Synergistic Effects of Combining Anti-Midkine and Hepatocyte Growth Factor Therapies Against Diabetic Nephropathy in Rats

BASIC INVESTIGATION

Synergistic Effects of Combining Anti-Midkine and Hepatocyte Growth Factor Therapies Against Diabetic Nephropathy in Rats Xiaojun Ren, MD, Zhaowei Meng, MD, PhD, Hui Yang, PhD, Hui Li, MD, PhD, Ke Xu, PhD, Wei Zheng, MD, PhD, Ping Feng, MD and Junwei Wang, MD

Abstract: Purpose: This study aimed to assess whether synergism could be achieved when combining midkine (MK) antisense oligodeoxynucleotides (anti-MK ODN) and recombinant human hepatocyte growth factor (HGF) in diabetic nephropathy (DN) rat models. Methods: Rats were randomized into 6 groups: control, DN rats without treatment, DN rats treated with scrambled ODN, DN rats treated with anti-MK ODN, DN rats treated with HGF and DN rats treated with anti-MK ODN plus HGF. DN models were created by intraperitoneal injection of streptozotocin. Two weeks later, treatments commenced. ODN (1 mg/kg) was intravenously injected weekly for 4 weeks. HGF (500 mg/kg) was subcutaneously injected daily for 4 weeks. Eight weeks later, rats were euthanized. Serum and urine parameters, kidney histopathological injury scores, immunohistochemistry and protein expressions were measured. Results: Blood glucose, creatinine, blood urea nitrogen and urine albumin were significantly elevated in DN rats. Any single treatment markedly reduced their levels, yet combined treatment decreased them significantly further. Any monotherapy could decrease renal injury score and immunohistochemistry positive percentage, although the most prominent change was displayed in combinational therapy. Western blot showed the expression of MK was significantly elevated in DN rats. Anti-MK ODN suppressed MK significantly. The protein expressions and serum concentrations of transforming growth factor-b1 and connective tissue growth factor between monotherapy and the combined therapy were significant. Conclusions: This study demonstrated that combining MK gene suppressing ODN and HGF protein synergistically attenuates renal injury in DN rats. This study may provide a novel avenue for designing future therapeutic regimens against DN. Key Indexing Terms: Diabetic nephropathy; Midkine; Hepatocyte growth factor; Transforming growth factor-b1; Connective tissue growth factor; Synergism. [Am J Med Sci 2015;350(1):47–54.]

From the Departments of Metabolic Diseases (XR, HL, PF, JW), Nuclear Medicine (ZM, WZ), and Anatomy (HY), Tianjin Medical University General Hospital, Tianjin, China; and Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment (KX), Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China. Submitted September 30, 2014; accepted in revised form February 11, 2015. The authors have no conflicts of interest to disclose. Supported by Tianjin Municipal Bureau Science Foundation Grant 09KZ113 (awarded to X.R.). This investigation was supported by the National Key Clinical Specialty Project (awarded to the Departments of Nuclear Medicine and Radiology). This study was also supported by Tianjin Medical University General Hospital New Century Excellent Talent Program; Young and Middle-aged Innovative Talent Training Program from Tianjin Education Committee and Talent Fostering Program (the 131 Project) from Tianjin Education Committee, Tianjin Human Resources and Social Security Bureau (awarded to Z.M.). Correspondence: Zhaowei Meng, MD, PhD, Department of Nuclear Medicine, Tianjin Medical University General Hospital, Anshan Road No. 154, Heping District, Tianjin, China 300052 (E-mail: [email protected]).

