Renal Connective Tissue Growth Factor Research Articles

P2 X 7 receptor is a critical regulator of extracellular ATP-induced profibrotic genes expression in rat kidney: implication of transforming growth factor-β/Smad signaling pathway

This study was designed to investigate the potential of extracellular adenosine 5′-triphosphate (ATP) via the P2 X 7 receptor to activate the renal fibrotic processes in rats. The present study demonstrates that administration of ATP rapidly activated transforming growth factor-β (TGF-β) to induce phosphorylation of Smad-2/3. Renal connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA and protein expressions were also increased following ATP administration. A decrease in TGF-β amount in serum as well as renal Smad-2/3 phosphorylation was noticed in animals pre-treated with the specific antagonist of P2 X 7 receptor, A 438,079. In addition, a significant reduction in mRNA and protein expression of CTGF and TIMP-1were also observed in the kidneys of those animals. Collectively, the current findings demonstrate that ATP has the ability to augment TGF-β-mediated Smad-2/3 phosphorylation and enhance the expression of the pro-fibrotic genes, CTGF and TIMP-1, an effect that is largely mediated via P2 X 7 receptor.

Fibroblast Growth Factor 21 Attenuates Diabetes-Induced Renal Fibrosis by Negatively Regulating TGF-β-p53-Smad2/3-Mediated Epithelial-to-Mesenchymal Transition via Activation of AKT.

BackgroundEpithelial-to-mesenchymal transition (EMT) is required for renal fibrosis, which is a characteristic of diabetic nephropathy (DN). Our previous study demonstrated that fibroblast growth factor 21 (FGF21) prevented DN associated with the suppressing renal connective tissue growth factor expression, a key marker of renal fibrosis. Therefore, the effects of FGF21 on renal fibrosis in a DN mouse model and the underlying mechanisms were investigated in this study.MethodsType 1 diabetes mellitus was induced in C57BL/6J mice by intraperitoneal injections of multiple low doses of streptozotocin. Then, diabetic and non-diabetic mice were treated with or without FGF21 in the presence of pifithrin-α (p53 inhibitor) or 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10-DEBC) hydrochloride (Akt inhibitor) for 4 months.ResultsDN was diagnosed by renal dysfunction, hypertrophy, tubulointerstitial lesions, and glomerulosclerosis associated with severe fibrosis, all of which were prevented by FGF21. FGF21 also suppressed the diabetes-induced renal EMT in DN mice by negatively regulating transforming growth factor beta (TGF-β)-induced nuclear translocation of Smad2/3, which is required for the transcription of multiple fibrotic genes. The mechanistic studies showed that FGF21 attenuated nuclear translocation of Smad2/3 by inhibiting renal activity of its conjugated protein p53, which carries Smad2/3 into the nucleus. Moreover pifithrin-α inhibited the FGF21-induced preventive effects on the renal EMT and subsequent renal fibrosis in DN mice. In addition, 10-DEBC also blocked FGF21-induced inhibition of renal p53 activity by phosphorylation of mouse double minute-2 homolog (MDM2).ConclusionFGF21 prevents renal fibrosis via negative regulation of the TGF-β/Smad2/3-mediated EMT process by activation of the Akt/MDM2/p53 signaling pathway.

Effect of herbal cake-partitioned moxibustion on renal function and expression of renal connective tissue growth factor, integrin-linked kinase and bone morphogenetic protein-7 in chronic renal failure rabbits

