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*Division of Clinical Immunology, Department of Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan; Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Science, Fukuoka, Japan.
Correspondence to Dr. Shoichi Maruyama, Division of Clinical Immunology, Department of Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya, Japan 466-8550. Phone: 81-52-744-2192; Fax: 81-52-744-2209;
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-smooth muscle actin (SMA), fibronectin-EDA, type I collagen, and collagen fibrils were significantly reduced in the 7ND-treated kidney on day 21. Furthermore, 7ND gene therapy significantly reduced MCP-1 and TGF-
1 mRNA expression. These results demonstrate that MCP-1 plays an important role in the development of tubulointerstitial inflammation, tubular damage, and fibrosis induced by proteinuria. The fact that 7ND gene therapy had little effect on the contralateral kidney indicates that 7ND acted locally. This strategy may have a potential usefulness as a gene therapy against tubulointerstitial renal injury. E-mail: marus@med.nagoya-u.ac.jp |
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B–dependent pathway (8). MCP-1 can also induce fibrosis through recruitment and activation of macrophages that synthesize TGF-1, a strong pro-fibrotic factor (9). The first objective of this study was to examine the role of MCP-1 in the progression of interstitial injury induced by proteinuria. For this purpose, we employed a rat model of protein overload nephropathy. This is a well-established model of non-immunological proteinuria. In this model, interstitial inflammation (mainly by macrophages) develops at one week, followed by the deposition of extracellular matrix proteins at two or three weeks (2,10). Tubular damage is also observed, with tubular cell apoptosis being suggested as a contributing factor (11). Upregulation of MCP-1 has previously been demonstrated in this model (2). However, the effect of a MCP-1 blockade has not been studied. To clarify this, we used 7ND, a deletion mutant of human MCP-1 (missing the amino-terminal amino acids 2 to 8) (12). Previous in vitro experiments showed that 7ND forms inactive heterodimers with wild-type MCP-1 and inhibits monocyte chemotaxis (13). The effectiveness of 7ND has also been shown in vivo by experiments infusing recombinant 7ND protein to MRL/lpr mice (14) or by experiments transferring the 7ND gene to a remote organ (muscle) in animals with atherosclerosis or pulmonary hypertension (15–17).
A second objective of this study was to demonstrate the effectiveness of a recently developed hydrodynamics-based gene transfer technique (18). With this procedure, naked plasmid can be transferred into the interstitial fibroblasts in the kidney, and the gene expression persists for more than 6 mo. This is the first usage of this technique in an animal model.
We report here that the 7ND gene was successfully transferred into the rat kidney and it acted locally. A blockade of MCP-1/CCR2 signaling reduced macrophage infiltration and subsequently inhibited tubular damage and interstitial fibrosis induced by protein-overload proteinuria.
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Expression Vector
FLAG-tagged (3' C terminus]) mutant MCP-1 (7ND) was constructed and cloned into the BamHI (5') and NotI (3') sites of the pCDNA3 expression vector plasmid (Invitrogen Corp., Carlsbad, CA) (17). The plasmid was prepared using a Qiagen EndoFree plasmid Giga kit (Qiagen GmbH, Hilden, Germany). An empty pCDNA3 vector plasmid was used as a control.
Introduction of 7ND Plasmid into Rat Kidney
Naked plasmid encoding 7ND was introduced into the left kidney of rats by means of a recently developed hydrodynamics based gene transfer technique (18). Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), and the left kidney was exposed by midline incision. After clamping the left renal vein and artery, 700 µl of Ringer solution containing plasmid DNA was injected retrogradely into the left renal vein within 5 s using a 24-gauge SURFLO intravenous catheter (Terumo, Tokyo, Japan). Blood flow was reestablished immediately after the injection.