The American Journal of the Medical Sciences



D

iabetic nephropathy (DN) is a major complication of diabetes mellitus, which is also the principle cause of endstage renal failure in many countries.1,2 With the worldwide increase in the prevalence of diabetes mellitus, DN has become a threat to human health and mortality. Approximately onethird of all diabetic individuals (either type 1 or type 2) are affected by DN, which produces significant social and economic burdens. Advances in understanding of the pathogenesis and etiology of DN (especially inflammatory status and oxidative stress) have identified additional risk factors for nephropathy, and novel therapeutic options are being explored.3–5 It is proven that longstanding hyperglycemia induces severe endothelial dysfunction, oxidative stress and inflammation, leading to changes in renal morphology and function. In diabetic rodent models and in diabetic patients, increased levels of transforming growth factor-b1 (TGF-b1)6–9 and connective tissue growth factor (CTGF)10–12 have been demonstrated as key factors during the process of DN. These growth factors, along with albuminuria and pathologic changes (glomerular hypertrophy, tubular dilation, tubular degeneration, interstitial expansion, extracellular matrix [ECM] accumulation and thickening of the glomerular basement membrane), are used as characteristic indicators of DN. The growth factor midkine (MK) has been implicated in neuronal survival and differentiation, cancer development and inflammation-related diseases.13 Recently, MK was identified as a key molecule not only in mesangial-mediated nephropathy but also as a direct activator of the tubulointerstitial inflammatory process associated with DN.14,15 MK antisense oligodeoxynucleotides (anti-MK ODN) can suppress tumor growth in nude mice,16 ameliorate arterial restenosis, ischemic reperfusion-induced renal damage and cisplatin-induced nephropathy.17–19 Hepatocyte growth factor (HGF) is a mesenchymederived cytokine with antifibrotic and regenerative properties in experimental models of chronic renal damage.20–25 HGF inhibition exacerbates renal fibrosis, whereas HGF supplementation reverses this progression.20–23 The antifibrotic activity of HGF was reported to work through the antagonistic action on TGFb1.26–28 The beneficial effect of a long-term treatment with recombinant HGF on streptozotocin-induced DN in mice has also been observed.29 In this study, the authors aimed to assess the effects of combining anti-MK ODN and HGF against DN in rat models, and tried to determine whether synergism can be achieved.

MATERIALS AND METHODS Chemicals, Reagents and Animals MK antisense ODN 59-AGGGCGAGAAGGAAGAAG39 corresponded to bases 15 to 32 in MK cDNA; scrambled ODN 59-GGGAAAAGAAACGGGAGG-39 was used as a

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control of the antisense DNA, as described before.16–18 Phosphorothioate-modified ODNs were synthesized from SBS Genetech Co (Beijing, China). These ODNs have been tested previously in murine models as effective in suppressing MK.16–18 Recombinant human HGF was purchased from R&D Systems (Minneapolis, MN). One hundred twenty male Sprague Dawley rats weighing (180 6 20) g, purchased from the Animal Center of Science Institute of China (Beijing, China), were used in this study. All the animals were kept in individual cages at a temperature of 18 to 20°C, humidity of 65% to 69%. The experimental procedures were approved by the animal ethics committee of Tianjin Medical University General Hospital. Experimental Protocol Rats were randomly divided into 6 groups (20 per group): control rats (group 1), DN rats without treatment (group 2), DN rats treated with scrambled ODN (group 3), DN rats treated with anti-MK ODN (group 4), DN rats treated with HGF (group 5) and DN rats treated with anti-MK ODN plus HGF (group 6). All DN rats were 1st intraperitoneally injected with streptozotocin (Sigma, St. Louis, MO) at a dose of 50 mg/kg diluted in citrate buffer (0.1 mol/L, pH 4.0), whereas rats in control group only received equal volume of citrate buffer. During the experimental period, body weight (BW) and fast blood glucose (FG) of rats were measured weekly, and all the diabetic rats were subcutaneously given long-acting insulin (Glargine; Sanofi-Aventis, Frankfurt-Höchst, Germany) to maintain the FG at about 30 mmol/L to avoid ketonemia and promote the well-being of animals. Two weeks after streptozotocin injection, different treatments commenced. One milligram per kilogram of ODN was intravenously injected through the tail vein every week for 4 weeks. Five hundred micrograms per kilogram of HGF was subcutaneously injected daily for 4 weeks. The dosages of ODN16–18 and HGF25–27 have already been tested in murine models as optimal when used as monotherapeutic modalities. Rats were euthanized at the end of 8 weeks after therapy initiation. Before the rats were euthanized, 24-hour urine samples were obtained by using metabolic cages and were centrifuged at 3,000 rpm for 10 minutes and stored at 280°C for subsequent examinations. Urine albumin (UAlb) concentrations were measured by Turbox microalbuminuria assay (Orion Diagnostica, Espoo, Finland). While the rats were euthanized, blood was collected in centrifuge tubes from the inguinal vein. Serum was isolated and stored at 280°C for subsequent examinations. Serum was assayed for values of FG, blood creatinine (Cr) and blood urea nitrogen (BUN) by an automatic biochemistry analyzer (Hitachi Model 7170 analyzer; Hitachi, Tokyo, Japan). Serum was also used for concentration measurements of MK, TGF-b1 and CTGF. The kidneys were quickly removed, 1 part of the tissue was fixed in neutral formalin for histological sectioning and the other part of the tissue was snap frozen into liquid nitrogen for protein extraction. Histopathology, Immunohistochemistry and Semiquantification Removed kidney samples were fixed in neutral buffered formalin (pH 7.4) and embedded in paraffin. Tissue sections from the rats were prepared at 4-mm thickness by a routine procedure. Sections were stained with hematoxylin and eosin for general histology. Kidney lesions, characterized by glomerular hypertrophy, tubular dilation, tubular degeneration, interstitial expansion, ECM accumulation and glomerular basement membrane thickening, were graded according to the extent of