To observe the effect of herbal cake-partitioned moxibustion on renal function and expression of connective tissue growth factor (CTGF), integrin-linked kinase (ILK) and bone morphogenetic protein-7 (BMP-7) in chronic renal failure (CRF) rabbits, so as to reveal its mechanisms underlying improvement of CRF. Twenty-four male New Zealand rabbits were randomly divided into control, model, medication and herbal cake-partitioned moxibustion (moxibustion) groups (n=6 rabbits in each group). The CRF model was established by gavage of suspension of Adenine (150 mg·kg-1·d-1) for 21 days. Herbal cake-partitioned moxibustion was applied to "Mingmen"(GV4) and bilateral "Shenshu"(BL23), "Pishu"(BL20) and for 5 moxa-cones every time. Rabbits of the medication group was treated by gavage of Losartan Potassium (2.33 mg·kg-1·d-1). All the treatments were conducted once daily,12 times a course for consecutive 3 courses with a two-day rest after each course of treatment. Serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urine protein contents were detected by using an automatic biochemical analyzer. The expression levels of CTGF, ILK and BMP-7 proteins and mRNA in the kidney tissue were determined by Western blot and quantitative real time-PCR, separately. Following modeling, Scr and BUN and 24 h urine protein contents were significantly increased in the model group in comparison with the control group (P<0.01). After the intervention, Scr and BUN contents were all significantly decreased in both the moxibustion and medication groups relevant to the model group (P<0.01), suggesting an improvement of the renal function. Compared with the control group, the expression levels of ILK and CTGF mRNAs and proteins were obviously up-regulated (P<0.01), and those of BMP-7 mRNA and protein significantly decreased in the model group (P<0.01). In comparison with the model group, the expression levels of ILK and CTGF mRNAs and proteins were significantly down-regulated in the two treatment groups (P<0.01, P<0.05), and those of BMP-7 mRNA and protein markedly increased in the two treatment groups (P<0.01). In comparison with the medication group, the expression level of ILK protein was significantly up-regulated (P<0.01) and BMP-7 protein was significantly down-regulated (P<0.01) in the moxibustion group. No significant differences were found between the medication and moxibustion groups in down-regulating the levels of Scr, BUN and 24 h urine protein and expression of ILK mRNA, CTGF mRNA and protein and BMP-7 mRNA(P>0.05). Herbal cake-partitioned moxibustion can improve renal function in CRF rabbits, which may be related to its effects in suppressing the expression of ILK and CTGF, and in up-regulating the expression of BMP-7 in the kidney tissue.

Suppression of connective tissue growth factor mediates the renoprotective effect of Sitagliptin rather than Pioglitazone in type 2 diabetes mellitus

AimDiabetic nephropathy (DN) is a leading cause of end-stage renal disease, and thus, the ability of antidiabetic drugs to ameliorate renal microvascular disease may be as important as their ability to control blood glucose. Therefore, we investigated the reno-protective effect of the antidiabetic drugs, Sitagliptin and Pioglitazone, versus combined Metformin/Enalapril in a rat model of type 2 diabetes. MethodMale Wistar rats were randomly assigned to be either normal control or diabetic. Induction of type 2 diabetes was done by intraperitoneal injection of\\ low dose streptozotocin (35mg/kg) on top of 2 weeks of high fat diet. Hyperglycemic animals were divided into 4 groups: untreated diabetic, Sitagliptin (10mg/kg), Pioglitazone (10mg/kg) and Metformin/Enalapril (500, 10mg/kg, respectively) treated. After 6 weeks, fasting blood glucose, plasma insulin, β-cell function, insulin resistance, serum lipids, urea & creatinine, albuminurea, kidney weight, renal oxidative stress, plasma connective tissue growth factor (CTGF) and renal histopathology were assessed. Key findingsSitagliptin decreased microalbuminurea, urea & creatinine, renal tropism, oxidative stress and CTGF to levels similar to Metformin/Enalapril group. It also preserved near normal renal histology. Although Pioglitazone treatment reduced urea, creatinine, renal tropism and oxidative stress, it did not improve renal pathological changes or significantly alter CTCF. SignificanceEarly Sitagliptin treatment in type 2 diabetes can equally ameliorate renal functions and structural changes as combined Metformin/Enalapril. Moreover Sitagliptin is a better renoprotective than Pioglitazone, probably due to its suppressor effect on CTGF, a key factor mediating diabetic renal injury.

Myeloid HIF-1 attenuates the progression of renal fibrosis in murine obstructive nephropathy

Hypoxia-inducible factors (HIFs) play an important role in the pathogenesis of renal fibrosis. Although the role of macrophage infiltration in the progression of renal fibrosis is well known, the role of macrophage HIF-1 remains to be revealed. To address this question, myeloid specific conditional HIF-1 knock out mice were subjected to unilateral ureteral obstruction (UUO). Renal interstitial deposition of collagen Ⅲ and mRNA expressions of collagen Ⅰ and collagen Ⅲ were markedly increased at 7 days after UUO and myeloid HIF-1 depletion significantly accelerated these increases. Immunohistochemistry and flow cytometric analysis revealed that renal infiltrating macrophages were increased with duration of UUO but myeloid HIF-1 depletion did not affect these changes. Myeloid HIF-1 depletion did not affect M1 and M2 macrophage phenotype polarization in obstructed kidneys. Renal connective tissue growth factor (CTGF) expression was markedly increased and myeloid HIF-1 depletion further enhanced this increase. Immunomagnetic separation of renal cells revealed that renal CTGF was expressed predominantly in renal cells other than macrophages. It is suggested that myeloid HIF-1 attenuates the progression of renal fibrosis in murine obstructive kidney. Alteration of CTGF expression in renal cells other than macrophages is one of possible mechanisms by which myeloid HIF-1 protected renal fibrosis.

