Chin Med J 2007;120(11):988-995 988

Original article

Effects of mycophenolate mofetil, valsartan and their combined therapy on preventing podocyte loss in early stage of diabetic nephropathy in rats ZHANG Yan, CHEN Bing, HOU Xiang-hua, GUAN Guang-ju, LIU Gang, LIU Hai-ying and LI Xue-gang Keywords: podocyte; diabetic nephropathy; mycophenolate mofetil; valsartan

Background Podocyte has inflammatory role in the development of diabetic nephropathy (DN). Mycophenolate mofetil (MMF), an anti-inflammatory agent, can suppress macrophage infiltration and reduce renal injury in streptozotocin-induced diabetic rats. Angiotensin II receptor blocker (ARB), another renal protecting agent, can decrease podocyte loss in DN. In this study, we detected the expression levels of monocyte chemoattractant protein-1 (MCP-1) and nephrin to evaluate podocyte’s role in inflammatory reaction in DN, observe and compare the effect of MMF alone and in combination with valsartan, on preventing podocyte loss in streptozotocin (STZ) induced diabetic rats. Methods Diabetic model was constructed in uninephrectomized male Wistar rats by single peritoneal injection of STZ (65 mg/kg). The successfully induced diabetic rats were randomly divided into four groups: diabetes without treatment group (DM), valsartan treated group (DMV), MMF treated group (DMM), and combined therapy group (DMVM). Normal rats of the same sibling were chosen as control (NC). At the end of the 8th week, serum biochemistry, 24-hour urinary protein (UP) and the ratio of kidney weight/body weight (RWK/B) were measured. The rats were sacrificed for the observation of renal histomorphology through light and electron microscope. Nephrin, desmin and MCP-1 levels were detected by semi-quantitative immunohistochemical assays. Real-time quantitative PCR was used to detect the mRNA levels of nephrin and MCP-1. Results Compared with group NC, serum glucose level, 24-hour UP and RWK/B in group DM were significantly higher (P<0.01), and the nephrin mRNA level in DM group was significantly lower (P<0.05). The nephrin mRNA expression levels in group DMV, DMM and DMVM were all higher than that of DM group (P<0.05) and no significant differences were found among the three treatment groups (P>0.05). Treatment with MMF, valsartan or their combination could significantly decrease the 24-hour UP and RWK/B, and suppress glomerulosclerosis and interstitial fibrotic lesions in diabetic rats. In diabetic rats, the high expressions of desmin and MCP-1 in kidney were suppressed by valsartan, MMF or their combination. Conclusions Podocytes are involved in the inflammatory reaction of diabetic rats. MMF could suppress MCP-1 and desmin expression, enhance nephrin expression, and attenuate proteinuria in diabetic rats. The combined therapy of valsartan and MMF did not show any superiority over monotherapies on renal protection. MMF may have renoprotective effect in early stages of diabetic nephropathy through preventing podocytes loss and anti-inflammatory activity.

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T

he incidence of diabetic nephropathy (DN) is on the tendency of steady rising in recent years. As one of

the main causes of end-stage renal disease (ESRD), the prevention and treatment of DN in early stage, and the retardation of DN development are attracting more and more attention from researchers. Among the three intrinsic cells in glomerulus, podocyte is one of the important ingredients of filtration barrier which has special cytobiological trait and physiological function. The injury of podocyte can inevitably lead to the occurrence of proteinuria. Studies have shown that podocyte damage and decrease of nephrin expression exist even at the early stage of DN and play a key role in accelerating the development of DN especially the decrease of nephrin and the increase of desmin expression.1,2 DN has been categorized as an immunological renal disease, but recently it has been reported to be related to inflammatory reaction. As with

other immunological renal diseases, the presence of monocyte chemoattractant peptide-1 (MCP-1), IL-1β, adhesion molecule, and macrophage infiltration in kidney has been reported in diabetic patients and animal diabetic models.3,4 MCP-1, a typical chemokine, attracts inflammatory cells to renal tissue and activates inflammatory processes such as the stimulation of other cytokines and growth factors, and ultimately leads to