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cortical involvement on a scale from 0 to 3: 0 5 normal; 1 5 involvement of less than 30%; 2 5 involvement up to 30% to 70%; 3 5 extensive damage involving more than 70% of the pertinent area. And they were expressed as injury scores.17,18 Immunohistochemistry was performed on paraffinembedded sections, as described previously.30 The measurement of positive staining percentage was conducted by counting average positive cells in 3 field areas. Protein Levels of MK, TGF-b1 and CTGF in Kidneys Assessed by Western Blot Protein extraction and Western blot were performed as described before.18,31,32 Briefly, kidney samples were pulverized in liquid nitrogen and resuspended in tissue lysis buffer on ice, and the supernatants were collected after centrifugation at 13,000g at 4°C for 20 minutes. Protein concentration was determined and then stored at 280°C until use Tissue lysates were 1st mixed with an equal amount of 23 SDS loading buffer. Then, equal amounts of protein were electrophoresed by 15% SDS-polyacrylamide gel electrophoresis. Semiquantification was performed by Quantity One Software Version 4.6.2 (BioRad, Hercules, CA). The intensity of the Western blot bands was quantified as the ratio of intensity of the protein of interest to that of b-actin 3 100%.33,34 Antibodies for MK, TGF-b1 and CTGF and b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). Serum Levels of MK, TGF-b1 and CTGF Measured by Enzyme-Linked Immunosorbent Assay Serum levels of MK, TGF-b1 and CTGF were measured by enzyme-linked immunosorbent assay method (Uscn Life Science Inc, Houston, TX). The assays were conducted according to the manufacturer’s instructions. Briefly, 100 mL of each serum sample or standard was incubated in precoated 96-well microplate 1st. Then, biotinylated anti-MK, TGF-b1 or CTGF antibodies were added and incubated. Afterward, streptavidinconjugated horseradish peroxidase was added and incubated. Later, substrate solutions (tetramethylbenzidine) were added. Finally, stop solutions (H2SO4) were added, and the optical densities of the wells were measured at 450 nm with a Multiskan MS Plate Reader (Labsystems, Helsinki, Finland). After creating a standard curve on log-log graph paper, concentrations of the samples were determined. Statistical Analysis All data were presented as mean 6 SD. Statistics were performed with SPSS 17.0 (SPSS Inc, Chicago, IL). Differences between groups were analyzed by 1-way analysis of variance (ANOVA). Least significant difference (LSD) test was used for multiple comparisons among groups. P value not exceeding 0.05 was considered statistically significant.