Inhibition of local aldosterone by eplerenone reduces renal structural damage in a novel model of chronic cyclosporine A nephrotoxicity.

The fact that mineralocorticoid receptor antagonists reduce structural and functional alterations induced by cyclosporine A (CsA) indicates that aldosterone plays a key role in chronic CsA nephrotoxicity. We and other researchers have reported local renal aldosterone synthesis. To investigate local renal aldosterone's role in chronic CsA nephrotoxicity, we evaluated the effect of eplerenone (Epl) on renal structural damage and renal dysfunction in adrenalectomized (ADX) rats, and assessed whether the therapeutic benefit was associated with reduction of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), plasminogen activator inhibitor type 1 (PAI-1) and collagen I (COL-I) expression. Male Sprague-Dawley rats fed a normal-sodium diet were divided in four groups: sham-ADX, ADX, CsA, or Epl. Rats in the ADX, CsA and Epl groups were adrenalectomized first. Aldosterone, sodium and potassium levels in serum and urine were measured on the second day. Two weeks later, vehicle (sham-ADX and ADX group), CsA (25mg/kg/d), or CsA and Epl (100 mg/ kg/d) combination was administrated, respectively. After six weeks, urinary protein, creatinine clearance (Ccr), tubulointerstitial fibrosis (TIF), aldosterone level in kidney, and renal aldosterone synthase CYP11B2, COL-I, TGF-β1, CTGF and PAI-1 gene expression levels were determined. On the second day after surgery, adrenalectomized rats showed undetectable aldosterone with natriuresis, hyponatremia, decreased urinary potassium excretion and hyperpotassemia. CsA reduced Ccr, induced urinary proteins and up-regulated COL-I, TGF-β1, CTGF and PAI-1 gene expression with a significant development of TIF. Eplerenone administration prevented TIF and COL-I, TGF-β1 and PAI-1 up-regulation but did not improve renal function. Our results suggest local renal aldosterone is an important mediator of renal injury induced by CsA.

Abstract 536: Resiniferatoxin-induced Disorders in Glucose Homeostasis and Hypertension: Role of Trpv1-positive Sensory Nerves

Resiniferatoxin (RTX), an ultrapotent agonist of the transient receptor potential vanilloid 1 (TRPV1) channels, has been used to relieve pain in humans in clinical trials that are ongoing, completed, or terminated due to its unwanted side effects that have not been well understood. We test the hypothesis that RTX causes disorders in glucose homeostasis, renal functional and structural impairment, and hypertension via insulin receptor-mediated increases in sympathetic nerve activity. Male Wistar rats were injected with RTX (0.2 mg/kg, i.p.) or vehicle. Pancreatic and renal TRPV1 levels were decreased 4 weeks after RTX treatment, confirming effective and lasting treatment of RTX (p&lt;0.05). RTX decreased plasma insulin levels (Control: 1.4±0.1 vs RTX: 0.8±0.1 ng/ml, p&lt;0.05) but improved glucose tolerance (p&lt;0.05). RTX increased the translocation of skeletal muscle glucose transporter 4 (Glut4) from cytosol to cell membrane indicated by increased membrane fraction but decreased cytosol fraction of Glut4 in RTX-treated rats (p&lt;0.05). RTX increased levels of phosphorylated insulin receptor substrate 1 in sympathetic ganglia (Control: 0.43±0.04 vs RTX: 0.65±0.08, p&lt;0.05), renal tyrosine hydroxylase contents, and urinary norepinephrine concentrations (Control: 352±70 vs RTX: 781±142 ng/day, p&lt;0.05). When TRPV1 expressed in sensory neurons and their nerve endings innervating the kidney was knocked down by intrathecal (i.t) injection of TRPV1 shRNA, insulin (3nmol/kg, i.t.)-induced increases in efferent renal nerve activity were further enhanced (Control: 40±3 vs TRPV1 shRNA: 53±2, p&lt;0.05). RTX decreased creatinine clearance (Control: 0.58±0.04 vs RTX: 0.46±0.04 ml/min/100 gbwt, p&lt;0.05) and increased levels of plasma urea, urinary albumin, renal connective tissue growth factor, and systolic blood pressure (Control: 128±1 vs RTX: 143±2 mmHg, p&lt;0.05). Thus, RTX treatment causes disorders in glucose homeostasis, renal functional and structural impairment, and hypertension, possibly via increasing sympathetic nerve activity resulting from enhanced stimulatory effects of the insulin receptors expressed in sympathetic nerves and weakened inhibitory effects of TRPV1-positive sensory nerves on sympathetic nerves.