Department of Nephrology, Second Hospital of Shandong University, Jinan 250033, China (Zhang Y, Chen B, Hou XH, Guan GJ, Liu G, Liu HY and Li XG) Department of Nephrology, Yuhuangding Hospital, Yantai 264000, Shandong Province China (Zhang Y) Correspondence to: Dr. GUAN Guang-ju, Department of Nephrology, Second Hospital of Shandong University, Jinan 250033, China (Tel: 86-531-85875004. Fax: 86-531-8962544. Email: guangj@sdu.edu.cn; zhangyan@medmail.com.cn) This study was supported by the National Natural Science Foundation of China (No. 30570866).

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kidney injuries by induction of α1-type IV collagen synthesis.5 Podocyte loss begins even in early DN, and this can accelarate the functional loss of glomerular permselectivity and aggravate proteinuria.6 However, podocyte involvement in the inflammatory response has not yet been reported. Considering the facts that podocytes are the major synthesis site of IL-1α and IL-1β, and have receptors for various chemokines, we may suppose that podocytes also have a role in inflammatory reaction.7 Utimura et al8 have reported that mycophenolate mofetil (MMF), known for its anti-inflammatory and antiproliferative properties and widely used to prevent allograft rejection in recent years, could prevent renal disease progression by suppressing lymphocytes, macrophages infiltration and adhesion molecules expression.9 MMF can reduce renal injury in anti-Thy1.1-nephritis rats, and has the anti-oxidant effect by blocking fibrosis in streptozotocin (STZ)-induced diabetic rats.10,11 However, MMF’s effect on preventing podocytes loss at early stage of DN remains poorly investigated. We detected MCP-1 and nephrin expression to evaluate podocyte’s role in inflammatory reaction. It has long been proved that angiotensin II receptor blockers (ARB) have the anti-proteinuria effect and can decrease podocytes damage in DN.12 Therefore, with ARB as control, we observed the effect of MMF on MCP-1 expression and podocytes loss, and investigated whether the effect of combined therapy of MMF with valsartan is better than that of monotherapies in reducing podocyte injury and proteinuria.

METHODS

Animals Male Wistar rats weighting 180–200 g were purchased from the Animal Experimental Center, Medical College of Shandong University, and housed at constant room temperature (21˚C) and humidity (75%) under a controlled light to dark cycle. Experimental protocol According to Anderson’s method,13 the rats went through lateral nephrectomy at the right side after being anesthetized by peritoneal injection of pentobarbital sodium (40 mg/kg body weight). Two rats died 48 hours after the surgery. Eight of the lateral nephrectomized rats were randomly chosen as control (NC, n=8). Two weeks later when the incisal opening healed completely, 40 remaining rats were given single peritoneal injection of STZ (65 mg/kg) (Sigma Chemical Co. St. Louis, USA) with 0.1 mmol/L citric acid as buffer while the control rats just had an injection of same amount of citric buffer. Forty-eight hours later, the blood glucose (BG) and urine glucose (UGLu) were detected twice in a week. The diabetic model was successfully induced if BG≥16.7 mmol/L and UGLu “+++” three times. Then the diabetic rats were randomly divided into four groups: DM group without any treatment (n=10); DMM group treated with MMF (Roche Switzerland Pharmaceutical Co.; n=10);