RESULTS Effects of Different Treatments on Physical Behaviors, BW, Serum Indices and Urine Protein In the experiments, rats in group 1 had normal growth and behavior; rats in group 2 and group 3 had hypopraxia, cachexia, polyuria/polydipsia, yellowish and damp fur and kyphosis; rats in group 4 and group 5 were relatively vibrant, had gray to white fur; rats in group 6 had almost normal vibrancy and white fur. BWs of rats were different (Table 1), rats in group 2 and group 3 had the lowest BWs, after monotherapy BW was significantly elevated in group 4 and group 5 (P , 0.01), and significant weight gain was also Volume 350, Number 1, July 2015

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TABLE 1. Effects of different treatments on body weight, serum indices and urine protein Body weight Fasting blood glucose Blood creatinine Blood urea nitrogen Groupsa (g) (mmol/L) (mmol/L) (mmol/L) 1 2 3 4 5 6 F (P)b P(1):(2)c P(2):(3)c P(2):(4)c P(2):(5)c P(4):(6)c P(5):(6)c

463.85 6 34.19 328.70 6 41.18 336.35 6 42.56 382.15 6 59.49 381.20 6 66.04 426.75 6 67.34 18.95 (,0.01) ,0.01 0.57 ,0.01 ,0.01 0.03 0.04

Urine albumin (mg/24 hr)

6.41 6 0.82

30.97 6 3.01

5.75 6 1.11

76.1 6 10.21

31.04 6 3.82

81.21 6 6.01

33.86 6 2.58

729.70 6 59.66

31.06 6 3.80

80.95 6 5.20

33.12 6 2.83

719.75 6 75.53

28.48 6 4.69

55.15 6 8.87

19.31 6 3.41

446.20 6 81.24

28.62 6 4.65

57.59 6 7.95

19.24 6 3.18

488.50 6 83.45

27.06 6 5.86

37.07 6 6.14

8.80 6 1.68

183.65 6 45.34

99.66 (,0.01) ,0.01 0.99 0.07 0.08 0.40 0.36

213.24 (,0.01) ,0.01 0.89 ,0.01 ,0.01 ,0.01 ,0.01

411.93 (,0.01) ,0.01 0.39 ,0.01 ,0.01 ,0.01 ,0.01

348.36 (,0.01) ,0.01 0.65 ,0.01 ,0.01 ,0.01 ,0.01

a Group 1, control group; group 2, DN rats without treatment; group 3, DN rats treated with scrambled ODN; group 4, DN rats treated with antiMK ODN; group 5, DN rats treated with HGF; group 6, DN rats treated with anti-MK ODN plus HGF. b Analyzed by 1-way analysis of variance. c Analyzed by least significant difference test.

observed in combinational therapy in contrast to single therapies (P , 0.05). Table 1 showed that FG levels were significantly increased in DN rats compared with control group (P , 0.01). Although after treatments, especially after combinational therapy, FG levels were lowered, yet, no significance has been achieved (P . 0.05). The levels of Cr, BUN and UAlb in rats of the DN groups were significantly higher than group 1 (P , 0.01), suggesting that the rodent DN models were successful. Any single treatment markedly reduced Cr, BUN and UAlb levels in the DN rats (P , 0.01), yet their levels decreased significantly further by combined treatment (P , 0.01). ANOVA unveiled significant differences among the groups (P , 0.01), whereas LSD certified the differences between any monotherapy group and the combined therapy group were significant (P , 0.01). Comparison of Histopathology and Immunohistochemical Indices Semiquantitative estimation of morphology-based renal damage showed significantly greater renal injury in DN rats than in control rats (Table 2 and Figures 1A–F). Any monotherapy could decrease injury score significantly, although the most prominent change was displayed in group 6. Immunohistochemical analyses of key DN indicators (TGF-b1 and CTGF) showed significant increases in DN rats than control group. Any monotherapy could decrease positive staining percentage significantly, although the most significant change was shown in group 6. ANOVA demonstrated significant differences among the groups (P , 0.01), whereas LSD proved the differences between any monotherapy group and the combined therapy group were significant (P , 0.01), indicating the best therapeutic effect and synergism in group 6 (Table 2 and Figures 1G–R). Copyright © 2015 by the Southern Society for Clinical Investigation.