Abstract 537: Segment-specific Intrathecal Injection of Metformin Protects Against High Fat Diet-induced Impairment in Afferent Renal Nerves and Renal Function

Metformin (Met), a drug for treating type 2 diabetes by suppressing glucose production and increasing insulin sensitivity, may possess neuroprotective and neurogenesis effects. We test the hypotheses 1) that high fat diet (HFD) intake impairs afferent renal nerves expressing transient receptor potential vanilloid 1 (TRPV1) channels, leading to increased efferent renal nerve activity (ERNA), decreased renal function, and hypertension; and 2) that Met given intrathecally (i.t.) to segments (T8-L3) supplying the kidney prevents HFD-induced impairment in afferent renal nerves, renal function, and hypertension. Met (1 ng/kg, i.t. daily) or vehicle was given to rats fed a HFD or normal fat diet (Con) for 8 weeks. Glucose or insulin tolerance was impaired to the same degree between rats fed HFD with and without Met treatment. HFD decreased renal TRPV1 expression, plasma levels of calcitonin gene-related peptide, and afferent renal nerve activity (ARNA, Con: 126±11, Met: 136±14, HFD: 77±7, HFD+Met: 121±8, p&lt;0.05) in response to intra-pelvis perfusion of capsaicin (a TRPV1 agonist at 4 mM, 3 min, 20 ml/min), which were prevented by Met. HFD increased renal tyrosine hydroxylase contents, ERNA responses to intrathecal injection of muscimol (a GABA-A receptor agonist at 3 nmol/kg), and urinary norepinephrine levels, which were prevented by Met. HFD decreased creatinine clearance (Con: 0.40±0.04, Met: 0.52±0.07, HFD: 0.12±0.05, HFD+Met: 0.30±0.04 ml/min/100 gbwt, p&lt;0.05) and urinary Na+ excretion, and increased levels of urinary albumin, plasma urea, renal collagen deposition, renal connective tissue growth factor, and systolic blood pressure (Con: 132±1, Met: 130±1, HFD: 145±3, HFD+Met: 132±1 mmHg, p&lt;0.05), which were prevented or attenuated by Met. Thus, HFD impairs TRPV1-positive afferent renal nerves and renal function, and increases renal sympathetic nerve activity and blood pressure. Segment-specific intrathecal injection of Met protects against HFD-induced impairment in afferent renal nerves and renal function, and prevents HFD-induced hypertension.

Abstract 451: Depletion of Macrophage Prevents Exacerbated Renal Injury and Hypertension Induced by Degeneration of Trpv1-positive Nerves in Rats Subjected to Renal Ischemic Reperfusion and Salt Load

High salt intake after recovery from renal ischemia-reperfusion (I/R) injury leads to hypertension and renal inflammation. This study tests the hypothesis that degeneration of transient receptor potential vanilloid type 1 (TRPV1)-positive nerves exacerbates salt-induced hypertension and renal inflammation after I/R via enhancing renal macrophage infiltration. Rats were fed a low sodium (0.4%) diet for 5 weeks after capsaicin pre-treatment (CAP, 100 mg/kg, s.c.) and renal I/R, followed by macrophage depletion with clodronate liposome (LC, 1.3 ml/kg/wk, i.v.) combined with a high sodium (4%) diet for 4 weeks. Western blot showed that CAP pretreatment further decreased renal TRPV1 levels after I/R (P&lt;0.05), which was not affected by LC treatment. Macrophage infiltration in the renal cortex and medulla and renal proinflammatory cytokine (TNF-α and IL-1 β) levels were increased in I/R rats and further intensified in I/R+CAP rats (P&lt;0.05), which were abolished by LC in both I/R and I/R+CAP groups. Mean arterial pressure (MAP) was elevated in I/R rats and further increased in I/R+CAP rats (P&lt;0.05), which was prevented by LC in both I/R and I/R+CAP groups. Decreased creatinine clearance (Sham: 0.54±0.06, I/R: 0.28±0.02, I/R+CAP: 0.16±0.02, Sham+LC: 0.54±0.03, I/R+LC: 0.51±0.04, I/R+CAP+LC: 0.49±0.04 ml/min/100 gbwt, p&lt;0.05) and increased levels of plasma urea, urinary 8-isoprostane, renal connective tissue growth factor (CTGF), and renal collage I and IV were observed in I/R rats and exacerbated in I/R+CAP rats (P&lt;0.05), which were restored to normal levels by LC in both I/R and I/R+CAP groups. Thus, degeneration of TRPV1-positive nerves aggravates hypertension and renal inflammation and fibrosis induced by I/R and high salt intake, possibly via enhancing renal macrophage infiltration and function. These data suggest that activation of TRPV1 after renal injury conveys renal protection against inflammation and fibrosis via inhibiting macrophage infiltration.

