Curcumin alleviates immune-complex-mediated glomerulonephritis in factor-H-deficient mice

Reagents

Curcumin was purchased from Calbiochem (Billerica, MA). Horse spleen apoferritin was procured from (Calzyme Laboratories, San Luis Obispo, CA).³¹ Sybr green and other PCR reagents were from Applied Biosystems Inc. (Foster City, CA). Oligonucleotides were generated by Integrated DNA Technologies (Coralville, IA). C3 and IgG antibodies were obtained from (Cappel, MP Biomedicals, Solon, OH). C9 antibody was a kind gift from Dr Scott Barnum (Birmingham, AL). All other antibodies used for FACS analysis were bought from BD-Pharmingen (Franklin Lakes, NJ) unless indicated otherwise. All reagents unless stated differently were from Sigma Chemical Co. (St Louis, MO).

Experimental protocol in Cfh^−/− mice

The Cfh^−/−8 mice on the C57BL/6 strain used were generously provided by Dr Marina Botto, Imperial College, London, UK. Cfh^−/− mice were divided into three groups of six mice each. Group 1: mice were treated with saline and served as controls; Group 2: immune complex glomerulonephritis was induced by immunizing 6- to 8-week-old male Cfh^−/− mice (n = 6) for 5 weeks with a daily intraperitoneal dose of 4 mg horse spleen apoferritin, as described previously.³² Group 3: mice were treated intraperitoneally with apoferritin and CMN obtained from Calbiochem,³³ 30 mg/kg. As CMN is non-polar it was suspended in 25 μl DMSO. Control Cfh^−/− mice (n = 5) were treated identically, except they received 25 μl vehicle alone. These animals were killed and a comprehensive assessment of disease phenotype was performed, as we have described previously in this model.³¹ All animal procedures were approved by the University of Chicago Institutional Animal Care and Use Committee.

Kidney function

Blood urea nitrogen (BUN) concentrations were detected with a Beckman Autoanalyzer (Beckman Coulter, Fullerton, CA). Serum creatinine was measured with Stanbio Creatinine Procedure No. 0400, (Stanbio Laboratory, Boerne, TX). Urinary albumin concentrations were measured with a mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX) and normalized to creatinine concentrations in the same urine.

Serum C3 levels, and circulating immune complex levels were determined by ELISA as described previously.³⁴ For serum C3 ELISA, plates were coated with goat anti-mouse C3 (Cappel Laboratories, Durham, NC). Serially diluted serum samples were added followed by horseradish peroxidase-goat anti-mouse C3 (Cappel Laboratories). Results are expressed as relative optical density at 450 nm values adjusted for standard wild-type C57BL/6 mouse serum samples included in all. The anti-mouse C3 antibody identifies native intact C3 along with the cleavage fragments iC3b and its component C3c to some extent.

To measure circulating immune complex levels, 96-well plates were coated with human C1q (Quidel, San Diego, CA). Serum samples were serially diluted and incubated for 2 hr at room temperature, followed by horseradish peroxidase-goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and o-Phenylenediamine dihydrochloride (OPD) peroxidase substrate (Sigma). Sera from several apoferritin treated Cfh^−/− mice were pooled and served as controls. Circulating immune complex levels were quantified by plotting against the standard curve.