DMV group treated with valsartan (Beijing Nuohua China Pharmaceutical Co., China; n=10); DMVM group (n=10) treated with valsartan and MMF. Through all experiments, rats took food and water freely. Total glucose levels of tail blood were measured by the glucose oxidase method. When BG≥21 mmol/L, insulin NPH neck hypodermic injection was applied to control the glucose level (1–2 U/d). Twenty-four hours after the successful construction of diabetic model, the rats in DMM group were given MMF (15 mg·kg-1·d-1), DMV group valsartan (40 mg·kg-1·d-1), and DMVM group a combination therapy of MMF (15 mg·kg-1·d-1) and valsartan (40 mg·kg-1·d-1). The medicine was dissolved in 5% dimethylsulfoxide and then olive oil,14 and given by intragastric administration with the volume within 1 ml every day. NC and DM groups were given corresponding amount of normal 5% dimethylsulfoxide and olive oil in the same way. The rats were sacrificed eight weeks later. Animal care followed the guidelines of the National Defense Medical College for the care and use of laboratory animals in research. Specimen collection Body weight, BG and blood pressure (by tail plethysmography) were measured regularly. At the end of 8th week, 24-hour urine was collected through metabolic cages for the measurement of the albumin concentration by radioimmunoassay as described previously.15 Then the rats were anesthetized by sodium pentobarbital (50 mg/kg, ip), and blood samples were collected for serum biochemistry. Serum creatinine level was measured by the enzymatic colorimetric method as described.16 Kidneys were completely perfused with normal saline before their removal. Part of the left kidney was immersed into 10% neutral formalin solution and 2.5% glutaraldehyde for pathomorphology analysis. The other part was divided into pieces and stored at –70℃ for future quantitative real-time polymerase chain reaction (PCR). RNA extraction and cDNA synthesis Frozen renal tissues were homogenized and total RNA was extracted with Trizol (Invitrogen, USA) one-step extraction protocol. The extracted RNA was measured by agarose gel electrophoresis for quality and spectrometry for quantity. The OD260/OD280 ratios were within the range of 1.8–2.0. Reverse transcription reaction system (TakaYa, DRR035A) had a total volume of 20 μl and contained 4 μl 5×ExScriptTM buffer, 1 μl dNTP mixture (10 mmol/L), 1 μl random hexmers (100 μmol/L), 0.5 μl ExScriptTMRtase (200 U/μl), 0.5 μl RNase inhibitor (40 U/μl), 5–10 μl of total RNA, and RNase free dH2O. Reaction condition was 42℃10–15 minutes, 95℃ 2 minutes. Quantitative real-time PCR Fluorescence real-time quantitative PCR: 1000 ng mRNA sample was accurately added into the reverse

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transcription system with the resulting cDNA as standard. It was diluted with EASY dilution in 10-fold series for building standard curve. The real-time PCR was carried out in ABI PRISM 7000 HT (Applied Invitrogen 11744-100, USA) in 25 μl reaction system: 12.5 μl SYBR Premix Ex TaqTM

(2×Conc.), 0.5 μl ROX reference dye (50×), 0.5 μl PCR forward primer (10 μmol/L), 0.5 μl PCR reverse primer (10 μmol/L, commercial primer kits from Shanghai Sagon Biological Engineering Technology Co., China). Nephrin: sense: 5’-CTGAACCTGGACACATGGAGA-3’; antisen: 5’-AGCCTGGGATAGCTTGGCTTG-3’ (97 bp). MCP-1: sense: 5’-CAGCCGACTCATTGGGATCA-3’; antisen: 5’-CTATG-CAGGTCTCTGTCACGCTTC-3’ (146 bp); Gapdh: sense: 5’-AAACCCATCACCATCTT- CCA-3’; antisense: 5’-GTGGTTCACACCCATCACAA-3’ (198 bp). Two-step PCR procedure was applied: pre-denaturation at 95℃ for 10 seconds and 45 cycles for 5 seconds at 95℃ and 31 seconds at 60℃–63℃. Standard curves for target genes and internal reference gene were built under the same condition, and the cycle threshold (Ct) values of the samples were used to calculate the corresponding gene copy number. The result was presented as the ratio of target gene copy over housekeeping gene (GAPDH) copy. 17

