Identification of inflammatory biomarkers in IgA nephropathy using the NanoString technology: a validation study in Caucasians.

OBJECTIVE AND DESIGN: Immunoglobulin A nephropathy (IgAN) is a kidney disease characterized by the accumulation of IgA deposits in the glomeruli of the kidney, leading to inflammation and damage to the kidney. The inflammatory markers involved in IgAN remain to be defined. Gene expression analysis platforms, such as the NanoString nCounter system, are promising screening and diagnostic tools, especially in oncology. Still, their role as a diagnostic and prognostic tool in IgAN remains scarce. In this study, we aimed to validate the use of NanoString technology to identify potential inflammatory biomarkers involved in the progression of IgAN. SUBJECTS: A total of 30 patients with biopsy-proven IgAN and 7 cases of antineutrophil cytoplasmic antibody (ANCA)-associated pauci-immune glomerulonephritis were included for gene expression measurement. For the immunofluorescence validation experiments, a total of 6 IgAN patients and 3 controls were included. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded kidney biopsy specimens, and a customized 48-plex human gene CodeSet was used to study 29 genes implicated in different biological pathways. Comparisons in gene expression were made between IgAN and ANCA-associated pauci-immune glomerulonephritis patients to delineate an expression profile specific to IgAN. Gene expression was compared between patients with low and moderate risk of progression. Genes for which RNA expression was associated with disease progression were analyzed for protein expression by immunofluorescence and compared with controls. RESULTS: IgAN patients had a distinct gene expression profile with decreased expression in genes IL-6, INFG, and C1QB compared to ANCA patients. C3 and TNFRSF1B were identified as potential biomarkers for IgAN progression in patients early in their disease course. Protein expression for those 2 candidate genes was upregulated in IgAN patients compared to controls. Expression of genes implicated in fibrosis (PTEN, CASPASE 3, TGM2, TGFB1, IL2, and TNFRSF1B) was more pronounced in IgAN patients with severe fibrosis compared to those with none. CONCLUSIONS: Our findings validate our NanoString mRNA profiling by examining protein expression levels of two candidate genes, C3 and TNFRSF1B, in IgAN patients and controls. We also identified several upregulated mRNA transcripts implicated in the development of fibrosis that may be considered fibrotic markers within IgAN patients.

Tungsten toxicity on kidney tubular epithelial cells induces renal inflammation and M1-macrophage polarization.

Tungsten is widely used in medical, industrial, and military applications. The environmental exposure to tungsten has increased over the past several years, and few studies have addressed its potential toxicity. In this study, we evaluated the effects of chronic oral tungsten exposure (100 ppm) on renal inflammation in male mice. We found that 30- or 90-day tungsten exposure led to the accumulation of LAMP1-positive lysosomes in renal tubular epithelial cells. In addition, the kidneys of mice exposed to tungsten showed interstitial infiltration of leukocytes, myeloid cells, and macrophages together with increased levels of proinflammatory cytokines and p50/p65-NFkB subunits. In proximal tubule epithelial cells (HK-2) in vitro, tungsten induced a similar inflammatory status characterized by increased mRNA levels of CSF1, IL34, CXCL2, and CXCL10 and NFkB activation. Moreover, tungsten exposure reduced HK-2 cell viability and enhanced reactive oxygen species generation. Conditioned media from HK-2 cells treated with tungsten induced an M1-proinflammatory polarization of RAW macrophages as evidenced by increased levels of iNOS and interleukin-6 and decreased levels of the M2-antiinflammatory marker CD206. These effects were not observed when RAW cells were exposed to conditioned media from HK-2 cells treated with tungsten and supplemented with the antioxidant N-acetylcysteine (NAC). Similarly, direct tungsten exposure induced M1-proinflammatory polarization of RAW cells that was prevented by NAC co-treatment. Altogether, our data suggest that prolonged tungsten exposure leads to oxidative injury in the kidney ultimately leading to chronic renal inflammation characterized by a proinflammatory status in kidney tubular epithelial cells and immune cell infiltration.

SMOC2 promotes an epithelial-mesenchymal transition and a pro-metastatic phenotype in epithelial cells of renal cell carcinoma origin.