FACS analysis

To enumerate and characterize the peripheral leucocytes and splenic cells, flow cytometry was performed. Splenocytes were isolated according to standard techniques and treated with ACK lysis solution (0·15 m NH₄Cl, 1 mm KHCO₃ and 0·1 mm Na₂EDTA, pH 7·2) for 5 min to deplete red blood cells. Cells were washed and resuspended in FACS buffer (2% fetal bovine serum, 0·05% NaN₃ and 2 mm EDTA in PBS) to a final concentration of 10⁷/ml. Blood was collected in EDTA followed by red blood cell lysis and cells were resuspended in FACS buffer. Then, 100 μl (10⁶ cells) of the splenocytes or peripheral blood cells were incubated with Fc block (1 μg monoclonal antibody 2.4G2) for 5 min. Cells were simultaneously stained for 1 hr with the following monoclonal antibodies: Brilliant Violet 421 αCD3 (145C211), allophycocyanin-Cy7 αCD19 (6D5), Alexafluor 647 αCD8 (KT15) (Molecular Probes, Inc., Eugene, OR), FITC αCD4 (W3/25), V500 αCD11b (M1/70), Peridinin chlorophyll protein-Cy5.5 αLy6C (HK1.4), phycoerythrin (PE) αCCR2 (475301), PE-Cy5 αCD62L (MEL-14), PE-Cy7 αF4/80 (BM8) (Biolegend, San Diego, CA), Alexafluor 700 αCD11c (N418). All antibodies were procured from BD PharMingen (San Diego, CA). Samples were washed and resuspended in FACS buffer and ran on an LSRII instrument (BD Biosciences, Franklin Lakes, NJ). Analysis was performed with FlowJo software (Tree Star, Inc., Ashland, OR).

Histology

To evaluate renal pathological changes, kidney tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Four-micrometer sections were stained with periodic acid-Schiff. Slides were scored in a blinded manner by a renal pathologist (AC) for the extent of glomerulonephritis and interstitial nephritis using scales of 0–4 (in increments of 0·5) as described previously.³¹

Kidney homogenate

The kidney was cut and immediately frozen in liquid nitrogen and kept at − 70° until use. The frozen kidney was ground to a powder and then mixed in ice-cold HEPES buffer (10 mm HEPES, 0·2% Triton X-100, 50 mm NaCl, 0·5 mm sucrose, 0·1 mm EDTA, protease and phosphatase inhibitors) and homogenized with an ice-chilled dounce homogenizer at 4°. The homogenate was stored as aliquots at − 70°.

Macrophages

Macrophages infiltrating the kidney were assessed using the myeloperoxidase detection kit from Cell Technology Inc. (Mountain View, CA), according to the manufacturer's instructions. In brief, homogenates were prepared and centrifuged at 12 000 g at 4°. The pellet was solubilized and subjected to freeze–thaw cycles. The reaction cocktail was prepared according to instructions and added to the samples. N-Ethylmaleimide (Sigma Cat.# E1271) was added to prevent interference due to the presence of glutathione and other reductants. The homogenates were incubated with 20 mm 3-amino-1,2,4-triazole (Sigma Cat.# A8056) for 60 min before running the assay to inhibit catalase activity, if present. Fluorescence was measured at excitation 530–570 nm and emission at 590–600 nm.

Immunofluorescence microscopy

For immunofluorescence (IF) microscopy, 4-μm cryostat sections were fixed in 1 : 1 ether : ethanol and stained with FITC-anti-mouse C3 (Cappel Pharmaceuticals, Aurora, OH), Alexa 647-anti mouse IgG and rabbit anti-mouse C9 followed by Alexa 594-anti-rabbit antibody (Molecular Probes). Primary antibodies were used at a dilution of 1 : 100 and secondary antibodies at 1 : 300. Slides were viewed with an Olympus BX-60 IF microscope (Carter Valley, PA). Representative photomicrographs were taken at identical settings with a Hamamatsu EM-CCD camera (Bridgewater, NJ). Mesangial staining intensity scores from 0 to 4 were assigned to the individual samples by observers who were masked to their origin. All assays included negative controls where the primary antibody was omitted.

Quantitative real-time RT-PCR analysis

RNA was isolated from kidneys using TRIzol reagent (Life Technologies, Grand Island, NY) as described previously.³⁵ Quantitative RT-PCR was performed as described previously. Real-time quantitative RT-PCR was performed for collagen, fibronectin, laminin, keratinocyte-derived cytokine (KC), CXCL2/macrophage inflammatory protein 2 (MIP-2), TGF-β, intercellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein 1 (MCP-1) and C3. All traces of genomic DNA were eliminated with RQ1 DNase (Promega, Madison, WI) at 37° for 30 min. The cDNA was produced from renal cortical RNA using a commercial kit (Superscript III; Invitrogen). Quantitative RT-PCR was performed with the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA) using an Applied Biosystems 7300 Real Time PCR System and the SybrGreen intercalating dye method with Hot-Star DNA polymerase (PE Applied Biosystems) according to the manufacturer's instructions. PCR was conducted with a hot start at 95° (5 min), followed by 45 cycles of 95° for 15 seconds and 60° for 30 seconds. For each sample, the number of cycles required to generate a given threshold signal (Ct) was recorded. Expression data were normalized to 18S RNA measured contemporaneously from the same samples. Primers were synthesized by Integrated DNA Technologies. The sequences of primers/probes are given below.