Immunohistochemical staining for nephrin, desmin and MCP-1 For immunohistochemical staining, renal tissues were fixed in 10% neutral buffered formalin, casted in paraffin, sliced into 4 μm sections, and placed onto microscope slides. After removal of the paraffin by xylene and dehydration by graded alcohol, slides were immersed into distilled water. Kidney sections were then transferred into a 10 mmol/L citrate buffer solution and heated at 80℃ for 5 minutes for antigen retrieval. After washing, 3.0% peroxide was applied for 20 minutes to block the activity of endogenous peroxidase. To avoid non-specific staining, slides were incubated with normal goat serum at room temperature for 20 minutes. The primary antibodies, monoclonal rabbit anti-rat MCP-1 antibody (Santa Cruz, CA, USA), polyclonal goat anti-rat nephrin antibody and monoclonal mouse anti-rat desmin antibody (Santa Cruz) were added separately at 1:200, 1:200, 1:150 dilution and kept at 4℃ overnight. Negative control sections were stained under the identical conditions by substituting the primary antibody with equivalent concentrations of normal rabbit IgG. With an LASB2 kit/HRP (DAKO, Carpinteria, CA, USA), kidney sections were sequentially treated with normal goat serum, primary antibody, link antibody, followed by horseradish peroxidase-conjugated streptavidin. Peroxidase activity was identified by reaction with 3, 3’-diaminobenzidine tetrahydrochloride (Santa Cruz USA) substrate. Sections were then counterstained with Mayer’s haematoxylin, dehydrated

and mounted. To evaluate MCP-1, nephrin and desmin staining, each glomerular or tubulointerstitial grid field was graded semiquantitatively. Each score reflects changes in the extent rather than the intensity of staining, and depends on the percentage of positive grid field or glomeruli. Cases in which more than 5% resident cells showed positive staining were regarded as positive. Four scores were awarded: 0, absent or less than 25% staining; 1, 25%–50% positive staining; 2, 50%–75% positive staining; and 3, more than 75% positive staining. For each sample, 30–40 glomeruli were counted and the average score was calculated.18 Each slide was scored by an observer who was unaware of the experimental details. Immunofluorescence staining for nephrin was performed with frozen kidney sections. Four-micrometer sections were fixed in acetone, blocked with 10% goat serum, and incubated overnight with polyclonal goat anti-rat nephrin antibody (Santa Cruz). Then, sections were stained with fluorescein isothiocyanate-conjugated anti-goat IgG for 30 minutes at room temperature. After washing with PBS, sections were observed through a confocal laser fluorescence microscope (LSM-510; Carl Zeiss, Jena, Germany). Histological study All formalin-fixed kidney sections (3 μm) were stained with hematoxylin (HE) and periodic acid-schiff (PAS). Forty full-sized glomeruli for each specimen were observed on PAS-stained sections under a high power field (×400) and the glomerulosclerosis index (GSI) in each glomerulus was scored semi-quantitatively as follows:19 0, no sclerosis; 1, sclerosis in <25% of glomerulus; 2, sclerosis in 25%–50% of glomerulus; 3, sclerosis in >50% of glomerulus. To evaluate interstitial fibrosis, 20 fields for each section were assessed on PAS-stained sections (×200). Semi-quantitative analysis in each field was assessed as follows: 0, no fibrosis; 1, fibrosis in <10% area; 2, fibrosis in 10%–25% area; 3, fibrosis in 25%–50% area; 4, fibrosis in >50% area. Averages of glomerulosclerosis and interstitial fibrosis scores were calculated. Transmission electron microscope (TEM): renal cortex pieces at the size of 1 mm3 were fixed, embedded, cut into 50-μm section, and observed under TEM. These microscopic evaluations were performed by histologists who were blind to the experimental groups. Statistical analysis All data were expressed as mean ± standard deviation (SD). Since GSI, interstitial fibrosis and 24-hour urinary protein followed a non-normal distribution, log transformation was performed prior to statistical analysis of these parameters. For statistical comparison between multiple groups, analysis of variance was performed followed by the q test. Correlations between GSI, interstitial fibrosis, MCP-1, nephrin mRNA and 24-hour

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urinary protein were assessed by Spearman’s correlation test. P<0.05 was considered statistically significant. All data were analyzed with the statistics software SPSS13.0 (SPSS Inc., Chicago, USA).

RESULTS Baseline characteristics of diabetic rats At the end of 8th week, blood glucose levels, 24-hour UP and hypertrophy index were higher than that of control rats (P<0.01). Valsartan reduced systolic blood pressure (SBP) by approximately 30 mmHg when compared with untreated diabetic rats (P<0.01). There was no statistically significant difference in blood pressure between the DMV and DMVM groups. MMF alone had no obvious effect on SBP. Increased proteinuria tended to decrease in diabetic rats treated with MMF, valsartan or combined therapy. There was no significant change in the serum creatinine in all groups (P>0.05, Table 1).

