at which neutrophils exhibited annexin V binding, indicating that externalization of phosphatidylserine was not accelerated by ANCAs. Furthermore, when ANCA-treated primed neutrophils were interacted with human or murine peritoneal macrophages after 12 hours there was significantly less phagocytosis by human macrophages and no difference in phagocytosis by murine peritoneal-derived macrophages when compared with N IgG-treated controls. In conclusion, ANCAs accelerate apoptosis and secondary necrosis in TNF-primed neutrophils by a mechanism dependent on the generation of reactive oxygen species, with uncoupling of nuclear and surface membrane changes, resulting in a “reduced window of opportunity” for phagocytic recognition and engulfment before disintegration