Statistical analysis

Numerical data from all experiments were first analysed using the ‘graphical summary’ function in Minitab 15 (State College, PA) to determine data normality (Anderson–Darling test) and 95% confidence intervals for mean, median and SD. Parametric and non-parametric data were analysed by one-way analysis of variance and Kruskal–Wallis tests, respectively. Comparisons within each group for a given parameter were made using paired Student's t-tests; P < 0·05 was considered statistically significant.

Curcumin protects against renal failure in Cfh-deficient mice with CSS

Studies were performed to examine the effect of curcumin on the functional and histopathological features of glomerulonephritis in Cf^−/− mice. Mice with 5 weeks of apoferritin treatment developed albuminuria (48·7 ± 6·4 versus 13·8 ± 1·5; P < 0·05) as seen in our studies earlier. Comparable to our earlier studies, BUN levels were elevated compared with control (45·4 ± 7·5 versus; 26·7 ± 2·6; P < 0·05). Curcumin treatment reduced both albuminuria (15·7 ± 7·1; P < 0·05) and the level of BUN (30·2 ± 2·9 mg/dl, P < 0·05 compared with apoferritin treated; Table 1) in Cfh^−/− mice, indicating that it was protective of kidney function in this setting. This was further substantiated by the significant reduction in serum creatinine in mice treated with CMN compared with those treated with apoferritin alone (Fig. 1c).

Curcumin alleviates glomerulonephritis in CfH-deficient mice with CSS

Formalin-fixed kidneys were embedded in paraffin wax and sections were stained with periodic acid-Schiff (Fig. 1a). Control Cfh^−/− mice receiving saline had mild to no evident glomerular pathology, while Cfh^−/− mice immunized with apoferritin developed significant glomerulonephritis, characterized by diffuse hypercellularity of the glomerular tufts as observed earlier. Sections were read by the pathologist in a blinded fashion to assess glomerular pathology (Fig. 1b). Treatment with CMN protected Cfh^−/− mice with CSS from renal pathology as the glomeruli looked closer to normal. Figure 1 shows these features in three representative examples of histological changes in each group.

We were then interested in whether CMN affected glomerular localization of C3 and IgG-containing immune complexes in this experimental serum sickness model (Fig. 2). As seen in all our earlier studies, immunofluorescence microscopy showed substantial linear C3 staining along glomerular capillary walls in Cfh^−/− C57BL/6 mice by 8 weeks of age, which continued to maintain the same profile after 5 weeks of apoferritin and even after CMN treatment (Fig. 2). Although the Cfh^−/− mice have marked consumption of circulating C3, these animals continued to deposit C3 within the kidney. This C3 may be derived from C3 produced intrinsically by glomerular epithelial and mesangial cells.^18,36 In line with our earlier observations, there was minimal IgG in saline-treated mice and substantial IgG deposits in mice with CSS.¹⁸ In Cfh^−/− mice with serum sickness, there was a qualitative and quantitative difference of IgG deposits (as scored in Fig. 2b), in that Cfh^−/− animals had more intense mesangial staining as well as greater involvement in peripheral capillary loops. The pattern of IgG and C3 deposition was different, so there was limited overlap and in regions of extensive deposits it even seemed to displace C3 (Fig. 2, arrow). Treatment with CMN reduced IgG deposits in these mice. To determine whether the late complement pathway (C5b-9) was affected in this setting, we determined C9 deposition by immunofluorescence. In Cfh^−/− mice with serum sickness, there was a significant increase in C9 deposits compared with saline-treated mice. Similar to the C3, IgG seemed to displace the C9 to the periphery. In CMN-treated Cfh^−/− mice with serum sickness, C9 deposits were reduced and, in line with the reduced IgG, were found distributed evenly across the glomerulus.

