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Published as doi: 10.1096/fj.06-6713com.
 (The FASEB Journal. 2007;21:3355-3368.)
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Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes

Mausumee Guha*,1, Zhong-Gao Xu{dagger}, David Tung{ddagger}, Linda Lanting{dagger} and Rama Natarajan{dagger}

* Department of Pharmacology, Kalypsys Inc., San Diego, California, USA;

{dagger} Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California, USA; and

{ddagger} Department of Pfizer Inc., St. Louis, Missouri, USA

1Correspondence: Department of Pharmacology (Metabolic Diseases), Kalypsys Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA. E-mail: mguha{at}kalypsys.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diabetic nephropathy (DN) remains a major complication in both type 1 and type 2 diabetes. Systemic administration of antitransforming growth factor-ß (TGF-ß) antibody has shown some promise in mouse models of DN. However, chronic blockade of the multifunctional TGB-ß could be problematic. Several downstream effects of TGF-ß are mediated by connective tissue growth factor (CTGF), which is up-regulated in several renal cells and secreted in the urine in the diabetic state. Using murine models of DN (type 1 and type 2) and a CTGF antisense oligonucleotide (ASO) of novel chimeric chemistry, we evaluated the specific role of this target in DN. In the type 1 model of DN, C57BL6 mice were made diabetic using streptozotocin injections and hyperglycemic animals were treated with CTGF ASOs (20 mg/kg/2 qw) for 4 months. ASO, but not mismatch control oligonucleotide, -treated animals showed significant reduction in target CTGF expression in the kidney with a concomitant decrease in proteinuria and albuminuria. Treatment with the CTGF ASO for 8 wk reduced serum creatinine and attenuated urinary albuminuria and proteinuria in diabetic db/db mice, a model of type 2 DN. The ASO also reduced expression of genes involved in matrix expansion such as fibronectin and collagen (I and IV) and an inhibitor of matrix degradation, PAI-1, in the renal cortex, contributing to significant reversal of mesangial expansion in both models of DN. Pathway analyses demonstrated that diabetes-induced phosphorylation of p38 MAPK and its downstream target CREB was also inhibited by the ASO. Our results strongly suggest that blocking CTGF using a chimeric ASO holds substantial promise for the treatment of DN.—Guha, M., Xu, Z-G., Tung, D., Lanting, L., Natarajan, R. Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes.


Key Words: diabetic nephropathy • CTGF • glomerular mesangial cells • antisense oligonucleotide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DIABETIC NEPHROPATHY (DN) ACCOUNTS FOR >30% of end-stage renal failure. The pathological hallmark of DN is glomerulosclerosis due to accumulation of extracellular matrix in the glomerular mesangium and tubulointerstitial fibrosis (1) . Hyperglycemia in diabetes contributes directly to the severity of the disease, and several mechanisms for hyperglycemia-induced cellular damage have been reported. It is now widely accepted that up-regulation of transforming growth factor-beta 1 (TGF-ß) is one of the key factors responsible for the increased fibrotic changes and scarring observed in response to diabetic renal injury (2 , 3) . The contribution of TGF-ß to the pathology of glomerulosclerosis has been established both by in vivo studies and by the analyses of glomerular cells in culture (2 3 4 5 6) . However, TGF-ß is a pleiotropic cytokine with beneficial anti-inflammatory properties that are not related to fibrosis and scarring. Therefore, therapeutic modalities that target TGF-ß may yield conflicting and confounding results. As a result, identification of causal factors downstream of TGF-ß, which are direct contributors to glomerular matrix expansion and tubulointerstitial fibrosis, could serve as better therapeutic targets for disease intervention in DN.

In recent years, connective tissue growth factor (CTGF) has been identified as a prosclerotic cytokine acting downstream of TGF-ß and shown to be involved in the regulation of matrix accumulation (7) . CTGF, a 36 to 38 kDa cysteine-rich secreted protein, is one of six distinct members of the CCN family and is encoded by an inducible immediate early gene. It has a heparin binding domain and interacts with integrin {alpha}Vß3 (8 9 10 11) . CTGF promotes chemotaxis, migration, differentiation, and formation of extracellular matrix. CTGF is induced mainly by TGF-ß, and the fibrogenic properties of TGF-ß may be mediated at least in part by CTGF. For example, TGF-ß, but not platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF), increases CTGF mRNA and protein in cultured normal human skin fibroblasts (12) . TGFß-induced CTGF expression is under transcriptional control, as a TGF-ß-responsive element was demonstrated in the CTGF promoter (13) . Several lines of evidence have implicated CTGF as a possible contributor to the various fibrotic complications observed in diabetes. CTGF is a potent inducer of extracellular matrix accumulation in various cell types, including glomerular mesangial cells (MC). Increased glucose concentration has been shown to induce CTGF mRNA and protein levels in human and rat MC in vitro, and CTGF has been implicated in the development of glomerulosclerosis and tubulointerstitial injury observed in the kidney cortex leading to disease progression in DN (14 15 16 17 18 19) . This cytokine has also been shown to play a critical role in the development of interstitial fibrosis in other kidney fibrosis models (19) . However, a direct role for CTGF in mediating the progression of DN has not yet been clearly demonstrated.

Evidence suggests that both TGF-ß and CTGF play key roles in the pathogenesis of fibrotic disorders in diabetic conditions. However, it is important to note that treatment with the anti-TGF-ß antibody did not attenuate albuminuria in the db/db mice despite its beneficial effects on glomerular matrix expansion and renal function (20) . Other studies suggest that TGF-ß may not be a key factor in the glomerular hemodynamic and permeability changes that result in proteinuria (21 22 23) . Furthermore, TGF-ß–independent induction of CTGF has also been described (14 , 21 , 23 24) . A positive correlation was demonstrated between progression from normoalbuminuria to microabuminuria and increased CTGF and collagen (IV) {alpha}2 expression in renal biopsies from type 1 DN patients (25 26 27) . CTGF therefore appears to be an attractive therapeutic target in DN, especially since urinary CTGF can be used as a biomarker for monitoring clinical outcomes (26) .

It was demonstrated recently that intravenous administration of CTGF antisense oligonucleotide (ASO) (phosphorthioate) significantly blocked CTGF expression in a proximal tubular epithelial cells in the remnant kidney of TGF-ß1 transgenic mice despite the sustained level of TGF-ß mRNA. There was also a concomitant decrease in the expression of genes involved in matrix expansion and matrix degradation, resulting in attenuation of renal interstitial fibrosis, which indicates that the TGF-ß1-independent CTGF pathway plays a role in the development of interstitial fibrosis (28) . These results suggest that CTGF-induced TGFß-dependent and TGFß-independent pathways may both contribute to the pathology of DN. The objective of the current study was to evaluate the specific role of CTGF in the progression of DN using murine models of type 1 and type 2 diabetes (29) . We have used a novel CTGF ASO with chimeric chemistry (phosphorthioate and phosphodiester) and a convenient biweekly dosing schedule, achieving maximized kidney targeting with good tolerability and no systemic toxicity.