Connective tissue growth factor gene expression and decline in renal function in lupus nephritis

In lupus nephritis (LN), kidney inflammation may be followed by fibrosis and progressive decline in function. Transforming growth factor (TGF)-β is a notable mediator of fibrosis, but it has other beneficial roles, thus indicating a need for alternate therapeutic targets for inhibition of fibrosis. Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-β in promoting fibrosis, without mediating the immunosuppressive effects of TGF-β. Animal studies show that CTGF may have important roles in renal fibrosis, but data are limited in human subjects. The present study tested the hypothesis that renal CTGF mRNA expression is related to TGF-β1 and collagen I expression and is predictive of renal function deterioration in patients with LN (n=39). Gene expression was measured using multiplex real-time quantitative RT-PCR and renal function was estimated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) glomerular filtration rate (GFR) equation. Decline in GFR was assessed by regression of GFR at biopsy to 1 year following biopsy. CTGF mRNA expression was significantly correlated with TGF-β1 and collagen I. GFR at biopsy was 89.2±39.2 ml/ min. Renal CTGF mRNA expression correlated inversely with baseline GFR. Renal CTGF mRNA was significantly higher in patients with moderate to severe CKD compared to those in the milder CKD group (low GFR 4.92±4.34 vs. high GFR 1.52±1.94, p<0.005). CTGF mRNA was also higher in patients with subsequent decline in GFR [GFR decline (5.19±4.46) vs. no GFR decline (1.79±1.97); P<0.01]. In conclusion, renal expression of CTGF was positively related to TGF-β1 and collagen I in patients with LN. Furthermore, high CTGF mRNA expression was associated with poor GFR at baseline and subsequent deterioration of kidney function. CTGF expression in the kidney may serve as an early marker for renal disease progression and could be evaluated as a target for therapeutic intervention to prevent renal failure in LN.

Effect of Rosmarinic Acid on Experimental Diabetic Nephropathy

Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Rosmarinic acid (RA) is a naturally occurring phenolic acid. This study was conducted to investigate the efficacy of RA on DN and to elucidate the potential mechanism. High glucose (HG)-stimulated cultured human renal proximal tubular epithelial cells (HK-2) analysed CTGF expression by western blotting, and it was investigated whether extracellular signal-regulated kinase (ERK) signalling pathway was involved. Using streptozotocin (STZ)-induced rat animal models, diabetic rats were randomized to receive intragastric (i.g.) doses of RA. Renal tissue, blood and urine samples were collected to determine biochemical index and analyse protein expression. In vitro study, RA reduced CTGF excretion in HG-induced HK-2 cells through the ERK signalling pathway. In an in vivo study, I.g. of RA 7.5 or 15 mg/kg significantly ameliorated renal function and increased body-weight. Meanwhile, RA reduced renal CTGF expression by immunohistochemical staining and reduced serum levels of CTGF. Besides, there were no significant differences in glycaemia levels between the RA groups compared with the STZ-treated group. Furthermore, RA ameliorated renal pathology. These results suggest that RA exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using RA in the treatment of DN.

Effect of loganin on experimental diabetic nephropathy

Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Loganin, an iridoid glucoside compound was isolated from Cornus officinalis Sieb. et Zucc. This study was conducted to investigate the efficacy of loganin on DN and to elucidate the potential mechanism. High glucose (HG) stimulated cultured human renal proximal tubular epithelial cells (HK-2) analyzed CTGF expression by Western blotting and investigated whether extracellular signal-regulated kinase (ERK) signaling pathway was involved. Streptozotocin (STZ)-induced experimental DN, randomized to receive intragastric (i.g.) of loganin. Renal tissue, blood and urine samples were collected to determine and analyze. In vitro study, loganin reduced CTGF excretion in HG-induced HK-2 cells through the ERK signaling pathway. In vivo study, I.g. of loganin 5mg/kg or 10mg/kg significantly ameliorated renal function and increased body weight. Meanwhile, loganin reduced renal CTGF expression by immunohistochemical staining, reduced serum levels of CTGF. Besides, there were no significant differences in blood sugar levels between the loganin groups compared to the STZ-treated group. Furthermore, loganin ameliorated renal pathology. These results suggested that loganin exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using loganin to treat DN.

Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: Implications for human efficacy and toxicity

Glucosamine is used for alleviating pain in osteoarthritis. Clinical trials have reported that glucosamine has equivocal efficacy. Glucosamine is also used in cell cultures to stimulate hexosamine flux and protein O-glycosylation, but at many-fold greater concentrations than those in human plasma following oral dosing. Lean Zucker rats were dosed orally for 6 weeks with glucosamine hydrochloride at doses (0–600 mg/kg/day) that produced peak serum concentrations of <1–35 μM, spanning the human exposure range. Relative expression of both TGFβ1 and CTGF mRNA were significantly increased up to 2.3-fold in liver, kidney and articular cartilage when evaluated 4 h after final dose. Apparent threshold serum glucosamine ( C max) concentration required to increase TGFβ1 expression in cartilage was 10–20 μM. These increases were associated with significant increases in UDP- N-acetylglucosamine concentrations suggesting increased hexosamine flux. Both TGFβ1 and CTGF are mediators of chondrocyte proliferation and cartilage repair. Study demonstrates that oral glucosamine doses that produce clinically relevant serum glucosamine concentrations can induce tissue TGFβ1 and CTGF expression in vivo and provides a mechanistic rationale for reported beneficial effects of glucosamine therapy. Induction of renal TGFβ1 and CTGF mRNA suggests that potential sclerotic side-effects may occur following consumption of potent glucosamine preparations.

Renal (pro)renin receptor contributes to development of diabetic kidney disease through transforming growth factor‐β1 – connective tissue growth factor signalling cascade

1. Transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) are expressed in renal glomeruli, and contribute to the development of diabetic nephropathy. Recently, we showed that (pro)renin receptor (PRR) is upregulated in the kidneys of the streptozocin (STZ)-induced diabetes rat model. We hypothesized that in the presence of hyperglycaemia, increased renal PRR expression contributes to enhanced TGF-β1-CTGF signalling activity, leading to the development of diabetic kidney disease. 2. In vivo and in vitro studies were carried out in Sprague-Dawley rats and rat mesangial cells (RMC). PRR blockade was achieved in vivo by treating STZ induced diabetes rats with the handle region peptide (HRP) of prorenin and in vitro by HRP or PRR siRNA in RMC. Angiotensin AT1 receptor blockade was achieved by valsartan treatment. 3. Results showed that expression of PRR, TGF-β1 and CTGF were upregulated in diabetic kidneys and RMC exposed to high glucose. Glucose exposure also induced PRR phosphorylation, a process that was inhibited by HRP, valsartan or PRR siRNA. HRP and valsartan significantly attenuated renal TGF-β1 and CTGF expression in diabetic animals and high glucose treated RMC. Similar results were observed in high glucose exposed RMC in response to PRR siRNA. TGF-β receptor blockade decreased CTGF expression in RMC. Combined administration of valsartan and PRR siRNA showed further reduction of TGF-β1 and CTGF expression in RMC. 4. In conclusion, PRR contributes to kidney disease in diabetes through an enhanced TGF-β1-CTGF signalling cascade.

Renal proximal tubular dysfunction is a major determinant of urinary connective tissue growth factor excretion

Connective tissue growth factor (CTGF) plays a key role in renal fibrosis. Urinary CTGF is elevated in various renal diseases and may have biomarker potential. However, it is unknown which processes contribute to elevated urinary CTGF levels. Thus far, urinary CTGF was considered to reflect renal expression. We investigated how tubular dysfunction affects urinary CTGF levels. To study this, we administered recombinant CTGF intravenously to rodents. We used both full-length CTGF and the NH(2)-terminal fragment, since the NH(2)-fragment is the predominant form detected in urine. Renal CTGF extraction, determined by simultaneous arterial and renal vein sampling, was 18 +/- 3% for full-length CTGF and 21 +/- 1% for the NH(2)-fragment. Fractional excretion was very low for both CTGFs (0.02 +/- 0.006% and 0.10 +/- 0.02%, respectively), indicating that >99% of the extracted CTGF was metabolized by the kidney. Immunohistochemistry revealed extensive proximal tubular uptake of CTGF in apical endocytic vesicles and colocalization with megalin. Urinary CTGF was elevated in megalin- and cubilin-deficient mice but not in cubilin-deficient mice. Inhibition of tubular reabsorption by Gelofusine reduced renal uptake of CTGF and increased urinary CTGF. In healthy volunteers, Gelofusine also induced an increase of urinary CTGF excretion, comparable to the increase of beta(2)-microglobulin excretion (r = 0.99). Furthermore, urinary CTGF correlated with beta(2)-microglobulin (r = 0.85) in renal disease patients (n = 108), and only beta(2)-microglobulin emerged as an independent determinant of urinary CTGF. Thus filtered CTGF is normally reabsorbed almost completely in proximal tubules via megalin, and elevated urinary CTGF may largely reflect proximal tubular dysfunction.