Protein Levels of MK, TGF-b1 and CTGF in Kidneys The expression of MK was significantly elevated in DN rats. Anti-MK ODN suppressed MK properly and significantly (Table 3 and Figure 2). Surprisingly, the HGF monotreatment could also decrease MK expression, which in

TABLE 2. Histopathological and immunohistochemical changes by different therapies Transforming Connective tissue growth factor-b1 growth factor (%) (%) Groupsa Injury score 1 2 3 4 5 6 F (P)b P(1):(2)c P(2):(3)c P(2):(4)c P(2):(5)c P(4):(6)c P(5):(6)c

0.55 6 0.51 2.80 6 0.41 2.75 6 0.44 1.85 6 0.49 1.90 6 0.55 0.95 6 0.51 70.41 (,0.01) ,0.01 0.71 ,0.01 ,0.01 ,0.01 ,0.01

3.45 6 1.47 58.40 6 7.18 58.10 6 6.93 39.80 6 6.30 42.25 6 6.80 15.75 6 2.75 308.99 (,0.01) ,0.01 0.89 ,0.01 ,0.01 ,0.01 ,0.01

2.90 6 1.37 58.80 6 7.02 56.75 6 6.69 38.45 6 6.31 38.30 6 6.07 14.45 6 2.63 336.43 (,0.01) ,0.01 0.35 ,0.01 ,0.01 ,0.01 ,0.01

a Group 1, control group; group 2, DN rats without treatment; group 3, DN rats treated with scrambled ODN; group 4, DN rats treated with anti-MK ODN; group 5, DN rats treated with HGF; group 6, DN rats treated with anti-MK ODN plus HGF. b Analyzed by 1-way analysis of variance. c Analyzed by least significant difference test.

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FIGURE 1. Therapeutic effects on histopathology and immunohistochemistry. Histopathology and immunohistochemistry were performed as described in Materials and Methods. A, hematoxylin/eosin stain of group 1 rat. B, hematoxylin/ eosin stain of group 2 rat. C, hematoxylin/eosin stain of group 3 rat. D, hematoxylin/ eosin stain of group 4 rat. E, hematoxylin/eosin stain of group 5 rat. F, hematoxylin/ eosin stain of group 6 rat. G, TGF-b1 immunohistochemistry of group 1 rat. H, TGF-b1 immunohistochemistry of group 2 rat. I, TGF-b1 immunohistochemistry of group 3 rat. J, TGF-b1 immunohistochemistry of group 4 rat. K, TGF-b1 immunohistochemistry of group 5 rat. L, TGF-b1 immunohistochemistry of group 6 rat. M, CTGF immunohistochemistry of group 1 rat. N, CTGF immunohistochemistry of group 2 rat. O, CTGF immunohistochemistry of group 3 rat. P, CTGF immunohistochemistry of group 4 rat. Q, CTGF immunohistochemistry of group 5 rat. R, CTGF immunohistochemistry of group 6 rat. Group 1 to 6 were control rats, DN rats without treatment, DN rats treated with scrambled ODN, DN rats treated with anti-MK ODN, DN rats treated with HGF and DN rats treated with anti-MK ODN plus HGF, respectively.

theory could be the result of improvement of DN after HGF therapy. Expressions of TGF-b1 and CTGF significantly increased in DN rats than in control rats. Any monotherapy

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could decrease their expressions significantly, although the most significant inhibition was shown in group 6 (P , 0.01), indicating synergy in group 6 (Table 3 and Figure 2). Volume 350, Number 1, July 2015

Synergizing Anti-MK ODN and HGF Against DN

TABLE 3. Quantitative estimation of the protein bands in kidney samples from Western blot Transforming Connective growth tissue growth Midkineb Groupsa factor-b1b factorb 1 2 3 4 5 6 F (P)c P(1):(2)d P(2):(3)d P(2):(4)d P(2):(5)d P(4):(6)d P(5):(6)d