Experimental Protocols
Bovine serum albumin (BSA) solution was prepared from Sigma preparation (fraction V, A4503), and protein overload nephropathy was induced as described previously (10). In experiment 1, rats were given 100 µg (n = 6), 300 µg (n = 6), or 1 mg (n = 3) of 7ND plasmid into the left kidney. Control rats were treated with 300 µg of empty vector (n = 6). Three days after gene transfer (day 0), intraperitoneal administration of BSA (10 mg/g body wt per day) was started and continued for 14 d. In experiment 2, 300 µg of 7ND plasmid (n = 6) or a control vector (n = 6) was transferred locally to the left kidney (day -3), followed by the daily injection of 10 mg/g body wt of BSA starting on day 0 and continuing for 21 d. Urine samples were collected on days 7, 10, and 13 in rats in the first series of experiments and on days 6, 13, and 20 in rats in the second series of experiments. Rats were sacrificed at the end of the experiments (day 14 or day 21), and kidney samples were collected for the study.
Histology and Immunohistochemistry
One part of the kidney was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 2-µm-thick sections. They were stained with periodic acid-Schiff reagent (PAS), periodic acid-methenamine-silver (PAM), or Sirius red (19).
Another part of the kidney was frozen in OCT compound (Miles, Elkhart, IN) and kept frozen at -80°C until use. Sections (2-µm-thick) were cut by a cryostat and fixed in acetone. Immunostaining was performed as described previously (20). Briefly, the sections were incubated with primary antibodies, followed by the fluorescein- or Rhodamine-conjugated secondary antibodies. The primary antibodies employed in this study were monoclonal antibodies against FLAG (M2; Sigma Aldrich, St. Louis, MO), ED1 (anti-rat-macrophage/monocyte, Serotec, Raleigh, NC), rat CD5 (Serotec, Raleigh, NC), -smooth muscle actin (SMA, 1A4; Dako, Carpinteria, CA), and cellular fibronectin-EDA domain (IST-9; Harlan Sera-lab, Belton, Loughborough, UK), and rabbit polyclonal antibody against rat collagen type I (Chemicon International, Temecula, CA). The secondary antibodies were fluorescein conjugates of goat anti-mouse IgG antibody (Zymed Laboratories, San Francisco, CA) and Rhodamine conjugates of goat anti-mouse IgG and goat anti-rabbit IgG antibodies (Sigma). Negative controls were performed by the replacement of primary antibodies with isotype-matched or species-matched antibodies. For the dual-staining study, anti-FLAG antibody, Rhodamine-conjugated goat anti-mouse IgG and fluorescein labeled anti-ED-1 antibody (Serotec) were used. Normal control rat kidneys (n = 4) were also stained to determine the baseline.
TUNEL Staining
Apoptotic cell death was determined on the 6-µm-thick paraffin-embedded kidney section by using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining with in situ Apoptosis Detection Kit (Takara, Shiga, Japan) according to the manufacturer’s instruction. Apoptotic cells were counted in at least ten randomly chosen non-overlapping cortical fields (x100; 0.58 mm2) for each kidney.
Morphometric Analysis of Histology and Immunohistology
To assess tubulointerstitial injury, kidney sections were analyzed using a semiquantitative grading (21). Briefly, the extents of tubular cast formation, tubular dilatation, and tubular degeneration (vacuolar change, loss of brush border, detachment of tubular epithelial cells, and condensation of tubular nuclei) were scored according to the following criteria by two observers in a blind manner: 0, normal; 1, less than 25%: 2, 25% to 70%; 3, more than 70% of the pertinent area. For the analysis of each type of immunostaining, 20 randomly selected non-overlapping cortical fields (x200; 0.146 mm2 each) were examined using a 20x lens with Zeiss microscope (Oberkochen, Germany) as described previously (21). Interstitial macrophage and T cell infiltration were assessed by counting ED-1 and CD5-positive cells in each field. Ratio of areas positively stained for SMA, fibronectin-EDA, or type I collagen to the total cortical area was measured by computer-aided planimetry using Mac Scope (Mitani, Fukui, Japan) and expressed as percentages (22). The staining for collagen fibrils was similarly quantified by calculating the ratio of area positively stained with Sirius red using a polarized light.