Kidneys of Alb/TGF-β1 Transgenic Mice Are Deficient in Retinoic Acid and Exogenous Retinoic Acid Shows Dose-Dependent Toxicity

Background: Alb/TGF-β₁ transgenic mice overexpress active transforming growth factor-β₁ (TGF-β₁) in the liver, leading to increased circulating levels of the cytokine and progressive renal fibrosis. This study was designed to explore if exogenous all-trans retinoic acid (tRA) prevents renal fibrosis in this animal model. Methods: The retinoid profile in kidney and liver of wild-type and Alb/TGF-β₁ transgenic mice was examined by high-performance liquid chromatography and slow-release pellets containing different amounts of tRA were implanted subcutaneously to treat the Alb/TGF-β₁ transgenic mice, starting at 1 week of age; mice were sacrificed 2 weeks later. Results: Kidneys of 3-week-old wild-type mice had abundant tRA, which was completely absent in kidneys of the transgenic mice. Low doses of tRA (6–10.7 mg/kg/day) failed to affect renal fibrosis although it tended to suppress the mRNA expression of some molecular markers of fibrosis and retinal dehydrogenase 2 (RALDH2), a gene encoding a key tRA-synthesising enzyme. These tendencies disappeared, mortality tended to increase and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased in the medium-dose group (12.7–18.8 mg/kg/day). High doses (20.1–27.4 mg/kg/day) showed even higher toxicity with increased renal fibrosis and significant mortality. Conclusions: Alb/TGF-β₁ transgenic mice are characterised by depletion of endogenous renal tRA. Exogenous tRA dose-dependently increases mortality and kidney fibrosis, which is associated with dose-dependent regulation of renal RALDH2 and CTGF mRNA expression.

Renal connective tissue growth factor correlates with glomerular basement membrane thickness and prospective albuminuria in a non-human primate model of diabetes: possible predictive marker for incipient diabetic nephropathy

Diabetic renal disease is characterized by accumulation of extracellular matrix, glomerulosclerosis, and tubulointerstitial fibrosis. Connective tissue growth factor (CTGF) is implicated in these changes, as it contributes to new matrix synthesis and is increased in the diabetic kidney. CTGF also inhibits mesangial matrix degradation through up-regulation of the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1). In a non-human primate model of diabetes, we determined whether the level of renal CTGF protein before development of albuminuria correlated with renal matrix and TIMP-1 changes and whether renal CTGF predicts progression to albuminuria. Methods In a group of diabetic ( n=9) and control ( n=6) baboons after a 5-year duration of diabetes, renal tissue CTGF and TIMP-1 were detected by immunohistochemistry and compared with glomerular basement membrane (GBM) thickness and mesangial volume measurements from electron photomicrographs of renal biopsies. Urinary albumin levels were measured at 5 and 10 years of diabetes. Results GBM thickness, CTGF protein, and TIMP-1 protein were increased after 5 years of diabetes (each P<.05). Tubular fibronectin scores correlated with tubular CTGF scores ( r=0.72, P=.002). In diabetic animals, GBM thickness correlated with tubular and total CTGF levels ( P=.002 and P=.04, respectively), whereas mesangial cell and total matrix volume correlated with glomerular TIMP-1 ( P=.02 and P=.01, respectively). Tubular CTGF scores ( P=.008) and GBM thickness ( P=.03) at 5 years in diabetes each predicted the degree of albuminuria at 10 years. Conclusions These results suggest that early increases in renal CTGF protein contribute to incipient diabetic nephropathy and that renal CTGF may have utility as an early marker for progression to dysfunction in the diabetic kidney.