Renal Cell Carcinoma (RCC) is the most common form of all renal cancer cases, and well-known for its highly aggressive metastatic behavior. SMOC2 is a recently described non-structural component of the extracellular matrix (ECM) that is highly expressed during tissue remodeling processes with emerging roles in cancers, yet its role in RCC remains elusive. Using gene expression profiles from patient samples, we identified SMOC2 as being significantly expressed in RCC tissue compared to normal renal tissue, which correlated with shorter RCC patient survival. Specifically, de novo protein synthesis of SMOC2 was shown to be much higher in the tubular epithelial cells of patients with biopsy-proven RCC. More importantly, we provide evidence of SMOC2 triggering kidney epithelial cells into an epithelial-to-mesenchymal transition (EMT), a phenotype known to promote metastasis. We found that SMOC2 induced mesenchymal-like morphology and activities in both RCC and non-RCC kidney epithelial cell lines. Mechanistically, treatment of RCC cell lines ACHN and 786-O with SMOC2 (recombinant and enforced expression) caused a significant increase in EMT-markers, -matrix production, -proliferation, and -migration, which were inhibited by targeting SMOC2 by siRNA. We further characterized SMOC2 activation of EMT to occur through the integrin ß3, FAK and paxillin pathway. The proliferation and metastatic potential of SMOC2 overexpressing ACHN and 786-O cell lines were validated in vivo by their significantly higher tumor growth in kidneys and systemic dissemination into other organs when compared to their respective controls. In principle, understanding the impact that SMOC2 has on EMT may lead to more evidence-based treatments and biomarkers for RCC metastasis.

Changes in Urinary and Serum Levels of Novel Biomarkers after Administration of Gadolinium-based Contrast Agents.

OBJECTIVE: The aim of our study is to describe the changes in urinary and serum levels of novel biomarkers after gadolinium contrast administration in patients with normal renal function. METHODS: We measured four biomarkers in 28 volunteers: interleukin-18 (IL-18), N-acetyl-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin, and cystatin C. Urinary and serum samples were collected at 0, 3, and 24 hours following gadolinium administration. RESULTS: Baseline serum creatinine was 57.8 ± 34.5 µmol/L and remained stable. Urinary IL-18 levels increased significantly at three hours (10.7 vs. 7.3 ng/mg creatinine; P < 0.05). Similarly, urinary NAG levels increased significantly at three hours (3.9 vs. 2.2 IU/mg creatinine; P < 0.001). For both these markers, the difference was no longer significant at 24 hours. No statistically significant differences were observed for urinary and serum neutrophil gelatinase-associated lipocalin levels and for serum cystatin C levels. CONCLUSIONS: Urinary IL-18 and NAG levels increased transiently after administration of gadolinium-based contrast agents in patients with normal renal function.

Effect of poly-L-lysine coating on macrophage activation by alginate-based microcapsules: assessment using a new in vitro method.

The characteristics of the microcapsule surface, which interacts directly with the host macrophages, may have a role in the biocompatibility of alginate-poly-L-lysine (PLL)-alginate (APA) microcapsule. The objectives of the study were: 1) to develop and validate a simple, rapid, and sensitive in vitro method for assessing microcapsule biocompatibility, based on microcapsule coincubation with macrophages and measurement, by reverse transcriptase-polymerase chain reaction, of cytokine mRNA expression, and 2) to evaluate the effect of alginate purification and PLL coating on macrophage activation. The mRNA expression of tumor necrosis factor-alpha and interleukin-1beta was significantly higher when macrophages were coincubated with beads made with nonpurified compared with purified alginate (p<0.01, p<0.05, respectively) and negative control (p<0.001) or with APA microcapsules compared with non-PLL-coated alginate beads and negative control (p<0.001). The mRNA expression of interleukin-6 differed significantly only when APA microcapsules were compared with a negative control (p<0.05). These results confirm that alginate purification improves microcapsule biocompatibility, and suggest that PLL is not completely covered and/or neutralized by the second alginate incubation and thus has a role in the host macrophage activation. The assay is sensitive to both alginate contaminants and microcapsule surface characteristics and may be a useful tool for the development of biocompatible microcapsules.