Chronic inflammation is a silent killer, contributing to death in patients with chronic kidney disease (CKD) and other chronic disorders, such as diabetes and obesity. Although the specific molecular pathways linking inflammation contribute to CKD pathogenesis still remain unclear, interleukin-6 (IL-6), the prototypic inflammatory biomarker and a potential mediator, is believed to be able to help us understand the mechanism that leads to CKD. We have been investigating the role of IL-6 family of genes using different animal models, such as the Unilateral Ureteral Obstruction (UUO), to induce interstitial fibrosis. The results from our studies have demonstrated that IL-6 and gp130 are profibrotic molecules. Therefore, our goal was to understand the mechanisms of IL-6 in myofibroblasts, the major contributors of collagen during fibrosis. For these studies, we used wild-type mice, IL6 (+/+), IL6 (-/-) and αSMA-Cre-IL-6(fl/fl), in the UUO model of interstitial fibrosis. We hypothesized that IL-6 regulates the crosslinking of collagen by regulating the lysyl-oxidase gene. Therefore, we assessed the Extracellular matrix expansion and collagen accumulation by measuring the changes in gene expression of Col-1, 3, and 4, IL-6, Lysyl-oxidase, Fli-1 and Hells at different time points during renal fibrogenesis. We consider that the change in collagen crosslinking is an important factor in the turnover rate of collagen and it changes its stability to be degraded/accumulated. The examination of the role of IL-6 could potentially provide a new therapeutic breakthrough for treatment and better detection/assessment of renal fibrosis.

Many occurrences of glomerulonephritis present recurrent acute inflammation episodes. With time these conditions often lead to progressive fibrosis and chronic renal failure. My project studies the role of Interleukin-6 (IL-6), one of the most proinflammatory cytokines, using the nephrotoxic serum (NTS) nephritis mouse model. The ubiquitous expression of IL-6 makes it a challenge to study this interleukin. We have genetically engineered mice with ablated IL-6 in the F4/80+ macrophages and the Ytgax (CD11c+) dendritic cells. We hypothesize a greater level of glomerular damage and disease in the wild type mice compared to the IL-6 depleted mice. IL-6 +/+ (IL6WT) and -/- mice (IL6KO) were injected with NTS in the tail vein and placed in metabolic cages for seven days. Urine was collected to measure renal failure and progression of the disease. Mice were sacrificed at 7 days after the injection and both kidneys harvested and sectioned. Extraction, quantification and quality analysis (RNA integrity number) from the IL6WT and IL6KO frozen kidney samples were conducted and later cDNA synthesized. Quantitative PCR (qPCR) reactions were carried out to analyze gene expression of IL-6 family genes and pathological analysis of frozen or paraffin embedded tissue was done to assess glomerular and interstitial damage. Preliminary results from histopathology analysis and qPCR suggest a significant role played by IL-6 in glomerular damage. IL6WT diseased mice show upregulated expression of IL-6 and greater degree of disease relative to the IL6KO. We plan to continue gene expression quantifications and perform immunostaining to compile more data and validate our results. These findings will help to establish the efficacy of IL-6 neutralization as a therapeutic option for kidney disease.

Mast cells regulate both inflammatory responses and tissue repair in human diseases but there are conflicting reports on the role of these cells in the pathogenesis of various kidney diseases. Interleukin-6 (IL-6) is an interleukin that acts as both a pro-inflammatory and anti-inflammatory cytokine. From our preliminary studies we have demonstrated that IL-6 and its receptor gp130 are pro-fibrotic in renal fibrogenesis. Here we measure mast cell function in unilateral urethral obstruction, a well-characterized model of renal fibrosis, using mice in which Cre recombinase is expressed under the control of a segment of the carboxypeptidase A3 (Cpa3) promoter bred to IL-6 flox/flox mice. Mast cells from the C57BL/6 Cpa3-IL-6 (fl/fl) mice do not express IL-6. We have groups of 10 for each of the following strains: control1; Cpa3-Cre, control2; IL-6fl/fl and experimental mice; Cpa3-IL-6. Mice under general anesthesia had the ureter ligated at 2 sites distally from the kidney and sacrificed after 14d. We measured the level of fibrosis by measuring hydroxyproline colorimetrically and by measuring area of Picro Sirius red in stained kidney sections, and quantified by pixel analysis. Our preliminary results indicate that the absence of IL-6 in mast cells is important in renal fibrosis. When IL-6 was ablated from mast cells the level of fibrosis measured by collagen and the expansion of extracellular matrix was reduced. These preliminary results suggest that mast cell-derived IL-6 promotes renal fibrosis after unilateral urethral obstruction.