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Pathophysiology of Renal Disease and Progression |
* Division of Nephrology, Department of Pediatrics, Powell Gene Therapy Center, and Genetics Institute, Departments of Ophthalmology and Molecular Genetics and Microbiology, and || Department of Pathology, University of Florida, Gainesville, Florida; Division of Nephrology, University of Alabama, Birmingham, Alabama; and ¶ Division of Nephrology, University of Maracaibo, Maracaibo, Venezuela
Address correspondence to: Dr. Wei Mu, Division of Nephrology, University of Florida, P.O. Box 100224, Gainesville, FL 32610. Phone: 352-392-3677; Fax: 352-392-5465; E-mail muw{at}medicine.ufl.edu
Received for publication March 17, 2005. Accepted for publication September 14, 2005.
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Abstract |
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and IL-2 in the kidney. The reduction in inflammatory cells was associated with a significant reduction in glomerulosclerosis and interstitial fibrosis. It is concluded that IL-10 blocks inflammation and improves renal function in this model of chronic renal disease. The feasibility of long-term overexpression of a gene using the AAV serotype 1 vector system in a model of renal disease is also demonstrated. |
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IL-10 has been reported to attenuate inflammation in a variety of animal models, including experimental vascular injury (7,8), autoimmune disease (9), and hepatic ischemia/reperfusion (10). IL-10 has also been reported to reduce inflammation and mesangial cell proliferation in acute glomerulonephritis induced with anti–Thy 1 antibody (11) and to suppress glomerulosclerosis formation in the FGS/Kist, a rat that develops spontaneous focal segmental glomerulosclerosis (12). To date, there have been no studies on the potential benefits of long-term IL-10 therapy in models of chronic renal disease.
In this study, we examined the effect of IL-10 in a model of chronic renal disease induced by 5/6 nephrectomy. Systemic expression of IL-10 was achieved using an adeno-associated virus serotype 1–IL-10 (rAAV1–IL-10) vector. We report a beneficial effect of IL-10 in the remnant kidney model, which is partly mediated by a reduction in the local inflammatory response.
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actin promoter/hybrid intron cassette (13).
Recombinant AAV Virus Production
rAAV production was performed by co-transfection of the rAAV1-ITR vector construct and a combined Ad/AAV helper construct. Individual helper constructs all express the Ad5 E2a, E4, and VA genes; the AAV Rep gene; and the specific AAV Cap gene desired for pseudotyping (most with AAV1). Transfections were performed by calcium phosphate co-precipitation in an Ad-E1a–and E1b-expressing permissive human cell line (HEK293). rAAV was purified from cells by iodixanol density gradient centrifugation. DNA dot blot assay was performed to quantify the titer of rAAV (14).
Animals
The study was approved by the Animal Care Committee at the University of Florida (Gainesville, FL). Male Sprague-Dawley rats (n = 20; Charles River Laboratories, Inc., Wilmington, MA) that weighed between 150 and 175 g received an intramuscular injection into the caudal muscle of the pelvic limb. These injections used 100 µl of saline that contained 1 x 1010 infectious units of either rAAV1-GFP (n = 10) or rAAV1–IL-10 (n = 10) per rat. Four weeks after injection, IL-10 levels in serum were measured by ELISA (see below). Two rats from the rAAV1–IL-10 group did not achieve serum IL-10 levels >100 pg/ml and therefore were excluded from the experiment. The remaining rats underwent the remnant kidney procedure.
Rats underwent a right subcapsular nephrectomy with surgical resection of the upper and lower thirds of the left kidney (n = 20), and tissues were kept for later analysis. To document equivalent reduction in renal mass, the resected kidney was weighed, and the remnant kidney weight was calculated as the weight of the resected whole kidney minus the resected sections of the other kidney. Only rats with a remnant kidney/body weight of 1 to 2% were used, resulting in nine rats in the rAAV1-GFP groups and six rats in the rAAV1–IL-10. Three rats in the rAAV1-GFP group died during the experiment; thus, six animals per group were left at the end of the study.
Serum samples were collected at 4, 6, 8, 10, and 12 wk for serum IL-10 measurement. Urine samples were collected at 2, 4, 6, and 8 wk after remnant kidney surgery for urine protein measurement. Eight weeks after nephrectomy, rats were killed for RNA, protein, and immunohistochemical analysis.