Using this ASO, we demonstrate that inhibition of diabetes-induced expression of CTGF in the kidney cortex attenuates progression of DN and holds substantial promise as a therapeutic tool in treating this debilitating disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotide chemistry and synthesis
2'-O-Methoxyethylribose (MOE) -modified phosphodiester/phosphorthioate (PO/PS) chimeric oligonucleotides and MOE-modified phosphorthioate (PS) oligonucleotides cross-reactive with murine, rat, and human CTGF were synthesized, purified, and provided by Isis Pharmaceuticals (Carlsbad, CA, USA) (30) . The oligonucleotides are 20-mer in length, with 4 bp on either end (wing) containing phosphate ester (PO) linkers; the 12 bp in the middle (gap) contain thioate ester (PS) linkers, resulting in an ASO with PO/PS mixed backbone (MBB) chemistry. The wings of these chimeric ASO consist of 2'-MOE modified PO linkages whereas the gap consists of 2'H/PS nucleotides demonstrating RNase H-dependent antisense activity. To demonstrate selective targeting of the chimeric ASO to the kidney vs. the liver, MOE-modified PS oligonucleotides with the same sequence as the chimeric CTGF ASO were also synthesized. The wings of the phosphorthioate ASO consist of 2'-MOE modified PS linkages and the gap consists of 2'H/PS nucleotides. Reduction of sulfur content in the CTGF chimeric (PO/PS) ASO compared with its CTGF PS counterpart allowed lower accumulation of the chimeric ASO in the liver compared with the full PS ASO, thereby altering its pharmacokinetic and tolerability properties. The sequences for CTGF ASO and the mismatch control oligonucleotide (7 bp mismatch) were 5'-CCACAAGCTGTCCAGT[b]CTAA3' and 5'-CCTCTAGCAGTCCTGT[b]CATA3', respectively. The four boldfaced nucleotides on each end of the ASOs represent the wings with the MOE modification, which increase the stability of the ASOs. Underlined nucleotides were modified in the mismatched ASO.

Reagents
Monoclonal antibodies for phosphor-specific and nonphospho-p38, -ERK1/2 mitogen-activated protein kinases (MAPKs), and cyclic AMP response element binding protein (CREB) were from Cell Signaling (Beverly, MA, USA); ß-actin antibody was from Sigma (St. Louis, MO, USA); CTGF antibody was obtained from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated secondary antibodies were from Cell Signaling; Supersignal chemiluminescence reagent was from Pierce (Rockford, IL, USA); urinary albumin and a creatinine ELISA Kit were from Exocell Inc. (Philadelphia, PA, USA). Relative multiplex reverse transcription-polymerase chain reaction (RT-PCR) kits and primers for Quantum RNA 18S internal standards were from Ambion Inc. (Austin, TX, USA); RNA-STAT 60 reagent was from Tel-Test (Friendswood, TX, USA).

Cell culture and transfections
A rat mesangial cell line (rMC) was obtained from ATCC (Manassas, VA, USA) and used to demonstrate EC50 of the ASO. Primary culture rMCs from Sprague-Dawley rats were obtained by the sieving method (23) and cultured in RPMI 1640 supplemented with 10% FBS at 37°C in 5% CO2. Cells between passages 5 to 8 were used for all studies. Transfections were performed using Lipofectamine 2000 reagent (Gibco BRL, Carlsbad, CA, USA) according to the manufacturer’s instructions. MCs were transfected with the CTGF-ASO or the mismatch control oligonucleotide (MM). After transfection (6 h), cells were washed and transferred to serum-free medium supplemented with 5.5 mM (normal) or with 25 mM (high) D-glucose. Cells were lysed for RNA and protein extraction at 48 and 72 h after transfection, respectively.

Animal models of diabetic nephropathy and ASO administration
All animal studies were conducted under a protocol approved by the Institutional Research Animal Care Committee. The animals were housed in a temperature-controlled room and given ad libitum access to water and standard rodent chow. C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) received 50 mg/kg of streptozotocin (STZ) intraperitoneally on 5 consecutive days to develop an experimental model of type 1 diabetes-induced DN (29) . In parallel, a similar number of mice were injected with the STZ diluent and served as control animals. Blood glucose levels were measured on day 5 after the last STZ injection to confirm the development of hyperglycemia. Experimental design consisted of a diabetic mice arm and a nondiabetic control mice arm. Each arm consisted of three groups (6 mice/group) treated subcutaneously with vehicle (0.8% physiological saline), CTGF ASO, or the mismatch control oligonucleotide (20 mg/kg per dose), using a biweekly dosing regimen for 16 wk. We defined diabetes as fasting glucose of >300 mg/dl. Body weights, blood chemistry, and 24 h urine analyses were performed monthly during ASO treatment. Blood glucose was measured with a glucometer, and 24 h urinary albumin and creatinine excretion were determined by ELISA according to the manufacturer’s instructions. All mice were sacrificed after 16 wk of treatment using general anesthesia and blood was drawn using cardiac puncture. Kidney weights were measured at the time of sacrifice. Cortical tissues were removed from the kidney and stored at –70°C for RNA and protein analyses. Kidney tissues were also fixed in buffered formalin for histology analyses using hematoxylin and eosin and periodic acid Schiff (PAS) staining.

Naturally developed diabetic nephropathy in db/db mice (>4 months of age) is a well-established model for type 2 diabetes, obesity, and insulin resistance (29) . Elevated serum creatinine, urinary albuminuria and proteinuria, increased fibrosis in the proximal tubule, and mesangial matrix expansion are all reminiscent of human disease in this model (28 , 30) . Therefore, we used this model to evaluate the pharmacological efficacy of CTGF ASO in reversing or attenuating the various indices of type 2 DN. Male db/db mice (BKS.Cg-m+/+leprdb/J) and age-matched db/m control mice were obtained from Jackson Laboratories. Mice were treated with the CTGF ASO (5, 10, or 20 mg/kg per dose) or the MM control oligonucleotide (20 mg/kg per dose) for 8 wk using a twice a week dosing regimen. Similar pharmacological measures were obtained as described for the STZ model (supplemental Fig. 1B). In separate studies, db/db mice were treated with the CTGF ASOs (chimeric or phosphorthioate) or the MM control oligonucleotide for 8 wk at 10 or 20 mg/kg using a 2 qw dosing regimen. Liver and kidney tissues were collected to measure ASO concentration (30) . In addition, kidney fractionation was performed to isolate proximal tubule, glomeruli, and distal tubule-enriched pellets. ASO concentration was measured in each subfraction, and CTGF mRNA level was determined in the proximal tubule and glomerular fractions (30 , 31) .

RNA isolation, RT-PCR, and quantitative RT-PCR (qRT-PCR)
Total RNA was isolated using RNA-STAT 60 reagent according to the manufacturer’s instructions. cDNA was synthesized with 1 µg of RNA using murine leukemia virus reverse transcriptase and random hexamers. Primers for the 18S ribosomal RNA (489 bp or 324 bp) were included in each reaction as an internal control. RNA was similarly reverse transcribed. The RT-PCR products were separated by electrophoresis using 1% agarose gels, and DNA band intensities were quantified using Quantitation One software (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to 18S, as described (32 , 33) .