Table 1. Basic characteristics of each grous (mean ± SD) Groups Bloodg lucose Ratio# Scr Systolic BP Upro (μmol/L) (×103) (μmol/L) (mmHg) (mg/24 hours)

NC 6.03±0.42 5.4±1.3 84.57±3.86 100±3 16.97±2.91 DM 18.20±1.26* 8.5±1.8* 85.87±9.44 138±9* 63.35±5.71*

DMM 17.96±1.58* 6.7±1.1△ 82.12±6.51 140±10* 23.49±7.02*△

DMV 18.66±3.54* 6.9±0.1△ 87.53±4.01 103±6△ 30.63±1.84*△

DMVM 18.98±3.21* 6.7±2.1 81.78±5.59 108±6△ 30.38±5.83*△

#Left kidney weight/body weight, *P<0.01 vs NC groups; △P<0.05 vs DM group.

Histological findings Glomerular lesions in diabetic rats were characterized by thickening of the basement membrane, mesangial expansion and sclerosis. The glomerulosclerosis scores in diabetic rats were significantly higher than that of control rats (1.05±0.06 vs 0.14±0.04, P<0.01). MMF, valsartan or their combined therapy could significantly reduce glomerulosclerosis in diabetic rats (0.78±0.08, 0.84±0.01, 0.81±0.07, P<0.01 vs untreated DM; Fig. 1). Interstitial fibrosis was focal and mild but the interstitial fibrosis

score was higher in untreated diabetic rats than that of control (0.49±0.01 vs 0.14±0.02, P<0.01; Fig. 1). Interstitial fibrotic lesions were significantly reduced by MMF, valsartan or combined therapy compared with that of untreated diabetic rats (0.36±0.03, 0.22±0.03, 0.32±0.02, vs 0.49±0.01, P<0.05). There were significant correlations among 24-hour urinary protein, glomerulosclerosis score (r=0.67, P<0.01) and interstitial fibrosis score(r=0.74, P<0.01).

Fig. 1. Semiquantitative analyses of glomerulosclerosis index (A) and interstitial fibrosis score (B) in all groups (*P<0.05 and**P<0.01 vs NC group; #P<0.05 and ##P<0.01 vs DM group).

Renal tissue sections were observed through an electronmicroscope. Compared with NC group, DM groupshowed thickened glomerular basement membrane,vacuolization in part of the renal tubular cells, foot

Fig. 2. Comparison of the foot processwidth between five groups (originalmagnification ×20 000). Average width ofthe foot process of the DM rat was fargreater than that of the control group. A.Non-diabetic control rat; B. Untreateddiabetic control rat; C. MMF treateddiabetic rat; D. Valsartan treated diabeticrat; E. Diabetic rat treated with acombination of MMF + valsartan.

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Fig. 3. Effect of MMF and valsartan on glomerular nephrin (A−E)/MCP-1(F−J) protein expression (Immunohistochemistry, SP, original magnification ×200). A, F. Non-diabetic control rat; B, G. Untreated diabetic control rat; C, H. Valsartan treated diabetic rat; D, I. MMF treated diabetic rat; E, J. Diabetic rat treated with a combination of valsartan + MMF.

Fig. 4. Immunofluorescent staining of nephrin in kidneys (original magnification ×300). A. Non-diabetic control rat; B. Untreated diabetic control rat; C. Valsartan treated diabetic rat; D. MMF treated diabetic rat; E. Diabetic rat treated with a combination of valsartan + MMF.