Analysis of mRNA for 7ND, MCP-1, and TGF-1
One hundred milligrams of renal cortical tissue was lysed in Trisol (Life Technologies, Frederick, MD), and total RNA was prepared according to the manufacturer’s protocol. One µg of total RNA was subjected to reverse transcriptase (RT) -PCR by using One Step RT-PCR kit (Takara). The samples were incubated at 50°C for 30 min and at 94°C for 5 min, followed by denature at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The final extension step was performed at 72°C for 5 min. The primers for 7ND were 5'-GAGACCTTCTGTGCCTGCTGCTCATAGCA-3' (forward) and 5'-GAGAGCTTGTCCAGGTGGTCCATGGAATC-3' (reverse); the primers for MCP-1 were 5'-ATGCAGGTCTCTGTCACG-3' (forward) and 5'-CTAGTTCTCTGTCATACT-3' (reverse); the primers for TGF-1 were 5'-CCTGCAAGACCATCGACATG-3' (forward) and 5'-CTGGCGAGCCTTAGTTTGGA-3' (reverse); and the primers for -actin were 5'-TTGTAACCAACTGGGACGATATGG-3' (forward) and 5'-GATCTTGATCTTCATGGTGCTAGG-3' (reverse). Thirty-five cycles of PCR were performed for 7ND. Expression of MCP-1 and TGF-1 mRNA was assessed semiquantitatively (23). Twenty-two cycles of PCR were performed for MCP-1, 24 cycles for TGF-1, and 16 cycles for -actin. PCR products were separated on 2% agarose gel containing ethidium bromide (0.5 µg/ml). The gels were imaged and the fluorescence intensities of the PCR products were analyzed using KODAK 1D Image Analysis Software (Eastman Kodak Company, Rochester, NY). Ratios of the values for MCP-1 or TGF-1 to -actin PCR products were determined and compared. To exclude the possibility of plasmid or genomic DNA contamination, experiments were carried out in the absence of RT.
Human MCP-1 ELISA
7ND protein levels in the sera of rats treated with 300 µg of 7ND were studied using a OptiEIA Human MCP-1 Kit (Phamingen, San Diego, CA) according to the manufacturer’s instruction.
Serum and Urine Measurements
Blood was taken from the aorta, and serum was collected. Blood urea nitrogen (BUN) and serum creatinine levels were measured using Daiya Auto UN or Daiya Auto Crea Kit (Daiya Shiyaku, Tokyo, Japan). Urinary protein was measured by a pyrogallol red method using Micro TP-Test Wako (Wako, Oasaka, Japan).
Statistical Analyses
All values are provided as mean ± SEM. Statistical analysis was performed by one-way ANOVA. When significant difference was present, statistical analysis was further performed using the Scheffe F test between two groups. Significant difference was set when the P value was less than 0.05.
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SMA expression was confined to blood vessel walls. On day 14, SMA staining was not different from that of a normal rat kidney. On day 21, however, enhanced SMA staining was observed in the cortex of the control rat kidney mainly in the inflamed interstitium or periglomerular area (Figure 6A). The staining was reduced by the 7ND treatment (Figure 6B). Computer-assisted quantitative analysis showed that 7ND gene transfer reduced the ratio of SMA-positive area by 40% as compared with the contralateral kidney and the kidney of the control vector–treated rats on day 21 (P < 0.01) (Figure 6I). The staining for fibronectin-EDA was observed in the control rat kidney on day 14 mainly in the peritubular and periglomerular area accompanied with massive macrophage infiltration. The staining was more evident on day 21 (Figure 6C). Fibronectin-EDA–positive areas in the 7ND-treated kidney were about 60% and 40% smaller than those in the controls on days 14 and 21, respectively (P < 0.01; Figure 6, D and J). The deposition of type I collagen was minimal in the control rat kidney on day 14, but it was enhanced on day 21 (Figure 6E). The type I collagen–positive area was decreased by about 30% by the 7ND gene transfer (P < 0.01; Figure 6, F and K). The deposition of collagen fibrils were studied by Sirius red staining. In the control rat kidney on day 14, minimal staining was observed, which was not different from that in the normal rat kidney. However, the staining was significantly enhanced on day 21 (Figure 6G). 7ND gene therapy attenuated the staining by 45% (Figure 6, H and L; P < 0.01).