For qRT-PCR, total RNA was extracted from the cortical slices and eluted into water using the RNAeasy 96-plate kit (Qiagen, Valencia, CA, USA). RNA levels were quantified using the Perkin-Elmer (Norwalk, CT, USA) ABI PRISM 7700 Sequence Detection System by real-time fluorescence PCR Detection. All primers and probes were synthesized by IDT, Inc. (Coralville, IA, USA). RT-PCR kits were purchased from Invitrogen (Carlsbad, CA, USA). The 25 µl PCR reaction contained 2.5 µl of 10 x PCR buffer, 5 mM MgCl2, 0.3 mM dNTP, 10 U RNase inhibitor, 0.625 U Taq, 6.25 U MuLV reverse transcriptase, 0.1 µM primers, 0.1 Fam-probe, and ~50–100 ng RNA. First-strand cDNA synthesis was performed at 48°C for 30 min followed by a 10 s heat inactivation step at 95°C. PCR denaturation was conducted at 95°C for 15 s, annealing/extension at 60°C for 1 min for 40 cycles. The primer probe sets used in the study are listed in Table 1 .


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  		Table 1. Primer sequences for amplification of various transcripts

Western blot
Cortical tissue samples were lysed in sodium dodecyl sulfate (SDS) sample buffer (2% SDS, 10 mM Tris-HCl, pH 6.8, 10% [v/v] glycerol). Lysates were centrifuged at 12,000 rpm for 15 min at 4°C and the supernatant was stored at –70°C. Protein (50 µg per lane) was separated on a 10% SDS-PAGE gels (Bio-Rad Laboratories), transferred onto a nitrocellulose membrane, and immunoblotted with antibodies phospho-p38 MAPK (1:1000), phospho-ERK1/2 (1:1000), phospho-CREB (1:1000), or anti-CTGF (1:500) as described (32 , 33) . The blots were stripped and reprobed with an antibody to ß-actin (1:5000), total p38 MAPK (1:1000), total ERK1/2 (1:1000) or total CREB (1:1000). Immunoblots were scanned using GS-800 densitometer and protein bands were quantitated with Quantitation One software (Bio-Rad).

Histology
Kidney slices were fixed in 10% buffered formalin overnight prior to paraffin embedding. Development of fibrosis in the tubular epithelium and mesangial matrix expansion were assessed in paraffin-embedded tissue sections (4 µm) stained with PAS. Parasagital tissue sections were analyzed by bright-field microscopy and collected images were analyzed using the ImagePro software. For analyses of matrix expansion in the glomeruli, the total matrix area (red) was normalized to the number of nuclei (blue) to account for the plane of sectioning through the glomeruli.

Statistical analysis
Data are expressed as mean ± SE from multiple experiments. One-way ANOVA along with Tukey’s post tests for multiple groups were used in the statistical evaluation of the data using PRISM software (Graph Pad, Prizm, San Diego, CA, USA). Statistical evaluation of the qRT-PCR and histology data was performed using a Student’s paired t test. Statistical significance was detected at the 0.05 level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CTGF ASO but not the MM control oligonucleotide inhibits high glucose-induced CTGF and ECM protein expression in an rMC
The ASOs were designed in silico and first screened for optimum inhibition of target mRNA in an rMC using a high throughput qRT-PCR approach. Lead compounds were characterized by dose response analyses, and the most potent ASO was evaluated for sequence-dependent down-regulation of target mRNA. The potent ASO selected and used in this study is cross-reactive with mouse, rat, and human CTGF. A series of mismatch (MM) oligonucleotides was designed for the CTGF ASO and a 7 bp MM oligonucleotide was selected for this study. We then evaluated the specificity of the ASO in reducing HG-induced CTGF expression in primary rMCs. Cells were transfected with the CTGF ASO or MM control oligonucleotide for 6 h, washed to remove the ASOs, then transferred to serum-free medium supplemented with high glucose (HG, 25 mM). Cyclophilin-normalized expression of CTGF in mock transfected cells was considered to be maximal (100%). The IC50 determined for the ASO was 53 nM, with no effect of the 7 bp oligonucleotide (Fig. 1 A). We also evaluated the effect of the ASO on CTGF expression (mRNA and protein) in rMCs cultured in NG (5.5 mM) or HG. Cells were transfected as described above, then transferred to serum-free medium supplemented with NG (5.5 mM) or HG glucose. Pretreatment of rMCs with CTGF ASO, but not the MM control oligonucleotide, specifically inhibited HG-induced CTGF mRNA (Fig. 1B ) and protein (Fig. 1C ) expression. Basal expression of CTGF in NG mesangial cells was also reduced by the ASO but not the MM control oligonucleotide.


Figure 1
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  		Figure 1. CTGF ASO down-regulates target and key matrix inducing genes. A) A rat mesangial cell line (rMC) was transfected with the CTGF ASO or the MM control oligonucleotide, and CTGF gene expression was evaluated by Taqman qRT-PCR to determine IC50 for the ASO. Data was normalized to cyclophilin B. The ratio of CTGF to cyclophilin B in the mock transfected cells represented control set to 100%. The concentration (IC50) at which the ASO inhibited 50% expression of CTGF compared with the control was 53 nM. B, C) Primary rMCs were transfected with the CTGF ASO or the MM control oligonucleotide for 6 h; the transfection medium was removed and transfected cells were cultured in NG (5.5 mM) or HG (25 mM) for 72 h. CTGF expression was evaluated by semiquantitative RT-PCR (B) with 18S internal control or by Western blot analyses (C). CTGF ASO, but not the MM control oligonucleotide, down-regulated endogenous expression (NG cells) as well as HG-induced expression of CTGF mRNA and protein. D, E) HG-induced expression of collagen I [{alpha}1], fibronectin, and MCP-1 was also evaluated by semiquantitative RT-PCR in ASO or MM control oligonucleotide transfected rMCs cultured in NG or HG. CTGF ASO, but not the MM control oligonucleotide down-regulated, HG-induced expression of collagen I [{alpha}1] and fibronectin. E) Expression of MCP-1 was induced by HG but was unaffected by the ASO or the MM control oligonucleotide. Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. mock transfected control.

Matrix expansion in the glomeruli is a hallmark of DN, collagen 1[{alpha}1] and fibronectin being two of the matrix-inducing genes (17) . We therefore evaluated the effect of the ASO on the expression of these matrix genes induced by HG. Expression of collagen 1[{alpha}1] and fibronectin mRNA, induced in rMCs by HG was also inhibited by the CTGF ASO, but not the MM control oligonucleotide (Fig. 1D ). The temporal sequence of HG-induced matrix gene up-regulation correlated well with HG-induced expression of CTGF. To confirm that ASO-mediated down-regulation of collagen 1[{alpha}1] and fibronectin mRNA levels were indeed a CTGF-specific effect, we evaluated the expression of other collagen and chemokine genes. Monocyte chemoattractant protein-1 (MCP-1) expression was induced in the mesangial cells by HG but was unaffected by the ASO or the MM control oligonucleotide (Fig. 1E ). Besides demonstrating specificity of the ASO, these results suggest that collagen 1[{alpha}1] and fibronectin may serve as biomarkers in monitoring the pharmacological efficacy of the ASO in rodent models of DN.

