A Mechanistic Study of Metal-Induced Nephrotoxicity: Metal-Induced Immune System Reactivity Towards Kidney ProteinsEPA Grant Number: U914752
Title: A Mechanistic Study of Metal-Induced Nephrotoxicity: Metal-Induced Immune System Reactivity Towards Kidney Proteins
Investigators: Matheson, Joanna M.
Institution: New York University Medical Center
EPA Project Officer: Broadway, Virginia
Project Period: January 1, 1995 through January 1, 1996
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1995) Recipients Lists
Research Category: Fellowship - Health , Health Effects , Academic Fellowships
The objective of this research project is to conduct in vivo (with animals) and in vitro experiments to address the following hypotheses:
1. Determine whether nephrotoxic metals can alter the immunogenicity of kidney proteins and induce an autoantibody response against native kidney proteins.
2. Determine whether nephrotoxic metals induce kidney damage by acting directly on the kidney or by activating an immune response against kidney proteins.
3. Determine the role of cytokines in the pathogenesis of metal-induced renal damage.
C57BL mice have been used for our preliminary studies, and will be used for the studies to investigate the three hypotheses above. C57BL mice also have been used by others for characterizing mouse glomerular basement membrane (GBM) in studies with metal exposures, and the cells from these animals have been used in various culture systems (including cultures with kidney cells). Mice are quarantined for 2 weeks and acclimated to a 12-hour light/dark cycle. The animals are housed in groups of three in polycarbonate boxes containing wood chip bedding with stainless steel lids. Food (Purina Rodent Chow) and water will be provided ad libitum. After the acclimation period, animals will be immunized, exposed to metals in the drinking water, or utilized for in vitro experiments. An n = 6 will be used for all exposure groups.
Immunizations (i.p.) will be conducted with metal-treated laminin, native laminin, cadmium, or vehicle (0.9 percent saline). The metals to be used are cadmium, mercury, chromium, and nickel. Metal treatment of the laminin will be prepared by incubating a 100:1 molar ratio (metal to protein) at 2-8°C for 24 hours. Twenty-five µg of protein will be administered at each immunization. (Preliminary studies have shown this to be an adequate dose to induce antibody production.) Two weeks after the initial immunization, the first booster is administered, with the second booster given 2 weeks after the first booster. Animals will be sacrificed 10 days (to correspond to peak antibody levels) after the second booster.
Mice will be exposed for 4 weeks to 100 ppm CdCl2, K2CrO4, HgCl2, PbOAc, or NiCl2 in drinking water. Preliminary studies with CdCI2 (100 ppm) and K2CrO4 (100 ppm) have demonstrated significant elevations of IL-6 levels and IgG antibodies to laminin. After the immunization and drinking water protocols have been conducted, animals will be injected with approximately 0.1 mL Nembutal and blood removed via cardiac puncture. Serum will be separated from the blood and stored at –70°C until used for the measurement of serum creatinine and serum urea nitrogen levels (Sigma). The serum also will be tested for the presence of autoantibodies to laminin, collagen IV, and GBM via enzyme-linked immunosorbent assay (ELISA). To determine specificity of the autoimmune response to renal proteins, control plates using a neural protein such as myelin basic protein also will be run. GBM will be isolated with modifications to the protocol outlined by Henry. Spleens and kidneys will be removed. Cell suspensions will be made from the spleens so that lymphoproliferative assays to LPS and ConA will be performed (MTT-tetrazolium assay, Sigma). Additionally, the supernatants of ConA- and LPS-stimulated lymphocytes will be analyzed by ELISA (Genzyme and PharMingen) for the presence and quantity of IL-2 (culture days 1-3) and IL-6 (culture days 2-4), respectively. IL-2 and IL-6 are being focused on because of their roles in promoting secretion of antibodies by activated B-cells and as a B-cell differentiation factor, respectively. To determine immune activity in situ in the kidney, the left kidney is bisected; half is homogenized in hot 1 percent SDS and stored at -70°C for use in measuring total protein (Pierce BCA assay) and acid-elution of antibodies to laminin, collagen IV, and GBM. The other half is fixed in formalin and embedded in paraffin for histology (hematoxylin and eosin, periodic acid-Schiff) to detect renal lesions.
Immunocytochemistry also will be performed on these sections for the detection of autoantibodies (VectaStain ABC method) and the cytokines IL-2 and IL-6 (APAAP method, fast red TR/naphthol AS-MX). Sections will be examined by light microscopy to determine intensity and localization of staining. The right kidney also is bisected; half is homogenized in hot 1 percent SDS and frozen at -70°C until use. The other half will have the cortex removed and homogenized in the hot 1 percent SDS and frozen. Total protein in the homogenates will be assayed (Pierce BCA assay). Cytokine levels (IL-2 and IL-6) also will be determined in these right kidney homogenates via a modification of the dot-immunobinding method utilized by O’Callaghan (Minifold II slot-blot).
Supernatants will be obtained by culturing splenic lymphocytes pooled from six C57BL/6J mice ± LPS and ConA in 24 well microtiter plates. Solutions of metal salts (CdCl2, K2CrO4, HgCl2, and NiCl2) using concentrations of 100 ppm, 10 ppm, 1 ppm, 100 ppb and 10 ppb will be added to wells. Supernatants will be collected on days 1, 2, and 3 for ConA-stimulated plates and on days 2, 3, and 4 for LPS-stimulated plates. Cytokine levels (IL-2 and IL-6) in the supernatants will be measured via ELISA.
A short-term coculture system will be established for the measurement of the interaction of renal glomerular cells with supernatants from spleen cells isolated from animals treated in vivo with one of three metal salts (CdCl2, HgCl2, PbOAc). Following the culture, supernatants will be collected, centrifuged, and stored at -70°C until assayed for cytokine levels (IL-2, IL-6) by ELISA. Kidney cell pathology will be assessed as a marker of splenocyte-initiated damage. The culture time period will be 8 days because the glomerular mesangial cells will overtake the culture after 9-10 days.
Significant increases in serum IgG and IgA antibodies against collagen IV were seen in mice immunized with Cd-treated laminin. Significant elevations of antibodies against laminin, GBM and collagen IV also were observed in the splenic cell supernatants of animals immunized with Cd-treated laminin.
Significant dose-response suppressions were seen in IL-2 and IL-6 production for spleen cell suspensions exposed to K2CrO4 and CdCl2 down to concentrations of 100 ppb (IL-6) and 10 ppb (IL-2) for K2CrO4; and, 2.5 ppm (IL-6) and 25 ppb (IL-2) for CdCl2 exposure. Spleen cells as indicated by IL-2 and IL-6 levels were more sensitive to the effects of K2CrO4 than CdCl2.