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1 mRNA Expression1 mRNA expression increased after 21 d of BSA administration compared with those in the normal rat kidneys. These values were significantly suppressed by the 7ND gene transfer (P < 0.05; Figure 7).
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(26), MIP-2 (27), and RANTES (28) also play a major role in animal models of renal injury. Although upregulation of MCP-1 has been demonstrated in the kidney after BSA administration (2), whether MCP-1 critically contributes to the inflammation in protein overload nephropathy remained to be determined. In the present study, infiltrating cells were mainly macrophages, and anti-MCP-1 gene therapy significantly reduced macrophage infiltration. A blockade of MCP-1 signaling also functioned to preserve the normal structure of the tubular epithelial cells. It has been shown that tubular epithelial cells are damaged by BSA, and the contribution of apoptosis to this process was suggested by in vitro and in vivo experiments (11,29). In our study, anti-MCP-1 therapy significantly suppressed the tubular damage and reduced the number of apoptotic cells induced by proteinuria. It is important to determine the levels of urinary protein in this model, because the degree of tubular injury was shown to be dependent on the amount of urinary protein excretion (10). The fact that the levels of urinary protein excretion were not different between the experimental groups suggests that the amount of BSA that reached the tubular epithelial cells was comparable. It is known that inflammatory macrophages produce a wide array of pro-inflammatory mediators and are capable of inducing apoptosis (30). Our data suggest that, besides the direct effect of BSA (29), infiltrated macrophages may contribute to the tubular damage by inducing apoptosis.
Moreover, the present study clearly showed that MCP-1 plays a significant role in the development of interstitial fibrosis. Computer-aided planimetry analyses revealed that anti-MCP-1 gene therapy significantly suppressed SMA expression (a marker of activated fibroblasts) and reduced the deposition of ECM proteins and collagen fibrils. Anti-MCP-1 therapy also decreased the expression of mRNA for TGF-1 (a strong pro-fibrogenic cytokine). Taken together, our results support the hypothesis that MCP-1 (induced by proteinuria) recruits and activates monocytes/macrophages to produce inflammatory and fibrogenic mediators, leading to tubulointerstitial renal fibrosis.
In the present study, as in the past report (10), serum creatinine levels did not show the increase one would expect from the pathologic changes in the kidney. This may have been because the interstitial inflammation and fibrosis were not severe enough to significantly affect renal function. Volume overloading, as was performed in this experiment, could also account for the lower than expected creatinine levels. It was therefore difficult to judge the effects of 7ND by measuring serum creatinine levels. Furthermore, the fact that we did not remove the right kidneys may have masked the effects of anti-MCP-1 gene therapy on the renal function. In contrast, BUN levels in the experimental groups were higher than those in the normal rats, presumably because of the infused albumin catabolism.
Of interest is that anti-MCP-1 gene therapy decreased the levels of MCP-1 mRNA expression in our study. This result suggests that, directly or indirectly, MCP-1 enhances MCP-1 production in the kidney. MCP-1 is known to be expressed by infiltrating macrophages as well as intrinsic renal cells (e.g., tubular cells and mesangial cells) (6,24). Macrophages have a MCP-1 receptor (CCR2) (31), and they produce MCP-1 upon activation (32). An amplification loop of inflammatory cytokine production in monocytes (33) and in mesangial cells (34) is suggested. Thus, an amplification cascade may also have a function in the process of interstitial nephritis induced by proteinuria. Hypothetically, MCP-1 secreted from the tubular epithelial cells stimulates monocytes/macrophages to produce and secrete inflammatory cytokines (including MCP-1 itself), and the secreted cytokines enhances MCP-1 expression leading to further recruitment and activation of monocytes/macrophages. Although direct proof of this hypothesis is beyond the present study, 7ND may have attenuated macrophage infiltration by blocking this amplification cascade.