Curcumin alters immunological features in Cfh^−/− mice with CSS

Humoral immunity was assessed in the three groups of Cfh^−/− mice, those treated with saline, apoferritin and CMN + apoferritin. As demonstrated in our earlier studies, Cfh deficiency leads to increased C3 consumption and therefore to reduced C3 in circulation. This feature was further aggravated by CMN treatment with C3 non-detectable in plasma (results not shown). To understand the effect of factor H and CMN on the synthesis of C3, mRNA expression was assessed. Interestingly, C3 synthesis is reduced significantly both in the liver (55·6 ± 9·8% reduction, P = 0·003) and the kidney (60·8 ± 1·8% reduction, P = 0·016) in mice treated with apoferritin compared with control (Fig. 3). However, the synthesis of kidney C3 mRNA occurs 30 times slower than synthesis of liver C3 mRNA. Curcumin reduces the decrease and improves C3 synthesis in this setting. Normal C57BL/6 mice were used as controls. On the other hand, circulating immune complexes were increased by apoferritin treatment. In addition to apoferritin, when the mice were given CMN the circulating immune complexes were significantly reduced (Fig. 4), P < 0·05.

Effect of curcumin on cellular autoimmunity was evaluated in Cfh-deficient mice with CSS

Lymphocytes isolated from spleens and blood were analysed by flow cytometry. The percentage of blood CD3⁺, CD11b⁺ cells remained static (results not shown). FACS analysis on splenic cells also demonstrated that mice treated with apoferritin or apoferritin + CMN do not differ in their CD4⁺ and CD8⁺ populations (Fig. 5a). However, % of CD19⁺ cells (Fig. 5b), activated B cells (CD19⁺ CD62L) and the ratio of CD19 : CD3 were significantly reduced by CMN treatment (Table 2). The monocyte population was not altered by the treatments, except the alternate, deactivated monocytes (CD11b⁺ Gr1-CCR2⁺), which were significantly reduced by apoferritin treatment (Fig. 5c). Treatment with CMN prevented the decrease in this subset of monocytes, suggesting a protective function in immune complex glomerulonephritis.

The possibility that macrophages are important inflammatory mediators in this setting was assessed using the MPO assay.³⁶ The MPO analysis of kidney homogenates revealed significant increase in macrophage infiltration (P > 0·05) after apoferritin treatment (Fig. 6). Macrophages infiltrating the kidney were increased by 356% in Cfh^−/− mice with serum sickness compared with control, which was reduced to 156% in CMN-treated mice compared with control.

Curcumin reduces mediators of renal inflammation in Cfh^−/− mice with CSS

Infiltration of inflammatory cells is regulated in part by cytokines or chemokines that modulate the activity of adhesion receptors and direct migration of cells to the tissue site. We therefore examined the effect of CMN on renal chemokine expression. Analysis of the expression of inflammatory mediators by quantitative RT-PCR indicated that mRNA expression of MCP-1, ICAM-1, TGF-β and MIP-2 were all increased in CSS whereas KC was unaltered (Table 3). Treatment with CMN prevented the increased expression of MCP-1, TGF-β and MIP-2, while ICAM-1 showed a downward trend.

Curcumin treatment prevents glomerulosclerosis in Cfh^−/− mice with CSS

Consistent with our earlier studies in apoferritin-immunized Cfh^−/− mice, there was an increase in mRNA for collagen IV (0·88 ± 0·04 versus 5·47 ± 0·82), fibronectin (3·08 ± 0·42 versus 6·38 ± 0·54) and laminin (0·95 ± 0·16 versus 3·00 ± 1·22) compared with controls as measured by quantitative RT-PCR (Table 4). Treatment with CMN prevented the increase in mRNA of extracellular matrix (ECM) proteins, maintaining them closer to normal.