42.95 6 7.34 143.99 6 21.25 141.94 6 19.89 21.83 6 4.21 120.56 6 19.95 20.10 6 3.16 323.77 (,0.01) ,0.01 0.66 ,0.01 ,0.01 0.71 ,0.01

37.30 6 7.18 140.53 6 21.15 140.40 6 25.10 89.33 6 13.64 92.04 6 12.16 53.74 6 8.14 143.82 (,0.01) ,0.01 0.98 ,0.01 ,0.01 ,0.01 ,0.01

35.49 6 7.40 132.96 6 15.23 134.98 6 18.52 89.51 6 11.15 91.65 6 12.63 55.90 6 7.45 198.27 (,0.01) ,0.01 0.62 ,0.01 ,0.01 ,0.01 ,0.01

a Group 1, control group; group 2, DN rats without treatment; group 3, DN rats treated with scrambled ODN; group 4, DN rats treated with antiMK ODN; group 5, DN rats treated with HGF; group 6, DN rats treated with anti-MK ODN plus HGF. b Band intensities were quantified as the ratio of intensity of the protein of interest to that of b-actin 3 100%. c Analyzed by 1-way analysis of variance. d Analyzed by least significant difference test.

Serum Levels of MK, TGF-b1 and CTGF MK, TGF-b1 and CTGF are secretory factors, which can be measured from peripheral blood samples. More importantly, TGF-b1 and CTGF have already been reported as very good serum biomarkers for DN.8–11 Therefore, in this study, the authors also intended to test whether serum parameter measurements could be used to indicate kidney changes in DN rats and found significant elevations of serum MK, TGF-b1 and CTGF levels in DN rats. Either anti-MK ODN or HGF could decrease serum levels of the parameters, yet the most significant suppression

FIGURE 2. Protein levels of MK, TGF-b1 and CTGF in kidneys. At the end of 8 weeks after therapy initiation, kidney tissues were subjected to protein extraction and then Western blot. (A–F) Referred to groups 1 to 6, namely, control rats, DN rats without treatment, DN rats treated with scrambled ODN, DN rats treated with anti-MK ODN, DN rats treated with HGF and DN rats treated with anti-MK ODN plus HGF, respectively. MK, TGF-b1 and CTGF were proteins of interest, and b-actin was used as control. Copyright © 2015 by the Southern Society for Clinical Investigation.

was observed in the combined therapy, which was also suggestive of synergistic therapeutic effects against DN in group 6 (Table 4).

DISCUSSION DN is one of the most problematic renal diseases because of the exponentially increasing number of patients entering chronic dialysis programs with renal failure resulting from diabetes, and the high mortality rates of these patients receiving dialysis.1,2 During initiation and the progression of DN, increase in the protein filtration, reabsorption and ECM accumulations contribute to tubulointerstitial damage and progressive loss of renal function. DN eventually leads to proteinuria and glomerular sclerosis, and it is directly related to a decline in renal function.35 The progression of DN is a complicated process in which many factors may play active roles. In this regard, it is not easy to find out an effective monotherapy. And no presently available treatment is effective in halting the progressive loss of renal function. It is necessary to develop novel treatment to deal with more than 1 target. This study was undertaken to test the hypothesis that a combined therapy by simultaneously targeting MK and HGF may be more effective in preventing the progression of DN. The results clearly demonstrated that MK gene suppression together with delivery of exogenous recombinant human HGF protein could lead to superior therapeutic outcomes. It is demonstrated that the growth factor MK played a critical role in the tubulointerstitial damage and inflammatory process associated with DN.15 MK evoked by high glucose in primary-cultured mesangial cells accelerated production of TGF-b1, and loss of MK may prevent kidney damage.15 MK enhanced both neutrophil and macrophage migrations in the tubulointerstitial regions, which were detrimental to kidneys.14,18 Several studies have shown that knockdown of MK gene prevented a variety of kidney damages, for example, onset of kidney disease in diabetes,15 ischemia-reperfusion17,36 and 5/6 nephrectomy-induced hypertension.37 MK antisense ODN