Another goal of this study was to demonstrate for the first time the effectiveness of a recently established kidney-targeted naked plasmid transfer technique (18) in an animal model of renal injury. Various method have been tried to transfer the therapeutic genes into the kidney (35,36). Plasmid DNA mixed with the HVJ liposome was introduced into the mesangial cells when infused via the renal artery or into the interstitial fibroblasts when infused via the ureter (37). Similar results were obtained when naked plasmid was injected into the kidney followed by electroporation (37,38). When an adenoviral vector was used, gene expression was observed in the tubules or glomerulus (39). In the present study, naked plasmid was used and the transgene was expressed in the interstitial cells. A previous study using an electron microscope suggested that these are interstitial fibroblasts and not endothelial cells. The fact that the gene transfer site differs in each method allows us to choose the proper one depending on the kind of renal injury. We assume that the technique employed in the present study will be useful to treat interstitial renal injury, but it may not be effective in treating the glomerular injury.
Various methods of gene transfer have been tried for the treatment of renal injury in animals (35,36). However to date, none of them have been applied to humans. The technique employed in the present study has several advantages over the previous methods. First of all, it is a safe technique, which does not require possibly toxic agents such as liposomes (37) or viral vectors (39). No damage was observed in the kidney from this procedure. Second, it is rather simple because it does not require special techniques such as electroporation (40). It is therefore theoretically possible to apply this method to the human kidney by using proper catheters. Third, it was demonstrated that the gene expression persists for a relatively long period with this technique. In this study, expression of 7ND mRNA and protein was detectable, and the therapeutic effects of 7ND were observed even at 24 d after gene transfer. Of note is the fact that the effects of the 7ND were confined to the gene-transfected kidney and that 7ND protein was not detectable in circulation by ELISA. These data indicate that 7ND expressed and acted locally in the targeted kidney. In the present study, we did not directly compare the effects of kidney-targeted gene transfer with those of systemic gene therapy such as intramuscular gene transfer using electroporation. Previous studies suggested that the serum levels of the transgene product (erythropoietin) obtained by these two gene transfer methods are comparable (18,41). We can therefore assume that a higher local concentration is likely to be achieved by transferring the gene directly into the target organs. Moreover, possible adverse effects may be minimized by transferring the gene locally. Because MCP-1 plays an important role in wound healing and host defense (42,43), systemic suppression of MCP-1 might be harmful. In this context, the kidney-targeted gene transfer technique is considered to be superior to systemic delivery.
In a previous study, rat erythropoietin was successfully expressed in pCAGGS vector by means of the same technique as was used in this study (18). However in that study, transgene expression was not detected when pCI was used. Although pCDNA3 and pCI share the same CMV promoter, factors other than the promoter may affect the transgene expression. In the present study, 7ND was expressed using the pCDNA3 vector. The efficiency of 7ND expression in the different types of expression vectors (e.g., pCAGGS, pCDNA3, and pCI) bears further study.
In summary, we were able to successfully introduce 7ND, a MCP-1 antagonist, using a hydrodynamics-based kidney targeted gene transfer technique. 7ND was expressed and acted locally in the target kidney. The results demonstrated that MCP-1/CCR2 signaling plays an important role in the development of tubulointerstitial inflammation, tubular damage, and interstitial fibrosis induced by proteinuria. Our results showed that the present strategy has potential therapeutic application for tubulointerstitial renal injury in the clinical setting.
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