The chimeric CTGF ASO accumulates preferentially in the kidney in vivo, holding promise for superior therapeutic tolerability
To demonstrate selective targeting of the chimeric ASO (2'-MOE-PO/PS) to the kidney vs. the liver, distribution of the chimeric ASO was compared with its 2'-MOE/PS counterpart. Diabetic db/db mice used for efficacy studies were treated with the chimeric or the 2'-MOE/PS CTGF ASO for 8 wk. Tissues (kidney and liver) were collected on the day of sacrifice 4 h after the last dose. The concentration of ASO was determined using capillary gel electrophoresis (31) . A dose-dependent accumulation of CTGF ASO was observed in the kidneys, the concentration being very similar in the chimeric as well as the 2'-MOE/PS ASO-treated mice (Fig. 2 A, chimeric and phosphorthioate). However, the concentration was 50% lower in the liver than in the kidneys in chimeric ASO-treated mice. The phosphorthioate ASO-treated mice had a 3-fold higher concentration of the ASO in the liver compared with the chimeric ASO-treated mice. Reduction in the sulfur content in the CTGF chimeric ASO compared with its 2'-MOE/PS counterpart allowed for lower accumulation in the liver, thereby altering its pharmacokinetic (PK) and tolerability properties. We next evaluated the distribution of the ASO in the different regions of the kidney using a kidney fractionation protocol as described in Materials and Methods. ASO concentration was determined in fractions enriched for proximal and distal tubules as well as the glomeruli (Fig. 2B ). ASO distribution was mainly localized to the outer cortex (proximal tubule and glomerule-enriched fractions), with levels below detection in the medullary region (distal tubule-enriched fraction). To further correlate the concentration of the ASO in the proximal tubule and glomeruli with target reduction, we evaluated mRNA levels of CTGF in each fraction by qRT-PCR (Fig. 2C ). Maximal CTGF reduction (>83% at 10 mg/kg) was observed in the proximal tubular fraction, demonstrating a good correlation between pharmacokinetic (PK, ASO concentration) and pharmacodynamics (PD, CTGF expression) end points.


Figure 2
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  		Figure 2. A–C) Chimeric (PO=PS) CTGF ASO has selective pharmacokinetic distribution in the kidney cortex and down-regulates the target in proximal tubular epithelial and the mesangial cells. A) ASO concentration was determined using capillary gel electrophoresis (CGE) analyses in liver and kidney tissues obtained from db/db mice treated with the chimeric (PO/PS), the phosphorthioate (PS) CTGF ASO (5, 10, or 20 mg/kg/dose given using a 2 qw regimen), or the MM chimeric oligonucleotide for 8 wk. Compared with the PS ASO, the chimeric ASO demonstrated reduced distribution in the liver. B) Concentration of the chimeric CTGF ASO was determined by CGE analyses in different fractions of the kidney to evaluate distribution of the ASO. The ASO was distributed primarily to the cortical region of the kidney, levels being undetectable in the medullary region. The majority of the ASO was distributed to the proximal tubular fraction, with low to moderate levels in the glomerular fraction. C) CTGF expression was determined by Taqman qRT-PCR in the proximal tubular and glomerular fractions of the kidney cortex. Expression of CTGF normalized to cyclophilin B in vehicle-treated mice served as the control, marked as 100%. The CTGF ASO demonstrated superior target reduction in the tubular cells and moderate target reduction in the glomerular compartment.

CTGF ASO inhibits various indices of renal disease in an STZ-induced mouse model of type 1 diabetes and DN
To determine the pharmacological and therapeutic potential of the CTGF ASO, we evaluated its efficacy in ameliorating the progression of nephropathy in STZ-challenged diabetic mice. The mice were treated with CTGF-ASO or the MM oligonucleotide for 16 wk after the establishment of diabetes. We monitored blood glucose levels several times during the study to ascertain sustained hyperglycemia in the STZ-treated group. The experimental design is outlined in supplemental Fig. 1 . Fasting blood glucoses were maintained at normoglycemic levels in the nondiabetic group (127±13 mg/dl), but at robustly hyperglycemic levels in the STZ-treated group (572±67 mg/dl) throughout the 16 wk treatment period. The CTGF ASO had no effect on blood glucose levels during the duration of the study. The diabetic mice had normal weight gain over the 16 wk experimental period, although the gain was moderately higher in nondiabetic control mice (Table 2 ). There was, however, a significant increase in the ratio of kidney weight to body weight (72%, P<0.01) in the vehicle-treated diabetic mice compared with their nondiabetic counterparts, demonstrating the development of renal hypertrophy characteristic of DN in the STZ-challenged animals (Table 2) . Furthermore, compared with vehicle-treated diabetic mice, treatment with the CTGF ASO, but not the MM control oligonucleotide, significantly reduced the diabetes-induced increase in kidney weight (32%, P<0.02). ASO treatment had no effect on kidney weight in the nondiabetic control animals.


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  		Table 2. Effect of CTGF ASO on kidney hypertrophy (kidney weight/body weight) in STZ-induced diabetic micea

We next evaluated the effect of CTGF ASO on two key pathological features of DN: serum creatinine and urinary albumin excretion. Serum creatinine (Fig. 3 A) and urinary albumin excretion (Fig. 3B ) were significantly higher in the vehicle-treated diabetic group vs. control group (2.8-fold, P<0.001 and 2.7-fold, P<0.005, respectively). Treatment with CTGF ASO but not the MM control oligonucleotide significantly reduced the diabetes-induced increase in levels of serum creatinine (56%, P<0.005; Fig. 3A ) and urinary albumin (52%, P<0.05; Fig. 3B ). These pharmacological end points remained unaffected in all three treatment groups in the control nondiabetic mice.


Figure 3
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  		Figure 3. A–C) Treatment with the CTGF ASO but not the MM control oligonucleotide significantly ameliorated features of DN in STZ-challenged diabetic mice. STZ-challenged diabetic mice or nondiabetic control mice were treated with vehicle, CTGF ASO (20 mg/kg using a 2 qw regimen), or the MM control oligonucleotide for 16 wk. Compared with control mice, diabetic mice showed increased serum creatinine levels (A) and 24 h urinary albumin excretion (B). PAS staining of kidney sections (6 µM) demonstrated significant mesangial matrix expansion in vehicle-treated diabetic kidneys compared with vehicle-treated normal kidneys (C). Treatment with the CTGF ASO but not the MM control oligonucleotide significantly attenuated each diabetes-induced pathology (Fig. 3 A–C). Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. vehicle-treated diabetic mice.

To evaluate the effects on diabetes-induced matrix expansion and fibrosis, PAS staining of parasagital sections from the diabetic vs. normal kidneys was performed, followed by quantitation using the ImagePRO software. Our results demonstrated that diabetic kidneys (STZ) had profound mesangial matrix expansion (3.2±0.9-fold; P<0.001) compared with normal kidneys (Fig. 3C , control vs. STZ). Treatment with CTGF ASO, but not the MM control, significantly attenuated diabetes-induced mesangial matrix expansion (43%, P<0.05; Fig. 3C , bar graph).

To demonstrate that the antisense mechanism of action was responsible for the pharmacology described above, we evaluated the expression of CTGF mRNA in tissue lysates made from the cortical regions of the kidneys, since distribution of the ASO was primarily limited to this region (Fig. 2B, C ). Compared with control kidneys, CTGF mRNA was strongly induced (2.3±0.5-fold, P<0.005) in diabetic kidneys (Fig. 4 A, lanes 1–3 vs. lanes 10–12). Treatment with the CTGF ASO but not the MM control oligonucleotide significantly attenuated CTGF mRNA expression (Fig. 4A , panel 1; 59%; P<0.01, Fig. 4B ).