process denudation, exposed GBM and shedded podocytes. In treatment groups foot process fusion was less serious, and shedded podocytes were rare (Fig. 2). Immunohistochemical staining for nephrin, desmin and MCP-1 Immunohistochemistry demonstrated the localization of podocyte-specific nephrin in NC rats and decreased as well as altered expression pattern mainly in the central aspect of glomeruli in untreated diabetic rat (Fig. 3). MMF and valsartan treatment restored the nephrin expression with dominant localization along the glomerular capillary area (Fig. 3). MCP-1 and desmin barely expressed in the control rats. In the diabetic kidney, MCP-1 was mainly observed in the tubulointerstitial areas (Fig. 3). As with MCP-1, desmin synthesis also increased rapidly in the diabetic kidney. Different from MCP-1, desmin was mainly observed in the glomeruli area. Interestingly, MMF and valsartan treatment could significantly suppress tubulointerstitial MCP-1 synthesis (0.47±0.44, 0.63±0.31 vs 1.73±0.64, P<0.01) and

intraglomerular desmin synthesis (0.69±0.30, 0.34±0.15, 0.54±0.30 vs 1.46±0.60, P< 0.01) (Table 2).

Table 2. Semiquantitative analysis of desmin, nephrin and MCP-1

by immnohistochemistry (mean ± SD) Groups Desmin Nephrin MCP-1 NC 0.25±0.12 2.70±0.60 0.22±0.14 DM 1.46±0.60** 0.17±0.16** 1.73 ±0.64**

DMM 0.69±0.30* △ 1.95±0.60** △ 0.47±0.44 △ DMV 0.34±0.15 △ 1.13±0.30** # △ 0.63±0.31△

DMVM 0.54±0.30 △ 1.05±0.20** # △ 0.52±0.50△

*P<0.05, **P<0.01 vs NC group; △P<0.01 vs DM group; #P<0.01 vs MMF treated diabetic group (DMM). Immunofluorescent staining also revealed the fact that untreated diabetic rats had a decreased nephrin expression as well as altered the expression pattern which mainly locates in the central area of glomeruli rather than in peripheral area (Fig. 4). MMF and valsartan treatment can stop nephrin expression decrease along the glomerular capillary area (Fig. 4). These results suggest that MMF and valsartan have a supportive role in maintaining nephrin and thus may be the glomerular filtration barriers.

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Quantitative real-time PCR Nephrin mRNA expression in diabetic rats was lower than that of control rats (78%, P<0.05). Valsartan, MMF and combined therapy can significantly restore nephrin mRNA expression in diabetic rats (Fig. 5). MCP-1 mRNA expression in the renal cortex was significantly increased in diabetic rats compared with that of control rats (251%, P<0.01). Treatment with valsartan had reduced MCP-1 mRNA expression by 66.9% (P<0.05). Over-expression of MCP-1 in diabetic rats was significantly suppressed by treatment with MMF or combined therapy of valsartan and MMF (50.2%, 60.9%, P<0.01). There were significantly negative correlations between nephrin and MCP-1 mRNA (r=–0.86, P<0.01), 24-hour UP and mRNA levels of MCP-1 (r=0.56, P<0.01) or nephrin (r=–0.78, P<0.01).

Fig. 5. Glomerular nephrin and MCP-1 mRNA in experimental diabetic nephropathy. mRNA was quantified using real-time PCR in glomerular from control rats (NC) and diabetic rats receiving MMF(DMM),valsartan (DMV),combined with MMF and valsartan (DMVM) or diabetic rats untreated (DM). Results are expressed as threshod cycle (Ct) for nephrin/MCP-1 standardized to Ct for the Gapdh. Values are means ± SE relative to controls, which were arbitrarily assigned a value of 1 (*P<0.05 and **P<0.01 vs NC group; #P<0.05 and ##P<0.01 vs DM group).