TABLE 4. Serum levels of the parameters Transforming Midkine growth factor-b1 a Groups (ng/mL) (ng/mL) 1 2 3 4 5 6 F (P)b P(1):(2)c P(2):(3)c P(2):(4)c P(2):(5)c P(4):(6)c P(5):(6)c

15.06 6 3.68 51.02 6 11.82 50.49 6 12.23 25.23 6 4.40 44.87 6 6.68 22.95 6 4.55 76.31 (,0.01) ,0.01 0.85 ,0.01 0.02 0.37 ,0.01

21.01 6 4.00 74.31 6 9.50 71.09 6 10.70 51.51 6 5.91 52.28 6 6.88 37.14 6 3.61 155.01 (,0.01) ,0.01 0.16 ,0.01 ,0.01 ,0.01 ,0.01

Connective tissue growth factor (ng/mL) 19.86 6 4.46 68.66 6 6.49 65.39 6 9.29 49.36 6 7.72 51.38 6 6.24 34.54 6 3.17 160.30 (,0.01) ,0.01 0.12 ,0.01 ,0.01 ,0.01 ,0.01

a Group 1, control group; group 2, DN rats without treatment; group 3, DN rats treated with scrambled ODN; group 4, DN rats treated with antiMK ODN; group 5, DN rats treated with HGF; group 6, DN rats treated with anti-MK ODN plus HGF. b Analyzed by 1-way analysis of variance. c Analyzed by least significant difference test.

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was proven to be effective in inhibiting renal damage. And delivery of the reagent to the kidney presented no problems.17 ODN, administered into the systemic circulation, has been demonstrated to be selectively taken up by the renal tubular epithelial cells in mice and rats.38,39 Sato et al17 demonstrated that MK antisense ODN was efficiently taken up in the proximal tubules. The side effects are also issues of concern in antisense therapeutics. The adverse effects common to phosphorothioate ODN are independent of the molecular target, which may include lymphoid proliferation, mononuclear cell infiltration to multiple tissues and degeneration of the liver and renal proximal tubules. However, the side effects of phosphorothioate ODN are generally observed at doses above 50 mg/kg.40–42 And 10 mg/kg dose clinical trials showed good tolerance.43–45 The dose of MK antisense ODN used in the present experiment was 1 mg/kg, which was tested previously as having optimal effects in murine monotherapy modality.16–18 And good efficacy and no apparent side effects were observed. HGF has numerous merits, such as protective, regenerative and antifibrotic effects in nephron tissues.20–25 In renal disease progression, TGF-b1 and HGF exert reciprocal and essential functions.26,28 They inhibit the synthesis of each other and counteract biological activities. TGF-b1 is a key modulator in renal fibrosis and HGF is a protective and antifibrotic factor, in addition, HGF has a potent antiapoptotic effect, whereas TGF-b1 induces destructive changes in parenchymal cells.26,28 Blocking endogenous HGF signaling with neutralizing antibody markedly promotes renal tissue fibrosis and dysfunction.26 Conversely, administration of recombinant HGF protein or its gene prevents the development and progression of renal dysfunction, accompanied by a decrease in TGF-b1 expression.29,31,46 It was reported that a high dose of HGF attenuated the induction of CTGF by TGF-b1 in tubular epithelial cells and that this was responsible for the significant suppression of renal interstitial fibrosis.47 HGF signaling also has a critical role in protecting podocytes against dedifferentiation and apoptosis triggered by injurious stimuli under pathological conditions,48 and administration of HGF protein or its gene was effective in ameliorating proteinuric kidney diseases.48–50 Moreover, it has even been proven that HGF can improve islet b-cell mass and function in transplant models of type 1 and type 2 diabetes.51 Therefore, in this study, HGF was used as one method to treat DN, and the results with HGF administration plus MK gene inhibition combination therapy were compared. Among many potential cytokines involved in tubulointerstitial injury, TGF-b1 is generally accepted as a critical indicator in renal diseases.52 TGF-b1 stimulates the deposition of ECM through direct upregulation of matrix protein genes, inhibits matrix degradation by suppressing proteases and increasing the synthesis of protease inhibitors, increases cell surface expression of integrins to promote attachment to newly synthesized matrix and autoinduces its own production.53 TGF-b1 mRNA and protein levels are increased in both glomerular and tubular compartments of various rat and mouse experimental models of type 1 and type 2 diabetes.53 CTGF expression is TGF-b responsive and is considered the major downstream effector of TGF-b, which acts as an extracellular mediator of cross-talk between various growth factor signaling pathways implicated in DN.10 CTGF is the key factor in stimulating connective tissue cell proliferation, ECM production and other profibrotic properties of TGF-b1.54 CTGF and TGF-b1 exhibit shared fibrogenic and angiogenic properties in vivo, as they both promote cell adhesion, migration, proliferation and differentiation.54 Therefore, TGF-b1 and CTGF (their expressions in