Figure 4
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  		Figure 4. A–D) Expression of CTGF and key fibrotic markers in the diabetic kidney were reduced by CTGF ASO treatment. A, B) CTGF, TGF-ß1, collagen 1[{alpha}1], fibronectin, and PAI-1 mRNA levels were determined by relative RT-PCRs, with 18S as an internal control. Compared with vehicle-treated normal mice, mRNA levels of each gene were significantly up-regulated in the kidney cortex of the diabetic mice. Treatment with CTGF ASO, but not MM control oligonucleotide, markedly attenuated each change. C) Compared with normal kidneys, CTGF protein level was only modestly up-regulated in cortical lysates from diabetic kidney, and CTGF ASO had a minimal effect on tissue protein levels. D) Urinary CTGF, however, was strongly induced in vehicle-treated diabetic mice compared with normal mice, and treatment with CTGF ASO, but not the MM control oligonucleotide, significantly attenuated urinary CTGF excretion. Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. vehicle-treated diabetic mice.

Immunoblotting of cortical lysates with anti-CTGF antibody demonstrated increased protein expression in vehicle-treated diabetic mice vs. nondiabetic control mice (Fig. 4C , lanes 7, 8 vs. lanes 1, 2). However, ASO-treated diabetic mice demonstrated only a marginal reduction in these tissue CTGF protein levels (Fig. 4C ; lanes 7, 8 vs. lanes 11, 12). Higher levels of urinary CTGF have been demonstrated in rodent models of diabetic nephropathy (25 26 27) ; such an increase was observed in our study as early as 3 wk after the establishment of diabetes. Urinary CTGF (2 months after treatment) was immunoprecipitated with heparin Sepharose beads, followed by immunoblotting with anti-CTGF antibody. Strikingly high levels of CTGF protein were observed in the urine from diabetic but not from the nondiabetic control mice (Fig. 4D , lanes 5–8 vs. lanes 1–4). Semiquantitative immunoblot analyses demonstrated that treatment with the CTGF ASO but not the mismatch control oligonucleotide reduced urinary CTGF levels (69%, Fig. 4D ; P<0.05) in the diabetic animals. Volume of the sample used in each lane was normalized for urine creatinine for an accurate comparison of CTGF protein levels.

We next examined the expression levels of key fibrotic markers and ECM-related genes that have been implicated in mesangial matrix expansion (16 , 20) and development of tubulointerstitial fibrosis (18 19) under diabetic conditions. Compared with nondiabetic mice, there was a significant up-regulation of collagen 1[{alpha}1] (1.82±0.25-fold) and PAI-1 (2.2±0.38-fold) mRNAs in the cortical regions of vehicle-treated diabetic mice (Fig. 4A , panels 2 and 5; and Fig. 4B ). Up-regulation of fibronectin (1.6±0.23-fold) and TGF-ß1 (1.73±0.3-fold) expression was also observed in the vehicle-treated diabetic but not the nondiabetic mice (Fig. 4A , panels 3 and 4; Fig. 4B ). Treatment with the CTGF ASO but not the MM control oligonucleotide significantly attenuated diabetes-induced expression of collagen 1[{alpha}1] (49%; P<0.05), fibronectin (29%; P<0.05), and TGF-ß1 (54%; P<0.03) (Fig. 4A, B ). Although PAI-1 expression was robustly augmented by diabetes, the CTGF ASO demonstrated only a trend in the reduction of PAI-1, which was not significant.

Pathway analyses have demonstrated that activation of the MAPKs and CREB plays key roles in the progression of DN and in the induction of the profibrotic and ECM markers discussed above (32 33) . We therefore compared the activation of key growth- and stress-related MAPKs and CREB in the cortical tissues of diabetic mice treated with CTGF ASO vs. the MM control oligonucleotide. Compared with nondiabetic control animals, STZ-challenged mice demonstrated diabetes-induced robust activation of p38 (4.34±1.54-fold), ERK1/2 (2.78±0.5-fold), and CREB (3.4±1.4-fold) proteins, as assessed by an increase in the levels of the corresponding phosphorylated proteins (pp38, pERK1/2, pCREB) in the cortical lysates (Fig. 5 A, panels 113; lanes 1–3 vs. lanes 7–9; Fig. 5B ). Treatment with the CTGF ASO but not the MM control oligonucleotide attenuated the activation of p38 kinase (54%; P<0.03) and CREB (74%; P<0.01), but not ERK1/2 (Fig. 5A , panels 1–3; lanes 7–9 vs. lanes 10–12; Fig. 5B ). The lack of change in levels of the nonphosphorylated form of these proteins indicated that CTGF ASO treatment specifically blocked diabetes-induced activation of p38 MAPK and CREB, components of a key pathway known to induce matrix accumulation in DN.


Figure 5
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  		Figure 5. A, B) Diabetes-induced activation of key MAPK pathway members was significantly attenuated after treatment with the CTGF ASO. Compared with vehicle-treated normal mice, phosphorylation of p38 MAPK, ERK1/2, and CREB was significantly increased in the cortical tissues lysates of vehicle-treated diabetic mice. CTGF ASO, but not MM oligonucleotide, markedly attenuated phosphorylation of p38 MAPK and CREB, but not ERK1/2 MAPK. Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. vehicle-treated diabetic mice.

CTGF expression is induced in diabetic kidneys from db/db mice, and urinary CTGF levels can be used as a biomarker to monitor disease progression in this model
It has been documented that db/db mice represent a model that faithfully simulates various feature of type 2 DN. Anti-TGFß antibody could partially improve renal function in this model by attenuating glomerular sclerosis and mesangial matrix expansion, but had no effect on urinary albumin excretion (20) . We demonstrated that CTGF mRNA is strongly induced (3.4±0.6-fold; Fig. 6 A) in cortical kidney slices from diabetic mice compared with age-matched db/m control mice (5 months age). We demonstrated earlier (Fig. 4D ) that CTGF protein is excreted in the urine of STZ-induced diabetic mice (model of type I DN), and may serve as a biomarker for disease progression. We therefore evaluated levels of the CTGF protein in urine samples of diabetic db/db mice and the control db/m mice. CTGF protein was immunoprecipitated with heparin Sepharose beads and immunoblotted with anti-CTGF antibody. Semiquantitative densitometric analyses of immunoblots demonstrated that urinary CTGF excretion was robustly induced in the vehicle-treated db/db mice compared with age-matched db/m control mice (Fig. 6B , 3.8±1.3-fold, lanes 1–8). Histological evaluation of frozen kidney sections from diabetic db/db mice stained with anti-CTGF antibody demonstrated increased expression of CTGF in the proximal tubules (Fig. 6C , middle panel) and up-regulated expression of CTGF in the mesangial cells and podocytes of db/db mice (Fig. 6C , right panel). On the other hand, we observed very low levels of CTGF protein only in the mesangial cells of control db/m mice, with no expression in the proximal tubular epithelial cells (Fig. 6C , left panel). Treatment of db/db mice with the CTGF ASO but not the MM control oligonucleotide dose-dependently reduced the CTGF mRNA (75% at the highest dose; P<0.01) in kidney cortical tissues (Fig. 6A ). Levels of urinary CTGF protein in the ASO and MM oligonucleotide-treated diabetic db/db mice were also evaluated as described in the STZ study and normalized to their urinary creatinine levels. Treatment with the CTGF ASO but not the mismatch control oligonucleotide dose-dependently reduced diabetes-induced urinary CTGF levels (57% at the highest dose; P<0.03; Fig. 6B , lanes 9–20). Immunoblot analyses of cortical slices from vehicle vs. ASO-treated mice demonstrated a modest decrease in CTGF protein expression (27%, data not shown), suggesting that urinary CTGF protein may represent an important depot during disease progression, thereby serving as a dependable biomarker of disease severity.