DISCUSSION

Podocyte is one of the glomerular inherent cells with the characteristics of high differentiation and limited dividing ability. Progressive podocyte damage in diabetic rats occurs due to three potential causes: (1) hyperlipidaemia, (2) monocyte/macrophages glomerular infiltration, and (3)

glomerular hypertrophy.20 These potential causes could make the podocyte lose its normal structure which then leads to foot process fusion, separation of podocyte from glomerular basement membrane (GBM) and GBM exposure. All these changes ultimately lead to the loss of GBM normal structure, occurrence of proteinuria, and high level of proteinuria can in return cause unreversible structural abnormalities and renal disfunction. Due to its limited ability to proliferate, podocyte can not recover the already exposed GBM and this condition finally leads to glomerular sclerosis. Nephrin, a recently discovered podocyte-specific protein, is crucial for the integrity of podocyte slit membrane structure and maintenance of an intact filtration barrier.21 In diabetic nephropathy, protein level of nephrin decreases due to the loss of nephrin into urine because the splice variant isoform of nephrin lacks a transmembrane domain.1,22 Desmin, the intermediate filament protein, which presents in mesangial cells in normal glomerulus, is highly expressed in podocytes in vivo exclusively in the setting of glomerular injury, and then is studied as a general marker of podocytes function perturbation and injury.23-25 Toyoda et al26 reported that nephrin mRNA expression was in close relation with the occurrence and development of proteinuria in DN patients. The more severe of proteinuria, the less nephrin mRNA is expressed. In our study, we observed foot process denudation, exposed GBM and shedded podocytes in diabetic rats. The harder the podocyte is damaged, the more the nephrin expression is down-regulated. As the consequences of nephrin expression down-regulation, the structure of slit membrane in podocyte is destroyed and the kidney state begins to deteriorate. All this demonstrated that the disorganization and decrease of slit membrane in podocyte can accelerate the development of DN. Podocyte damage can be illustrated by increased desmin or decreased nephrin as a sign for a switch-over to intermediate filament expression.27,28 MMF can slow down the development of glomerular injury in STZ induced diabetic rats.8 Our study proved that MMF and valsartan could prevent the loss of podocytes and subsequently reduce urinary albumin excretion. MMF or valsartan or their combined therapy could reduce glomerular expression of desmin /MCP-1 and increase the expression of nephrin. Podocyte damage occurs in early diabetic nephropathy and is related to the severity of proteinuria.6,29 Podocytes are known in synthesis of major proteins to be related to the progression of renal disease such as transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF).30 Han et al31 reported that MCP-1 was also synthesized by glomerular podocytes, and was detectable in the tubulointerstitial tissues. Thus, podocytes panticipate in the synthesis of MCP-1, an inflammatory mediators known as the strongest chemotactic factor for monocytes and is upregulated in many renal diseases,32 including DN.33 Our study demonstrated that MMF could suppress MCP-1 synthesis, alleviate glomerulosclerosis,

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and lighten interstitial fibrotic lesions in STZ induced diabetic kidneys. These results indicate that podocytes play an important role in the inflammatory process under diabetic conditions. In addition, an increased 24-hour UP was observed during the early stage of DN in STZ-induced diabetic rats. It is of interest that MMF could alleviate proteinuria and inflammatory reactions, and is shown by significant inhibition of renal MCP-1 staining in diabetic renal tissues. To the best of our knowledge, these results demonstrate that podocytes can actively take part in the inflammatory process under diabetic conditions and MMF has a beneficial role in early DN due to its anti-inflammatory action. Our experiment showed that MCP-1 expression rapidly increased after induction of DM, and the mRNA levels of MCP-1 and desmin significantly increased in the early stage of DN. Our findings are in agreement with previous reports that urinary MCP-1 excretion increases in patients of early DN, and increases more as albuminuria becomes severe.34 Our results show that MMF or ARB can suppress renal MCP-1 and desmin expression and attenuate renal podocyte injury without altering glycaemic level in diabetic rats，but the combination of valsartan and MMF showed no superiority over monotherapies on renal protection. These drugs may function by suppression of inflammation to protect podocytes. Therefore, the application of these agents at the early stage of DN is recommended. In conclusion, podocytes are involved in the inflammatory process in diabetic rats, and MCP-1 synthesis is upregulated under diabetic conditions and is significantly suppressed by MMF or valsartan treatment. This study helps us to understand the multifactorial nature of DN and suggests that therapeutic intervention for this condition may require an approach involving different agents to inhibit various putative pathogenic pathways. These results suggest that MMF may be a new therapeutic agent in the treatment of early DN.

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(Received February 15, 2007)

Edited by WANG Mou-yue and LIU Huan