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.

Subchronic oral exposure of tungsten induces myofibroblast transformation and various markers of kidney fibrosis.

Tungsten is a naturally occurring transition element used in a broad range of applications. As a result of its extensive use, we are increasingly exposed to tungsten from our environment, including potable water, since tungsten can become bioaccessible in ground sources. The kidneys are particularly susceptible to tungsten exposure as this is the main site for tungsten excretion. In this study, we investigated the prolonged effects of tungsten on the kidneys and how this may impact injury and function. When mice were exposed to tungsten in their drinking water for 1 mo, kidney function had not significantly changed. Following 3-mo exposure, mice were presented with deterioration in kidney function as determined by serum and urine creatinine levels. During 3 mo of tungsten exposure, murine kidneys demonstrated significant increases in the myofibroblast marker α-smooth muscle actin (αSMA) and extracellular matrix products: fibronectin, collagen, and matricellular proteins. In addition, Masson's trichrome and hematoxylin-eosin (H&E) staining revealed an increase in fibrotic tissue and vacuolization of tubular epithelial cells, respectively, from kidneys of tungsten-treated mice, indicative of renal injury. In vitro treatment of kidney fibroblasts with tungsten led to increased proliferation and upregulation of transforming growth factor ß1 (TGFß1), which was consistent with the appearance of fibroblast-to-myofibroblast transition (FMT) markers. Our data suggest that continuous exposure to tungsten impairs kidney function that may lead to the development of chronic kidney disease (CKD).

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.

Alk1 haploinsufficiency causes glomerular dysfunction and microalbuminuria in diabetic mice.

Endothelial dysfunction has been shown to play an important role in the pathogenesis of glomerular damage during diabetic kidney disease (DKD). As such, a better understanding of the molecular mechanisms involved in glomerular endothelial dysfunctions could provide novel therapeutic strategies for the prevention of DKD. We have previously shown that Alk1/BMP9 signaling plays an important function to maintain vascular integrity in diabetic animals. As such, we evaluated the effects of Alk1 suppression on glomerular endothelial function in diabetic mice. In the present study, we used mice with conditional heterozygote deletion of Alk1 in the endothelium (Alk1ΔEC) to evaluate the role of Alk1 on kidney function during STZ-induced diabetes. DKD was investigated in diabetic control and Alk1ΔEC mice euthanized eight weeks after the onset of diabetes. We showed that Alk1 expression is reduced in the glomeruli of human DKD patients. While renal function was not altered in Alk1ΔEC non-diabetic mice, we showed that Alk1 haploinsufficiency in the glomerular endothelium leads to microalbuminuria, thickening of the glomerular basement membrane, glomerular apoptosis and podocyte loss in diabetic mice. These data suggest that Alk1 is important for the proper function of glomerular endothelial cells and that decreased Alk1 combined with chronic hyperglycemia can impair renal function.

Characterization of Matricellular Protein Expression Signatures in Mechanistically Diverse Mouse Models of Kidney Injury.

Fibrosis is the most common pathophysiological manifestation of Chronic Kidney Disease (CKD). It is defined as excessive deposition of extracellular matrix (ECM) proteins. Embedded within the ECM are a family of proteins called Matricellular Proteins (MCPs), which are typically expressed during chronic pathologies for ECM processing. As such, identifying potential MCPs in the pathological secretome of a damaged kidney could serve as diagnostic/therapeutic targets of fibrosis. Using published RNA-Seq data from two kidney injury mouse models of different etiologies, Folic Acid (FA) and Unilateral Ureteral Obstruction (UUO), we compared and contrasted the expression profile of various members from well-known MCP families during the Acute and Fibrotic injury phases. As a result, we identified common and distinct MCP expression signatures between both injury models. Bioinformatic analysis of their differentially expressed MCP genes revealed similar top annotation clusters from Molecular Function and Biological Process networks, which are those commonly involved in fibrosis. Using kidney lysates from FA- and UUO-injured mice, we selected MCP genes from our candidate list to confirm mRNA expression by Western Blot, which correlated with injury progression. Understanding the expressions of MCPs will provide important insight into the processes of kidney repair, and may validate MCPs as biomarkers and/or therapeutic targets of CKD.

Mechanism of insulin resistance in a rat model of kidney disease and the risk of developing type 2 diabetes.