Proteinuria and Renal Function
Urinary protein excretion (24 h) was measured using the Bio-Rad DC Protein Assay Kit II (Bio-Rad, Hercules, CA), and blood urea nitrogen and creatinine were determined by Alfa Wassermann’s VetACE biochemistry machine (Alfa Wassermann, West Caldwell, NJ); urine creatinine was measured using the Sigma Creatinine kit (Sigma, St. Louis, MO). Creatinine clearance (Ccr) rates were calculated as U x V/[P], where U and P denote urinary and serum creatinine concentrations, respectively, and V represents ml urine/min.
Renal Pathologic and Histologic Studies
Renal tissues were fixed in Methyl Carnoy’s fixative, and 4-µm paraffin sections were stained with periodic acid-Schiff and hematoxylin. Immunohistochemistry was performed using the following affinity-purified primary antibodies: mAb against CD4, CD8, ED1, and OX62 (BD Pharmingen, San Diego, CA). Horseradish peroxidase–conjugated goat anti-mouse IgG antibodies (Rockland Immunochemicals, Inc., Gilbertsville, PA) were used as secondary antibodies.
For immunohistochemistry, renal tissues were fixed in 4% (wt/vol) buffered paraformaldehyde and frozen at –70°C. Cryosections (4 µm) were incubated with first antibody as described above overnight at 4°C. Sections were washed in PBS, inactivated with endogenous peroxidase in 0.3% H2O2 in methanol, labeled with second antibody as described above followed by mouse peroxidase anti-peroxidase, and developed with DAB substrate kit (Vector Laboratories, Burlingame, CA) to produce a brown color.
For quantification of immunohistochemistry staining, stained sections were imaged using a Axioplan 2 imaging microscope (Carl Zeiss Inc., Thornwood, NY), CR5 digitized color camera, and image-analyzed using Zeiss AutoMeasure software (Axiovision 4.1). The kidney cortex area was evaluated by an investigator without previous knowledge of the experimental group of the animals from which the tissue was taken. Single image frames (700 x 550 µm) were captured at x400 magnification, and 20 frames per sample were used to count the number of CD4+, CD8+, ED-1+, and OX62+ cells (expressed as cell number/[700 x 550 µm]).
Glomerulosclerosis and tubulointerstitial fibrosis scores were also measured in all renal biopsies. For glomerulosclerosis, the percentage of glomeruli that exhibited focal or global glomerulosclerosis was determined by evaluation of all glomeruli present in the biopsy. Glomerulosclerosis was defined as segmental increases in the glomerular matrix, segmental collapse, obliteration of capillary lumina, and accumulation of hyaline, often with synechial attachment to Bowman’s capsule (15). Twenty glomeruli per sample were calculated for glomerulosclerosis scores. Tubulointerstitial injury was defined as inflammatory cell infiltration, tubular dilation and/or atrophy, or interstitial fibrosis. Injuries were graded semiquantitatively by a blinded observer, who examined at least 20 cortical fields (magnification x200) in periodic acid-Schiff–stained biopsies. Only cortical tubules were included in the following scoring system (16): 0 = changes <10% of the cortex; 1+ = changes in up to 20% of the cortex; 2+ = changes in up to 40% of the cortex; 3+ = changes in up to 60% of the cortex; 4+ = changes in up to 80% of the cortex; 5+ = changes in >80% of the cortex sections. Each score per biopsy was based on 20 frames (representing 700 x 550 µm2 tissue areas). Interstitial fibrosis was also assessed by using an Axioplan 2 microscope with Zeiss’s AutoMeasure software to quantify the positive staining areas of interstitial collagen I and collagen III immunostaining on 20 frames per sample tissue area (700 x 550 µm)2
RNA Isolation, Reverse Transcription, and Real-Time PCR
Total RNA was isolated using the SV Total RNA Isolation kit (Promega, Madison, WI) according to the manufacturer’s protocol. The RNA was eluted with 50 µl of RNase-free water. All RNA was quantified by spectrophotometer, and the optical density 260/280 nm ratios were determined. Reverse transcription was performed in a one-step protocol using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s protocols. Reactions were incubated at 25°C for 5 min, and 42°C for 30 min, 85°C for 5 min and cooled at 4°C in a Thermocycler (Eppendorf, Hamburg, Germany). Primers (Table 1) were designed by Genetool software (BioTools Inc., Edmonton, Canada), and oligonucleotides were synthesized by Sigma Genosys (Sigma-Genosys Ltd., Woodlands, TX).