Figure 6
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  		Figure 6. Diabetes-induced up-regulation of CTGF mRNA and urinary CTGF protein in db/db mice is attenuated after treatment with the CTGF ASO. Compared with age-matched db/m mice, diabeteic db/db mice (5 months) demonstrate up-regulation of CTGF mRNA in the cortical tissue (A) and CTGF protein in the urine (B). Immunostaining of frozen sections of kidney with anti-CTGF antibody demonstrated diabetes-induced up-regulation of CTGF protein in the proximal tubular epithelial and mesangial cells in the kidney cortex (C). Low levels of CTGF expression were seen only in the glomerulii in db/m mice (C, left panel). Treatment with CTGF ASO (5, 10, or 20 mg/kg per dose using a biweekly dosing regimen), but not the MM control ASO (20 mg/kg), dose-dependently attenuated CTGF mRNA in the kidney cortex compared with vehicle-treated db/db mice (A). Urinary CTGF protein was also dose-dependently down-regulated in the ASO but not the MM oligonucleotide-treated group (B). Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. vehicle-treated db/db mice.

CTGF ASO improves functional and histopathological changes related to diabetic nephropathy in the db/db mouse model of type II diabetes
Compared with age-matched db/m control mice, aged vehicle-treated db/db mice develop DN as demonstrated by increased serum creatinine (3.2±0.7-fold), urinary protein (3.6±1.3-fold), and urinary albumin (2.7±1.1-fold) excretion, cumulative indications of both glomerular and tubular damage (Fig. 7 A). In this study, we demonstrate that treatment with the CTGF ASO but not the mismatch control oligonucleotide dose-dependently reduced serum creatinine (37% at 20 mg/kg; P<0.01), urinary total protein (41% at 20 mg/kg; P<0.03), and urinary albumin (48% at 20 mg/kg; P<0.05) excretion in the db/db mice (Fig. 7A ). Reduction in serum creatinine and urinary total protein was significant even at a lower dose of ASO treatment (10 mg/kg), whereas the reduction in urinary albumin excretion was significant only with the highest dose (20 mg/kg) of the ASO.


Figure 7
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  		Figure 7. A–C) Treatment with CTGF ASO demonstrates dose-dependent improvement in key indices of diabetic nephropathy in db/db mice. A) Treatment with CTGF ASO (5, 10, or 20 mg/kg per dose) but not the MM control oligonucleotide (20 mg/kg per dose) for 8 wk, using a biweekly dosing regimen, demonstrated a dose-dependent reduction in serum creatinine, urinary protein, and urinary albumin levels in db/db mice with established DN. B, C) Compared with age-matched db/m mice, vehicle-treated db/db mice also showed significant mesangial matrix expansion as demonstrated by quantitative PAS staining. Compared with vehicle-treated db/db mice, treatment with the CTGF ASO (10 mg/kg per dose) but not the MM control oligonucleotide (20 mg/kg per dose) showed significant attenuation of mesangial matrix expansion. Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. vehicle-treated db/db mice.

Functional hallmarks of disease progression in diabetic nephropathy are mesangial matrix expansion and tubulointerstitial fibrosis, leading to impaired glomerular filtration and scarring of the kidney (34 35 36 37) . Compared with db/m control mice, vehicle-treated db/db mice demonstrated a significant increase in mesangial matrix expansion as revealed by quantitative densitometry of PAS-stained kidney sections (Fig. 7B, C ). Treatment with the top two doses of CTGF ASO (10 and 20 mg/kg) demonstrated a moderate but similar reduction (33% and 41%, respectively), whereas the MM control oligonucleotide was without effect on mesangial matrix expansion (Fig. 7B , C). Although CTGF protein was clearly up-regulated in the tubular epithelial cells of the db/db mice compared with age-matched db/m mice, PAS or trichrome staining showed that the amount of collagen deposition in the tubulointerstitial spaces was minimal. Therefore, we were unable to measure the effect of CTGF ASO on interstitial fibrosis in this model by using histological tools. Quantitative RT-PCR was therefore performed on cortical slices from the kidneys to evaluate the effect of the ASOs on genes involved in mesangial matrix expansion and tubulointerstitial fibrosis. Analyses using gene-specific primers and probes demonstrated that both collagen 1[{alpha}1] (3.3±1.2-fold) and collagen IV ({alpha}2) (3.2±1.4-fold) were significantly up-regulated in the kidney cortex of the vehicle-treated db/db mice compared with the db/m control mice (Fig. 8 ). Treatment with CTGF ASO but not the MM oligonucleotide significantly down-regulated both types of collagen expression. In this model, fibronectin expression was only modestly induced in the diabetic mice but was dose-dependently down-regulated to control levels by the ASO but not the MM oligonucleotide treatment. Finally, expression of PAI-1 was dramatically induced (3.4±1.1-fold) in the db/db mice similar to the STZ-induced diabetic mice. Although the ASO had only a marginal effect on PAI-1 in the STZ model, there was a dose-dependent significant reduction of PAI-1 in the db/db model. MCP-1 was strongly up-regulated in the vehicle-treated db/db mice compared with the db/m control mice, but the CTGF ASO was without effect on MCP-1 gene expression. These results suggest that down-regulation of CTGF specifically attenuates matrix expansion and fibrosis via down-regulation of genes involved in matrix synthesis as well as inhibition of matrix degradation.


Figure 8
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  		Figure 8. Treatment with CTGF ASO results in significant down-regulation of genes involved in matrix expansion in the kidneys of db/db mice. Compared with db/m control mice, kidneys from the diabetic db/db mice showed significant up-regulation in the expression of a cluster of genes involved in matrix expansion (collagen I [{alpha}1], collagen IV [{alpha} 2], fibronectin, and PAI-1). Gene expression was analyzed by qRT-PCR, and expression of each gene normalized to cyclophilin in the vehicle-treated db/db mice was marked as control (100% expression). Treatment with the CTGF ASO but not the MM control oligonucleotide attenuated expression of each of these genes. Expression of MCP-1 was also up-regulated in the db/db mice, but was unaffected by the ASO or the MM control oligonucleotide. Results were analyzed by 1-way ANOVA, followed by Tukey’s post test; *P < 0.05 vs. vehicle-treated db/db mice.

The toxicological profile and tolerability of the therapy were evaluated by monitoring serum transaminase levels (AST and ALT) during the course of the treatment in both the type 1 and type 2 models of DN. No increase in serum transaminase was observed at any of the ASO doses tested (data not shown). We therefore conclude that specific down-regulation of CTGF expression using ASO shows promise as a nontoxic therapeutic tool in models of both type 1 and type 2 DN. Furthermore, the chemistry of the ASOs used in this study allowed maximal pharmacological efficacy using an infrequent dosing schedule (2 qw), demonstrating an appropriate pharmacokinetic profile in the kidney with no systemic toxicity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
High glucose-induced CTGF expression in vitro is observed in key cell types involved in the development and progression of DN (4 5 6 7 , 14 , 16) . In addition, up-regulated and induced expression of CTGF has been demonstrated using various rodent models of nephropathy (32 33 34 35 36) . Urinary albumin excretion is used to identify diabetic patients at risk of developing nephropathy (27) . CTGF is undetectable in urine collected from patients with normoalbuminuria but is readily detectable in diabetic patients, with levels correlating well with the degree microalbuminuria (25 26 27) . In this study we demonstrate that CTGF expression (mRNA and protein) is robustly up-regulated in HG cultured primary mesangial cells, and ASO-mediated specific down-regulation of CTGF attenuates HG-induced expression of key genes critical to the pathology of DN. Specific down-regulation of the target by CTGF ASO but not the MM control oligonucleotide in vivo demonstrated pharmacological efficacy in models of both type 1 and type 2 DN. This included improvement in almost all the pathological features of DN including increased kidney weight/body weight ratio, elevated serum creatinine, microalbuminuria, mesangial matrix expansion, and fibrosis. Compared with the vehicle or MM oligonucleotide-treated group, ASO treatment resulted in a concomitant decrease in diabetes-induced expression of genes involved in matrix synthesis (fibronectin, collagens 1 and IV) and inhibition of matrix degradation (PAI-1) in cortical tissues.