SGK1-Dependent Upregulation of Connective Tissue Growth Factor by Angiotensin II

Angiotensin II has previously been shown to trigger fibrosis, an effect involving connective tissue growth factor (CTGF). The signaling pathways linking angiotensin II to CTGF formation are, however, incompletely understood. A gene highly expressed in fibrosing tissue is the serum- and glucocorticoid-inducible kinase SGK1. The present study explored whether SGK1 is transcriptionally regulated by angiotensin II and participates in the angiotensin II-dependent regulation of CTGF expression. To this end, experiments have been performed in human kidney fibroblasts and mouse lung fibroblasts from gene-targeted mice lacking SGK1 (sgk1^–/–) and their wild-type littermates (sgk1^+/+). In human renal fibroblasts, SGK1 and CTGF protein expression were enhanced by angiotensin II (10 nM) within 4 h. In sgk1^+/+ mouse fibroblasts, SGK1 transcript levels were significantly increased after 4 h of angiotensin II treatment. Angiotensin II stimulated both transcript and protein abundance of CTGF in fibroblasts from sgk1^+/+ mice, effects significantly blunted in fibroblasts of sgk1^–/– mice. In conclusion, angiotensin II stimulates the expression of SGK1, which is in turn required for the stimulating effect of angiotensin II on the expression of CTGF. Thus, SGK1 presumably contributes to the profibrotic effect of angiotensin II.

Protective Effects of Sirolimus by Attenuating Connective Tissue Growth Factor Expression in Human Chronic Allograft Nephropathy

Background Chronic allograft nephropathy (CAN) remains a great challenge for the transplant clinician. The introduction of sirolimus (RAPA) with cyclosporine (CsA) reduction maybe shed new light to improve graft survival. The aim of this study was to investigate the overall effects of sirolimus conversion on biopsy-proven CAN. Methods One hundred and ten renal transplant recipients with biopsy-proven CAN were randomized into two groups: 54 for CsA reduction and 56 for sirolimus conversion treatment. After 24-month follow-up, the outcome variables included graft function and survival as well as CAN Banff grading and intrarenal expression of connective tissue growth factor (CTGF) via repeated biopsy. Results Graft function and survival rate were significantly better in the RAPA group. CAN grading worsened in the CsA group, whereas they were stable in the RAPA group. There was weak expression in the RAPA group but significant, increased expression of CTGF in glomeruli and interstitial area in the CSA group ( P < .01) both by immunhistochemical staining or real-time polymerase chain reaction detection. Conclusion Sirolimus conversion provided a beneficial strategy to improve long-term graft survival in CAN. Attenuation of renal CTGF expression maybe one of its antifibrotic and antiproliferation effects.

Interactions between aldosterone and connective tissue growth factor in vascular and renal damage in spontaneously hypertensive rats

The aim of the present study was to investigate possible inter-relationships between connective tissue growth factor (CTGF) and aldosterone in vascular and renal damage associated with hypertension. Spontaneously hypertensive rats (SHR) were treated with two doses (100 and 30 mg/kg per day) of the mineralocorticoid receptor antagonist eplerenone, or with antihypertensive therapy (HHR) (20 mg/kg per day hydralazine + 7 mg/kg per day hydrochlorothiazide + 0.15 mg/kg per day reserpine). CTGF mRNA expression and protein levels in the aorta of SHR were upregulated (P < 0.05) compared with Wistar-Kyoto rats. Both doses of eplerenone similarly and significantly diminished CTGF upregulation, correlated with amelioration of aortic remodelling and endothelium-dependent relaxations. Only high-dose eplerenone and HHR significantly reduced arterial blood pressure. HHR treatment also diminished CTGF overexpression, suggesting a blood-pressure-mediated effect in CTGF regulation. This reduction, however, was lower (P < 0.05) than that produced by eplerenone (100 mg/kg per day). The direct effect of aldosterone on vascular smooth muscle cells was also studied. Incubation of cultured vascular smooth muscle cells with aldosterone increased CTGF production in a dose-related manner, but was reduced (P < 0.05) by the mineralocorticoid receptor antagonist spironolactone. Renal CTGF mRNA and protein levels were higher in SHR than in Wistar-Kyoto rats (P < 0.05), and were similarly diminished by all treatments (P < 0.05). These data show that aldosterone and haemodynamic stress from elevated blood pressure levels regulate vascular and renal CTGF in SHR. The results suggest that aldosterone, through CTGF stimulation, could participate in vascular and renal structural alterations associated with hypertension, describing a novel mechanism of aldosterone in hypertensive target organ damage.