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Measurement of Cytokine and Chemokine by ELISA
At each time point mentioned above for serum and tissue preparation, IL-10 and monocyte chemoattractant protein-1 (MCP-1) were measured by ELISA (Rat IL-10 BD OptEIA ELISA Set and Rat MCP-1 BD OptEIA ELISA Set; BD Biosciences Pharmingen, San Diego, CA). Briefly, wells of polystyrene microtiter plates (Polysorp F96; Nunc, Glostrup, Denmark) were coated with Capture antibody of IL-10 or MCP-1 in carbonate bicarbonate buffer (pH 9.6) overnight at 4.0°C. The next day, the well was washed three times with PBS that contained 0.5% Tween 20 (PBST) and blocked with PBS that contained 5% BSA for 1 h at room temperature. After three washes, 100 µl of standard and rat serum samples were added into wells. After 2 h of incubation at room temperature and five washes, 100 µl of detection antibody and enzyme reagent mixture were added to each well and the plate was incubated for 1 h at room temperature. Unbound detection antibody and enzyme mixture was removed by seven consecutive washings with PBST. A total of 100 µl of substrate solution was added to each well, and the plate was incubated in dark at room temperature for 30 min. The reaction was stopped by addition of 50 µl of stop solution, and absorbance was measured at 405 nm with a microplate scanning spectrophotometer (Powerwave 200; BIO-TEK Instruments, Winooski, VT)
Statistical Analyses
All data are presented as mean ± SD. Differences in the various parameters between groups were evaluated by single-factor ANOVA. Differences in parameters at each time point after remnant kidney surgery were compared by paired t test. Correlation between different factors was evaluated by Pearson correlation, Kendall 
-b, and Spearman 
correlation. Significance was defined as P < 0.05.
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which has been reported to augment macrophage-mediated renal injury (17), and mRNA expression of the cytokine IL-2 which has an important role in the activation of T cells (18). Quantitative real-time PCR of whole kidney samples obtained at killing demonstrated a reduction in MCP-1, RANTES mRNA, and IFN- and IL-2 mRNA expression in rAAV1–IL-10–treated rats compared with rAAV1-GFP rats (Figure 5). Kidney tissue lysates were also assayed for MCP-1 (measured by ELISA) and confirmed a lower expression of MCP-1 protein in rAAV1–IL-10–treated rats (Figure 6). The level of MCP-1 protein in individual animals correlated closely with the number of macrophage (ED-1+) cells (r = 0.63), dendritic cells (r = 0.47), CD4+ cells (r = 0.73), and CD8+ cells (r = 0.66) present (Figure 7). Similarly, the level of RANTES mRNA correlated with the number of CD4+ (r = 0.68), CD8+ (0.66), and OX62+ dendritic cells (r = 0.58; P < 0.05 for all comparisons; Figure 7). Serum IL-10 levels also correlated inversely with both renal tissue MCP-1 levels (r = –0.64) and renal RANTES mRNA (r = –0.73; P < 0.05; Figure 8).
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, collagen I, and collagen III compared with control rats as evaluated by real-time PCR (Figure 5), and this was associated with an approximately 50% reduction in collagen staining (Table 3).
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Discussion |
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Chronic systemic overexpression of IL-10 was achieved by using rAAV gene delivery. This gene delivery system has been used by others to suppress the development of type 1 diabetes (21–24). It has superiority over many gene delivery approaches in that elevated levels of the cytokine of choice can be achieved for the lifetime of the animals (25). Most studies suggest that elevated blood levels of the gene of interest are not achieved until 2 to 4 wk after gene delivery. We therefore waited 4 wk to ensure increased blood levels of IL-10 before initiation of the remnant kidney disease model. At this time point, circulating IL-10 levels were 12-fold higher compared with control rats, and these levels were maintained for all 8 wk after induction of the remnant kidney model. The levels of IL-10 achieved are well within the range of its known biologic effects on cultured cells (26). We also observed no toxicity in our model, and there was no difference in weight or behavior between the IL-10–and GFP-overexpressing rats. We thus could address accurately the effect of chronic IL-10 on the inflammatory cell response in this model.
The primary observation was that IL-10 treatment improved the remnant kidney model, as assessed functionally by better Ccr and lower proteinuria and histologically by less glomerulosclerosis and interstitial fibrosis. The improvement was substantial and resulted in a 30% improvement in renal function and a 50% or greater effect on renal scarring.
The potential mechanism by which IL-10 is renoprotective is likely complex, as IL-10 has multiple actions. Its primary effect is to inhibit activation and effector function of T cells, monocytes, and macrophages. IL-10 binds to its receptor (IL-10R), where it activates several signaling pathways depending on the cell type, including stat 3–dependent and –independent pathways (27), and NF-
B and AP-1 (28,29). A major target of IL-10 is the monocyte, where it blocks its activation and proliferation (30–34). IL-10 potently inhibits production of cytokines, such as IL-1 and TNF, and chemokines, such as MCP-1 and RANTES in activated monocytes/macrophages (33–37). IL-10 also inhibits dendritic cell function (38–40) and T cell activation, the latter in part by its effect on macrophages (41–44). IL-10R1 expression has also been observed on nonhemopoietic cells (45), raising the possibility that some of the actions of IL-10 could be mediated by direct effects on resident renal cell populations.