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kidneys and serum concentrations), along with morphologybased kidney injury scores as indicators, were used to measure therapeutic effects in this research. The present work validated a hypothesis that synergistic effect existed in a combined therapy targeting multiple key molecules of DN. This approach of mechanism could be necessary for designing an effective treatment of diabetic renal disease. Although MK gene suppression and delivery of exogenous HGF protein alone attenuated renal lesions, their combination showed dramatically synergized efficacy. The authors found that the combined therapy not only diminished TGF-b1 and CTGF but also instructed kidney cells, leading to a constructive repair process to restore the nephron structure and function. Among the myriads effects of the dual targets in this investigation, MK is the upstream activator of TGF-b1 and HGF is the antagonist of TGF-b1. The authors assume that suppression of TGF-b1 could be one of the major mechanisms at play in making the outcomes of the dually treated animals better. Therefore, the combined therapy could have the real potential to halt the progression of chronic renal disease. In fact, the similar combinational effects superior to monotherapy has also been observed in a previous study; Yang et al31 demonstrated that combining HGF gene therapy and reninangiotensin system blockade synergistically attenuated renal interstitial fibrosis in obstructive nephropathy in mice. Above all, because no monotherapy is sufficient to manage DN, the combined therapeutic strategy is very attractive in future clinical trials. There is 1 additional issue deserves comments. The animals in groups 4 to 6, which were treated with either monotherapy or combined therapy, had lower FG and higher BW than animals in the untreated groups. The authors considered that the improved metabolic milieu and body condition in treated animals were the result of the therapies. For instance, HGF was proven to improve the quantity and function of engrafted b-cells, resulting in corrected FG.51 For the case of MK, its signaling cascade is mediated by cell surface receptors, and then kinases in mitogen-activated protein kinase (MAPK) pathway.55,56 It was reported that inhibition of MAPK pathway could decrease plasma glucose and b-cell apoptosis, and increase plasma insulin level and glucose homeostasis as well.57 MAPK inhibition could also improve diabetic peripheral neuropathy,58 diabetic cardiomyopathy59 and endothelial functions.60 Therefore, the authors deduce that MK antisense ODN acting on targets in various organs and tissues contributed to the lowered FG and elevated BW in the animals through the suppressed MAPK pathway. In other words, the better metabolic condition of the treated animals should be the consequences of the therapies. In fact, the same phenomenon has also been observed by Kosugi et al.15 In Kosugi’s report, lower FG was demonstrated in MK-deficient mice DN models than in MK wild-type mice DN models. The authors will test this hypothesis in their next step investigations. While the authors believe that there is a direct effect of these therapeutic agents in the diabetic rat kidney, it is likely that these agents had an indirect effect through the improved metabolic milieu observed with their use. It is widely accepted61–63 that high glucose is the initial culprit that sets off a chain of events that leads to the enhanced production and laying down of ECM seen in DN.

CONCLUSIONS This study demonstrated that a combined therapy with MK gene suppressing ODN and HGF protein synergistically attenuates renal interstitial fibrosis in DN rats. This study may Volume 350, Number 1, July 2015

Synergizing Anti-MK ODN and HGF Against DN

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