Pathway analysis has demonstrated that diabetes-induced p38 MAPK activation is profibrotic (38 , 39) . Therefore, in further elucidating possible mechanisms for the pharmacological response in the ASO-treated group, we demonstrate that specific down-regulation of CTGF attenuates diabetes-induced activation of p38 MAPK and its downstream target transcription factor CREB, which has been implicated in regulating matrix proteins.

Urinary CTGF has been demonstrated as a biomarker for disease progression in human DN (25 26 27) . Therefore, CTGF is an attractive therapeutic target since urinary CTGF can easily be measured using noninvasive techniques. Our study supports the observation made by Gilbert et al. (25) that urinary CTGF protein can serve as a biomarker for disease progression in DN. Since distribution of the ASO was restricted mainly to the cortical regions of the kidney (tubular and the glomerular compartment), with little or no distribution in the medullary region, it appears that down-regulation of CTGF in the kidney cortical cells is sufficient to control progression of DN at least in the rodent models.

It is well established that PS ASOs have preferential distribution to the liver (31) , sometimes resulting in reduced tolerability and increased liver toxicity (elevated serum transaminases) at pharmacologically efficacious doses. In this study we used a chimeric CTGF ASO with mixed backbone chemistry that resulted in greater ASO distribution to the kidney compared with the liver. Pharmacokinetic analyses using the chimeric CTGF ASO demonstrated that reduction in the sulfur content minimized distribution of the ASO to the liver whereas kidney distribution remained unchanged. This approach should improve tolerabilty in a chronic dosing paradigm, and we did not observe any increase in serum transaminases (ALT or AST) even at the highest dose (20 mg/kg) of the ASO tested. The infrequent dosing schedule (twice a week) and the tolerability of the chimeric ASO in a chronic dosing paradigm make this class of CTGF ASO an attractive therapeutic tool for treatment of DN.

The profibrotic state observed in DN results from an imbalance between signaling activities of the different growth factors involved in renal matrix homeostasis (32 33 34 35 , 40 41) . TGFß-dependent and independent up-regulation of CTGF and its downstream signaling pathways appear to be crucial profibrotic events resulting in both increased matrix synthesis and decreased turnover. Antisense oligonucleotide and neutralizing antibody to TGF-ß block the fibrotic process in animal models of DN (19 , 20) . Neutralizing antibody to TGFß, however, failed to block the microalbuminuria observed in these models. Studies suggest that CTGF may enhance the profibrotic effects of TGFß by enhancing the ability of TGFß to bind to its receptor at low TGFß concentrations (40) . Downstream effector signals regulating the effects of CTGF are still not fully resolved. Although CTGF or TGFß alone causes only a transient fibrotic response in vivo, simultaneous administration causes a persistent response for at least 1 wk after cessation of treatment (36 37 , 41) . Our study therefore provides critical proof-of-concept that down-regulation of diabetes-induced CTGF expression in the kidney cortex contributes to the improvement of all aspects of DN, including elevated serum creatinine, microalbumiuria, and progression of fibrosis. To our surprise, we observed that down-regulation of CTGF partially attenuated the expression of its upstream regulator TGF-ß in the kidney cortex. Although the mechanism is not clearly understood, we believe that this may be an indirect effect of CTGF down-regulation. We therefore propose a model (Fig. 9 ) for the role of CTGF in DN and provide evidence that specific down-regulation of this target using the chimeric CTGF ASO holds significant promise in the treatment of DN.


Figure 9
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  		Figure 9. Model outlining the specific role of CTGF in diabetic nephropathy. Diabetes-induced up-regulation of CTGF occurs via TGFß-dependent and independent pathways, resulting in pathology in both type 1 and type 2 models of DN in mice. Specific down-regulation of CTGF improves all the parameters of DN.


   ACKNOWLEDGMENTS
 
This work was supported by grants from National Institutes of Health (RO1 DK58191) and the Juvenile Diabetes Research Foundation (to R.N.). The authors would like to thank ISIS Pharmaceuticals for providing the CTGF ASO and the MM oligonucleotides for the study and Stephen Scott for his technical assistance