Because IL-10 acts primarily on monocytes and other immune cell populations, we examined the effect of IL-10 on the monocyte, dendritic cell, and T cell infiltration in our model. Macrophages are known to be present in the remnant kidney model, where they correlate with fibrosis (46). T cells are also known to be present in human and experimental chronic renal disease, although their exact role in the fibrogenic response remains debated (47–50).
Indeed, we confirmed that there was an infiltration of monocyte/macrophages, T cells, and dendritic cells in our model. It is interesting that IL-10 was associated with an approximately 50% reduction in this inflammatory response. The possibility that this could be one of the renoprotective mechanisms for IL-10 in this model is supported by a similar study in which treatment of the remnant kidney model with mycophenolate mofetil also reduced macrophage and T cell infiltration with functional and histologic improvement (16).
We further identified a potential mechanism by which IL-10 can block the local inflammatory response. Thus, one of the most important chemokines for macrophages in the kidney is MCP-1, and MCP-1 is known to be expressed in the remnant kidney model, where it correlates with the monocyte infiltration (20,51,52). Inhibition of MCP-1 has also been found to result in less tubulointerstitial inflammation and fibrosis in several disease models (7,53,54). IL-10 is known to potently suppress MCP-1 in activated macrophages (33); hence, this is a likely mechanism to account for the reduction in monocyte infiltration observed in the IL-10–overexpressing rats. Although MCP-1 is also produced by tubular cells, we were not able to show an effect of IL-10 to suppress MCP-1 in proximal tubular (NRK52E) cells that express the IL-10R (data not shown). Thus, it is likely that the effect of IL-10 is via its suppression of MCP-1 in the inflammatory cell population and that this slowed the recruitment of monocyte/macrophages into the renal interstitium. It is also possible that some of the protective effects were due to inhibition of RANTES and the T cell infiltration; however, the role of these latter cells in chronic renal disease is still unclear.
Consistent with this hypothesis, we found that serum IL-10 levels correlated inversely with both renal tissue MCP-1 levels and renal RANTES mRNA (Figure 8), and the protein level of MCP-1 protein correlated closely with the number of macrophage, dendritic cells, and T cells. Furthermore, the mRNA level of RANTES correlated closely with the number of dendritic cells and T cells but not with macrophage number (Figure 7), which likely reflects that RANTES works primarily on the T cell population.
The reduction in renal inflammation was associated with significantly less renal fibrosis, as reflected by decreased TGF- mRNA and decreased collagen mRNA and protein (Figures 5 and 9; Table 3). The reduction in inflammation and fibrosis was also associated with less proteinuria and better preserved renal function. The lower proteinuria likely results from the less severe glomerular injury, which could also potentially result in less tubulointerstitial injury and inflammation because proteinuria is known to activate tubular cells.
Whereas the injection of AAV–IL-10 resulted in high circulating IL-10 levels, IL-10 mRNA levels that were assessed by real-time PCR were actually decreased in the kidneys of IL-10–overexpressing rats (data not shown). This is likely the consequence of the reduction in immune cell infiltration (because these cells are major sources of IL-10). This suggests that the effect that we observed was due to the high circulating levels of IL-10, which could have both systemic and intrarenal effects.
In conclusion, IL-10 treatment slowed renal progression in an animal model of chronic renal disease. It is interesting that the effect may be mediated by suppression of chemokines and reduction in infiltrating inflammatory cells, resulting in a less vigorous fibrotic response. The possibility that IL-10 may have direct suppressive effects on fibrosis also remains possible. Further studies are indicated to elucidate the protective mechanisms by which IL-10 acts in chronic renal disease.
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Acknowledgments |
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Footnotes |
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W.M. and X.O. contributed equally to this work.
W.W.H. and the University of Florida could be entitled to patent royalties for inventions related to this work, and both own equity in a company that may commercialize some of the technology described herein.
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) have increased serum IL-10 levels (pg/ml as measured by ELISA) compared with rAAV1-GFP control rats (•) beginning at 4 wk after rAAV1 injection, and levels were maintained until the rats were killed. Shown are mean ± SD; six rats are included in each group. ***P < 0.001.
, rAAV1-GAP control group; 
rAAV1-IL-10 treatment group.