Chronic kidney disease is associated with homeostatic imbalances such as insulin resistance. However, the underlying mechanisms leading to these imbalances and whether they promote the development of type 2 diabetes is unknown. The effect of chronic kidney disease on insulin resistance was studied on two different rat strains. First, in a 5/6th nephrectomised Sprague-Dawley rat model of chronic kidney disease, we observed a correlation between the severity of chronic kidney disease and hyperglycemia as evaluated by serum fructosamine levels (p<0.0001). Further, glucose tolerance tests indicated an increase of 25% in glycemia in chronic kidney disease rats (p<0.0001) as compared to controls whereas insulin levels remained unchanged. We also observed modulation of glucose transporters expression in several tissues such as the liver (decrease of ≈40%, p≤0.01) and muscles (decrease of ≈29%, p≤0.05). Despite a significant reduction of ≈37% in insulin-dependent glucose uptake in the muscles of chronic kidney disease rats (p<0.0001), the development of type 2 diabetes was never observed. Second, in a rat model of metabolic syndrome (Zucker Leprfa/fa), chronic kidney disease caused a 50% increased fasting hyperglycemia (p<0.0001) and an exacerbated glycemic response (p<0.0001) during glucose challenge. Similar modulations of glucose transporters expression and glucose uptake were observed in the two models. However, 30% (p<0.05) of chronic kidney disease Zucker rats developed characteristics of type 2 diabetes. Thus, our results suggest that downregulation of GLUT4 in skeletal muscle may be associated with insulin resistance in chronic kidney disease and could lead to type 2 diabetes in predisposed animals.

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.

Inflammatory response to peritoneal implantation of alginate-poly-L-lysine microcapsules.

A thorough understanding of the mechanisms involved in the host reaction to alginate-poly-L-lysine microcapsules (HRM) is important to design methods for the evaluation, selection, and development of biocompatible biomaterials and microcapsules or treatments to control this reaction. The objective of this study was to identify those immune cells and cytokines involved in the pathogenesis of the HRM. The total and differential cell counts were evaluated, and the mRNA expression of TNF-alpha, IL-1beta, IL-6 and TGF-beta1 was measured in peritoneal washings at 3, 17, 48, 96 and 168 h after saline or microcapsule injections. Neutrophil number and IL-1beta and IL-6 m-RNA expression presented an early transient increase, with no differences between saline and microcapsule injections, suggesting a reaction to the procedure. Macrophages, lymphocytes and TNF-alpha were significantly more activated over a longer period of time, after microcapsule implantation than saline injection. They are likely involved in transforming the reaction into a chronic inflammatory process. TGF-beta1 and IL-1beta presented a late (day 7) significant increase after microcapsule but not saline injections. They are likely involved in transforming the reaction into a fibrogenic process. These results suggest that macrophages, lymphocytes, TNF-alpha, IL-1beta and TGF-beta1 play a role in the pathogenesis of the HRM.

Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers.

Microencapsulation in semi-permeable membranes protects transplanted cells against immune destruction. Microcapsule strength is critical. We describe a method to microencapsulate living cells in alginate-poly-L-lysine (PLL)-alginate membranes with covalent links between adjacent layers of microcapsule membranes, while preserving the desired membrane molecular weight cut-off (MWCO) and microencapsulated cell viability. A heterobifunctional photoactivatable cross-linker, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) was used. The N-hydroxysuccinimide ester group of ANB-NOS was covalently linked to PLL. Islets of Langerhans were immobilized in alginate beads, incubated in PLL-ANB-NOS and again in alginate. Upon illumination with UVA, covalent links were created between the phenyl azide residue of ANB-NOS and alginate from both the core bead and the outer coating. Covalently linked microcapsules remained intact after 3 years in a strong alkaline buffer (pH 12), whereas standard microcapsules disappeared within 45 s in the same solution. A standardized mechanical stress broke 22-fold more standard than covalently linked microcapsules. The MWCO and microencapsulated cell viability were similar with standard and covalently linked microcapsules. These microcapsules, extremely resistant to chemical and mechanical stresses, will be useful in numerous applications.

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.

Insulin-like growth factor II allows prolonged blood glucose normalization with a reduced islet cell mass transplantation.

IGF-II has been reported to decrease neonatal islet cell apoptosis and in vitro adult islet cell necrosis and apoptosis, but the usefulness of IGF-II in a transplantation setting is unknown. We evaluated the effect of in vitro IGF-II incubations on microencapsulated rat islet survival both in vitro and in minimal mass transplantations into diabetic mice. After 6 d in culture, fresh examinations, histology, fluorescence microscopy, sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)-benzene sulfonic acid hydrate assay, and apoptosis studies all indicated that IGF-II significantly improves islet cell viability in a dose-dependent fashion. IGF-II 100 ng/ml and 500 ng/ml induced a 51% and 83% increase of viable islets (P = 0.052, P < 0.01). A 20%, 29%, and 33% reduction of the apoptotic index was observed with 50, 100, and 500 ng/ml incubations respectively (P < 0.05; P < 0.005; P < 0.001). Ten weeks after transplantation of 150 encapsulated rat islet equivalents incubated with IGF-II 500 ng/ml, 80% of diabetic mice were normoglycemic. Without IGF-II preincubation, only 8% of the recipients remained normoglycemic with the transplantation of 150 islets and 42% with 300 islets (P < 0.05). In conclusion, IGF-II promotes islet cell survival, and allows successful transplantation using a smaller number of islets.