Bruce L. Riser

Treatment with pyrophosphate inhibits uremic vascular calcification

Pyrophosphate, which may be deficient in advanced renal failure, is a potent inhibitor of vascular calcification. To explore its use as a potential therapeutic, we injected exogenous pyrophosphate subcutaneously or intraperitoneally in normal rats and found that their plasma pyrophosphate concentrations peaked within 15 min. There was a single exponential decay with a half-life of 33 min. The kinetics were indistinguishable between the two routes of administration or in anephric rats. The effect of daily intraperitoneal pyrophosphate injections on uremic vascular calcification was then tested in rats fed a high-phosphate diet containing adenine for 28 days to induce uremia. Although the incidence of aortic calcification varied and was not altered by pyrophosphate, the calcium content of calcified aortas was significantly reduced by 70%. Studies were repeated in uremic rats given calcitriol to produce more consistent aortic calcification and treated with sodium pyrophosphate delivered intraperitoneally in a larger volume of glucose-containing solution to prolong plasma pyrophosphate levels. This maneuver significantly reduced both the incidence and amount of calcification. Quantitative histomorphometry of bone samples after double-labeling with calcein indicated that there was no effect of pyrophosphate on the rates of bone formation or mineralization. Thus, exogenous pyrophosphate can inhibit uremic vascular calcification without producing adverse effects on bone.

Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy

Profibrotic growth factors and inflammatory chemokines have been implicated in the pathogenesis of diabetic nephropathy (DN). However, measurement of urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers has not previously been reported, and neither have two such molecules in urine been examined in a single study of DN. In this prospective observational study, 43 adult diabetic patients were studied, 40 were followed up for 6years. Urinary MCP-1/creatinine ratios were found to be significantly higher in patients with macroalbuminuria (3.3- and 2.1-fold higher (p<0.01) than normoalbuminuric and microalbuminuric patients, respectively). CCN2 exhibited a pattern different from that of urinary MCP-1. Urinary CCN2/creatinine ratios were greatly elevated in both microalbuminuric and macroalbuminuric patients (125- and 74-fold higher than normoalbuminuric patients, respectively, p<0.01 and p<0.05, respectively). Further, urinary CCN2, but not MCP-1, correlated with progression of microalbuminuria (R=0.49, p<0.05). In contrast, MCP-1, but not CCN2, correlated with the rate of eGFR decline for all patients (R=0.61, p<0.0001), reflective of its predictive value in patients with macroalbuminuria, but not for patients with microalbuminuria or normoalbuminuria. In conclusion, increased urinary CCN2 is associated with the early progression of DN, whereas MCP-1 is associated with later stage disease.

CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

Daily peritoneal administration of sodium pyrophosphate in a dialysis solution prevents the development of vascular calcification in a mouse model of uraemia

BACKGROUNDnThe high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) is a significant barrier to improved life expectancy. Unique in this population is the marked development and aggressive worsening of vascular calcification (VC). Pyrophosphate (PPi), an endogenous molecule, appears to naturally inhibit soft tissue calcification, but may be depressed in chronic kidney disease (CKD) and ESRD. Although once thought to be a promising therapeutic, PPis very short half-life in circulation curtailed earlier studies. We tested the possibility that a slow, continuous entry of PPi into the circulation and prevention of VC might be achieved by daily peritoneal dialysis (PD).nnnMETHODSnPharmacokinetic studies were first carried out in rats with renal impairment resulting from a 5/6 nephrectomy. Efficacy studies were then performed in the apolipoprotein E gene knockout mouse model overlaid with CKD. PPi was delivered by means of a permanent peritoneal catheter in a solution simulating PD, but without the timed removal of spent dialysate. von Kossas staining followed by semiquantitative morphological image processing, with separation of inside (intimal) and outside (presumed medial) lesions, was used to determine aortic root calcification.nnnRESULTSnIn comparison to an intravenous bolus, delivery of PPi in a PD solution resulted in a slower, extended delivery over >4 h. Next, the efficacy studies showed that a 6-day/week PD-simulated administration of PPi resulted in a dose-dependent inhibition of aortic calcification in both intimal and medial lesions. A dose-response effect on total aortic calcification was also documented, with a full inhibition seen at the highest dose. A limited peritoneal catheter-related inflammation was observed, as expected, and included the placebo-treated control groups. This inflammatory response could have masked a lower level PPi-specific adverse effect, but none was observed.nnnCONCLUSIONSnOur findings suggest potential for PPi, administered during PD, to prevent the development of VC and to potentially extend the life of ESRD patients.

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-β1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-β1. Conversely, TGF-β1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-β1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.

CCN2 plays a key role in extracellular matrix gene expression in severe hypertrophic cardiomyopathy and heart failure

Hypertrophic cardiomyopathy (HCM) is the most common inherited primary myocardial disorder. HCM is characterized by interstitial fibrosis and excessive accumulation of extracellular matrix (ECM) proteins. Fibrosis in HCM has been associated with impaired cardiac function and heart failure, and has been considered a key substrate for ventricular arrhythmias and sudden death. The molecular triggers underpinning ECM production are not well established. We have previously developed a double-mutant mouse model of HCM that recapitulates the phenotype seen in humans with multiple mutations, including earlier onset of the disease, progression to a dilated phenotype, severe heart failure and premature mortality. The present study investigated the expression of ECM-encoding genes in severe HCM and heart failure. Significant upregulation of structural Fn1, regulatory Mmp14, Timp1, Serpin3A, SerpinE1, SerpineE2, Tgfβ1, and Tgfβ2; and matricellular Ccn2, Postn, Spp1, Thbs1, Thbs4, and Tnc was evident from the early, pre-phenotype stage. Non-myocytes expressed ECM genes at higher levels than cardiomyocytes in normal and diseased hearts. Synchronous increase of secreted CCN2 and TIMP1 plasma levels and decrease of MMP3 levels were observed in end-stage disease. CCN2 protein expression was increased from early disease in double-mutant hearts and played an important role in ECM responses. It was a powerful modulator of ECM regulatory (Timp1 and SerpinE1) and matricellular protein-encoding (Spp1, Thbs1, Thbs4 and Tnc) gene expression in cardiomyocytes when added exogenously in vitro. Modulation of CCN2 (CTGF, connective tissue growth factor) and associated early ECM changes may represent a new therapeutic target in the treatment and prevention of heart failure in HCM.

CCN2 (CTGF) gene polymorphism is a novel prognostic risk factor for cardiovascular outcomes in hemodialysis patients.

Background: The very high cardiovascular (CV) mortality and morbidity rates in hemodialysis (HD) patients are greatly related to atherosclerosis. CCN2 (connective tissue growth factor/CTGF) is a profibrotic factor that is secreted by endothelial cells, involved in atherogenesis, promoting fibroblast proliferation and matrix production. CCN2 protein is significantly increased in complicated fibrous plaques and enhances monocyte migration into atherosclerotic lesions. The aim of this study was to investigate a possible association between CCN2 gene polymorphism and CV morbidity and mortality in HD patients. Methods: 98 HD patients, followed for 24 months, were genotyped for the common polymorphism on the CCN2 gene (G-945C). HD patient characteristics were: age 64 ± 13 years, males 64%, diabetes 24%, hypertension 62%, smokers 38%, dyslipidemia 28%, all undergoing standard HD three times weekly. Results: All-cause mortality was not associated with CCN2 polymorphism (G-945C). In contrast, however, the GG genotype was strongly associated with CV mortality: OR 13 (1.49–155), p = 0.0048. Interestingly, the GG genotype was also greatly associated with the serious CV events of stroke and myocardial infarction in surviving HD patients: OR 13.3 (2.5–87.08), p = 0.0001. Conclusions: We demonstrate for the first time that CCN2 gene polymorphism is a prognostic risk factor for CV morbidity and mortality in HD patients. These data may have important implications for better understanding the link between accelerated atherosclerosis and increased mortality in HD population.

Patients with Encapsulating Peritoneal Sclerosis Have Increased Peritoneal Expression of Connective Tissue Growth Factor (CCN2), Transforming Growth Factor-β1, and Vascular Endothelial Growth Factor

Introduction Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD). The pathogenesis is not exactly known and no preventive strategy or targeted medical therapy is available. CCN2 has both pro-fibrotic and pro-angiogenic actions and appears an attractive target. Therefore, we studied peritoneal expression of CCN2, as well as TGFβ1 and VEGF, in different stages of peritoneal fibrosis. Materials and methods Sixteen PD patients were investigated and compared to 12 hemodialysis patients and four pre-emptively transplanted patients. Furthermore, expression was investigated in 12 EPS patients in comparison with 13 PD and 12 non-PD patients without EPS. Peritoneal tissue was taken during kidney transplantation procedure or during EPS surgery. In a subset of patients, CCN2 protein levels in peritoneal effluent and plasma were determined. Samples were examined by qPCR, histology, immunohistochemistry, and ELISA. Results Peritoneal CCN2 expression was 5-fold higher in PD patients compared to pre-emptively transplanted patients (P<0.05), but did not differ from hemodialysis patients. Peritoneal expression of TGFβ1 and VEGF were not different between the three groups; neither was peritoneal thickness. Peritoneum of EPS patients exhibited increased expression of CCN2 (35-fold, P<0.001), TGFβ1 (24-fold, P<0.05), and VEGF (77-fold, P<0.001) compared to PD patients without EPS. In EPS patients, CCN2 protein was mainly localized in peritoneal endothelial cells and fibroblasts. CCN2 protein levels were significantly higher in peritoneal effluent of EPS patients compared to levels in dialysate of PD patients (12.0±4.5 vs. 0.91±0.92 ng/ml, P<0.01), while plasma CCN2 levels were not increased. Conclusions Peritoneal expression of CCN2, TGFβ1, and VEGF are significantly increased in EPS patients. In early stages of peritoneal fibrosis, only CCN2 expression is slightly increased. Peritoneal CCN2 overexpression in EPS patients is a locally driven response. The potential of CCN2 as biomarker and target for CCN2-inhibiting agents to prevent or treat EPS warrants further study.

Balanced regulation of the CCN family of matricellular proteins: a novel approach to the prevention and treatment of fibrosis and cancer

The CCN family of matricellular signaling proteins is emerging as a unique common link across multiple diseases and organs related to injury and repair. They are now being shown to play a central role in regulating the pathways to the initiation and resolution of normal wound healing and fibrosis in response to multiple forms of injury. Similarly, it is also emerging that they play a key role in regulating the establishment, growth, metastases and tissue regeneration in many forms of cancer via the interaction of cancer cells with the tumor stroma. Evidence has been recently provided that these proteins do not act independently but are co-regulated working in a yin/yang manner to alter the outcome of both normal physiological processes as well as pathology. The purpose of this review is to twofold. First, it will summarize work to date supporting CCN2 as a therapeutic target in the formation and progression of renal, skin, and other organ fibrosis, as well as cancer stroma formation. Second, it will highlight recent evidence for CCN3 as a counter-regulator and a potential therapeutic agent in these diseases with an exciting, novel potential to both treat and then restore tissue homeostasis in those afflicted by these devastating disorders.

Treatment with the Matricellular Protein CCN3 Blocks and/or Reverses Fibrosis Development in Obesity with Diabetic Nephropathy

Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).