Received for publication December 19, 2006. Accepted for publication May 3, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kreisberg, J. I., Ayo, S. H. (1993) The glomerular mesangium in diabetes mellitus. Kidney Int. 43,109-113[Medline]
  2. Ziyadeh, F. N., Han, D. C. (1997) Involvement of transforming growth factor-beta and its receptors in the pathogenesis of diabetic nephrology. Kidney Int. 60,S7-11
  3. Reeves, W. B., Andreoli, T. E. (2000) Transforming growth factor beta contributes to progressive diabetic nephropathy. Proc. Natl. Acad. Sci. U. S. A. 97,7667-7669[Free Full Text]
  4. Ziyadeh, F. N., Sharma, K., Ericksen, M., Wolf, G. (1994) Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-ß. J. Clin. Invest. 93,536-542[Medline]
  5. Han, D. C., Isono, M., Hoffman, B. B., Ziyadeh, F. N. (1999) High glucose stimulates proliferation and collagen type I synthesis in renal cortical fibroblasts: mediation by autocrine activation of TGF-beta. J. Am. Soc. Nephrol. 10,1891-1899[Abstract/Free Full Text]
  6. Hoffman, B. B., Sharma, K., Zhu, Y., Ziyadeh, F. N. (1998) Transcriptional activation of transforming growth factor-ß1 in mesangial cell culture by high glucose concentration. Kidney Int. 54,1107-1116[CrossRef][Medline]
  7. Sakharova, O. V., Taal, M. W., Brenner, B. M. (2001) Pathogenesis of diabetic nephropathy: focus on transforming growth factor-beta and connective tissue growth factor. Curr. Opin. Nephrol. Hypertens. 10,727-738[Medline]
  8. Bradham, D. M., Igarashi, A., Potter, R. L., Grotendorst, G. R. (1991) Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J. Cell Biol. 114,1285-1294[Abstract/Free Full Text]
  9. Bork, P. (1993) The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett. 327,125-130[CrossRef][Medline]
  10. Brigstock, D. R. (1999) The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr. Rev. 20,189-206[Abstract/Free Full Text]
  11. Lau, L. F., Lam, S. C. (1999) The CCN family of angiogenic regulators: the integrin connection. Exp. Cell Res. 248,44-57[CrossRef][Medline]
  12. Igarashi, A., Okochi, H., Bradham, D. M., Grotendorst, G. R. (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol. Biol. Cell 4,637-645[Abstract]
  13. Grotendorst, G. R., Okochi, H., Hayashi, N. (1996) A novel transforming growth factor ß response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ. 7,469-480[Abstract]
  14. Murphy, M., Godson, C., Cannon, S., Kato, S., Mackenzie, H. S., Martin, F., Brady, H. R. (1999) Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J. Biol. Chem. 274,5830-5834[Abstract/Free Full Text]
  15. Riser, B. L., Denichilo, M., Cortes, P., Baker, C., Grondin, J. M., Yee, J., Narins, R. G. (2000) Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J. Am. Soc. Nephrol. 11,25-38[Abstract/Free Full Text]
  16. Wahab, N. A., Yevdokimova, N., Weston, B. S., Roberts, T., Li, X. J., Brinkman, H., Mason, R. M. (2001) Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy. Biochem. J. 359,77-87[CrossRef][Medline]
  17. Uchio, K, Graham, M., Dean, N. M., Rosenbaum, J., Desmouliere, A. (2004) Down regulation of connective tissue growth factor and type 1 collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental fibrosis. Wound Repair Regen. 12,60-66[CrossRef][Medline]
  18. Wang, S., Denichilo, M., Brubaker, C., Hirschberg, R. (2001) Connective tissue growth factor in tubulointerstitial injury of diabetic nephropathy. Kidney Int. 60,96-105[CrossRef][Medline]
  19. Yokoi, H., Mukoyoma, M., Nagae, T., Mori, K., Suganami, T., Sawai, K., Yoshioka, T., Koshikawa, M., Nishida, T., Takigawa, M., et al (2004) Reduction in connective tissue growth factor by anstisense treatment ameliorates renal tubulointerstitial fibrosis. J. Am. Soc. Nephrol. 15,1430-1440[Abstract/Free Full Text]
  20. Ziyadeh, F. N., Hoffman, B. B., Han, D. C., Iglesias-De La Cruz, M. C., Hong, S. W., Isono, M., Chen, S., McGowan, T. A., Sharma, K. (2000) Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Proc. Natl. Acad. Sci. U. S. A. 97,8015-8020[Abstract/Free Full Text]
  21. Iglesias-de la Cruz, M. C., Ziyadeh, F. N., Isono, M., Kouahou, M., Han, D. C., Kalluri, R., Mundel, P., Chen, S. (2002) Effects of high glucose and TGF-ß1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes. Kidney Int. 62,901-913[CrossRef][Medline]
  22. De Vriese, A. S., Tilton, R. G., Elger, M., Stephan, C. C., Kriz, W., Lameire, N. H. (2001) Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. J. Am. Soc. Nephrol. 12,993-1000[Abstract/Free Full Text]
  23. Bloom, I. E., van Dijk, A. J., Wieten, L., Duran, K., Ito, Y., Kleij, L., Denichilo, M., Rabelink, T. J., Weening, J. J., Aten, J., Goldschmeding, R. (2001) In vitro evidence for differential involvement of CTGF, TGFß, and PDGF-BB in mesangial response to injury. Nephrol. Dial. Transplant. 16,1139-1148[Abstract/Free Full Text]
  24. Twigg, S. M., Chen, M. M., Joly, A. H., Chakrapani, S. D., Tsubaki, J., Kim, H. S., Oh, Y., Rosenfeld, R. G. (2001) Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor-binding protein-related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus. Endocrinology 142,1760-1769[Abstract/Free Full Text]
  25. Gilbert, R. E., Akdeniz, A., Weitz, S., Usinger, W. R., Molineaux, C., Jones, S. E., Langham, R. G., Jerums, G. (2003) Urinary connective tissue growth factor excretion in patients with type 1 diabetes and nephropathy. Diabetes Care 26,2632-2636[Abstract/Free Full Text]
  26. Riser, B. L., Cortes, P., DeNichilo, M., Deshmukh, P. V., Chahal, P. S., Mohammed, A. K., Yee, J., Kahkonen, D. (2003) Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: preliminary report. Kidney Int. 64,451-458[CrossRef][Medline]
  27. Adler, S. G., Kang, S. W., Feld, S., Cha, D. R., Barba, L., Striker, L., Striker, G., Riser, B.L., LaPage, J., Nast, C. C. (2001) Glomerular mRNAs in human type 1 diabetes: biochemical evidence for microalbuminuria as a manifestation of diabetic nephropathy. Kidney Int. 60,2330-2336[CrossRef][Medline]
  28. Okada, H., Kikuta, T., Kobayashi, T., Inoue, T., Kanno, Y., Takigawa, T., Sugaya, T., Kopp, J. B., Suzuki, H. (2005) Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis. J. Am. Soc. Nephrol. 16,133-143[Abstract/Free Full Text]
  29. Breyer, M. D., Bottinger, E., Brosius, F. C., 3rd, Coffman, T. M., Harris, R. C., Heilig, C. W., Sharma, K. (2005) Mouse models of diabetic nephropathy. J. Am. Soc. Nephrol. 16,27-45[Abstract/Free Full Text]
  30. Monia, B. P., Lesnik, E. A., Gonzalez, C., Lima, W. F., McGee, D., Guinosso, C. J., Kawasaki, A. M., Cook, P. D., Freier, S. M. (1993) Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268,14514-14522[Abstract/Free Full Text]
  31. Peng, B., Andrews, J., Nestorov, I., Brennan, B., Nicklin, P., Rowland, M. (2001) Tissue distribution and physiologically based pharmacokinetics of antisense oligonucleotide ISIS 1082 in rats. Antisense Nucleic Acid Drug Dev. 11,15-27[CrossRef][Medline]
  32. Reddy, M. A., Adler, S. G., Kim, Y. S., Lanting, L., Rossi, J. J., Kang, S. W., Nadler, J. L., Shahed, A., Natarajan, R. (2002) Interaction of MAPK and 12-lipoxygenase pathways in growth and matrix protein expression in mesangial cells. Am. J. Physiol. 283,F985-F994
  33. Kim, Y. S., Xu, Z-G., Reddy, M. A., Lanting, L., Sharma, K., Adler, S. G., Natarajan, R. (2005) Novel interactions between transforming growth factor-ß1 actions and the 12/15-lipoxygenase pathway in mesangial cells. J. Am. Soc. Nephrol. 16,352-362[Abstract/Free Full Text]
  34. Leask, A., Abraham, D. J. (2004) TGF-beta signaling and the fibrotic response. FASEB J. 18,816-827[Abstract/Free Full Text]
  35. Abdel-Wahab, N., Weston, B. S., Roberts, T., Mason, R. M. (2002) Connective tissue growth factor and regulation of the mesangial cell cycle: role in cellular hypertrophy. J. Am. Soc. Nephrol. 13,2437-2445[Abstract/Free Full Text]
  36. Van Nieuwenhoven, F. A., Jensen, L. J., Flyvbjerg, A., Goldschmeding, R. (2005) Imbalance of growth factor signaling in diabetic kidney disease: is connective tissue growth factor (CTGF, CCN2) the perfect intervention point. Nephrol. Dial. Transplant. 20,6-10[Free Full Text]
  37. McLennan, S. V., Wang, X. Y., Moreno, V., Yue, D. K., Twigg, S. M. (2004) Connective tissue growth factor mediates high glucose effects on matrix degradation through tissue inhibitor of matrix metalloproteinase type 1: