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Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in cell communication and microenvironmental signaling. Overexpression of CCN2 has been documented in various cardiovascular pathologies, wherein it may exert either deleterious or protective effects depending on the pathological context, thereby suggesting that its role in the cardiovascular system is not yet fully elucidated. In this study, we aimed to investigate the effects of Ccn2 gene deletion on the progression of acute cardiac injury induced by doxorubicin (DOX), a widely utilized chemotherapeutic agent. To this end, we employed conditional knockout (KO) mice for the Ccn2 gene (CCN2-KO), which were administered DOX and compared to DOX-treated wild-type (WT) control mice. Our findings demonstrated that the ablation of CCN2 ameliorated DOX-induced cardiac dysfunction, as evidenced by improvements in ejection fraction (EF) and fractional shortening (FS) of the left ventricle. Furthermore, DOX-treated CCN2-KO mice exhibited a significant reduction in the gene expression and activation of oxidative stress markers (Hmox1 and Nfe2l2/NRF2) relative to DOX-treated WT controls. Additionally, the deletion of Ccn2 markedly attenuated DOX-induced cardiac fibrosis. Collectively, these results suggest that CCN2 plays a pivotal role in the pathogenesis of DOX-mediated cardiotoxicity by modulating oxidative stress and fibrotic pathways. These findings provide a novel avenue for future investigations to explore the therapeutic potential of targeting CCN2 in the prevention of DOX-induced cardiac dysfunction.

Cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a downstream mediator of other profibrotic factors including transforming growth factor (TGF)-β and angiotensin II. However, recent evidence from our group demonstrated the direct role of CCN2 in maintaining aortic wall homeostasis and acute and lethal aortic aneurysm development induced by angiotensin II in the absence of CCN2 in mice. In order to translate these findings to humans, we evaluated the potential association between three polymorphisms in the CCN2 gene and the presence of a thoracic aortic aneurysm (TAA). Patients with and without TAA retrospectively selected were genotyped for rs6918698, rs9402373 and rs12526196 polymorphisms related to the CCN2 gene. Multivariable logistic regression models were performed. In our population of 366 patients (69 with TAA), no associations were found between rs6918698 and rs9402373 and TAA. However, the presence of one C allele from rs12526196 was associated with TAA comparing with the TT genotype, independently of risk factors such as sex, age, hypertension, type of valvulopathy and the presence of a bicuspid aortic valve (OR = 3.17; 95% CI = 1.30-7.88; p = 0.011). In conclusion, we demonstrated an association between the C allele of rs12526196 in the CCN2 gene and the presence of TAA. This study extrapolates to humans the relevance of CCN2 in aortic aneurysm observed in mice and postulates, for the first time, a potential protective role to CCN2 in aortic aneurysm pathology. Our results encourage future research to explore new variants in the CCN2 gene that could be predisposed to TAA development.

Diabetic nephropathy (DN) is the main leading cause of chronic kidney disease worldwide. Although remarkable therapeutic advances have been made during the last few years, there still exists a high residual risk of disease progression to end-stage renal failure. To further understand the pathogenesis of tissue injury in this disease, by means of the Next-Generation Sequencing, we have studied the microRNA (miRNA) differential expression pattern in kidneys of Black and Tan Brachyury (BTBR) ob/ob (leptin deficiency mutation) mouse. This experimental model of type 2 diabetes and obesity recapitulates the key histopathological features described in advanced human DN and therefore can provide potential useful translational information. The miRNA-seq analysis, performed in the renal cortex of 22-week-old BTBR ob/ob mice, pointed out a set of 99 miRNAs significantly increased compared to non-diabetic, non-obese control mice of the same age, whereas no miRNAs were significantly decreased. Among them, miR-802, miR-34a, miR-132, miR-101a, and mir-379 were the most upregulated ones in diabetic kidneys. The in silico prediction of potential targets for the 99 miRNAs highlighted inflammatory and immune processes, as the most relevant pathways, emphasizing the importance of inflammation in the pathogenesis of kidney damage associated to diabetes. Other identified top canonical pathways were adipogenesis (related with ectopic fatty accumulation), necroptosis (an inflammatory and regulated form of cell death), and epithelial-to-mesenchymal transition, the latter supporting the importance of tubular cell phenotype changes in the pathogenesis of DN. These findings could facilitate a better understanding of this complex disease and potentially open new avenues for the design of novel therapeutic approaches to DN., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Opazo-Ríos, Tejera-Muñoz, Soto Catalan, Marchant, Lavoz, Mas Fontao, Moreno, Fierro Fernandez, Ramos, Suarez-Alvarez, López-Larrea, Ruiz-Ortega, Egido and Rodrigues-Díez.)

Background: CCN2 (cellular communication network factor 2) is a matricellular protein involved in cell communication and microenvironmental signaling responses. CCN2 is known to be overexpressed in several cardiovascular diseases, but its role is not completely understood., Methods: Here, CCN2 involvement in aortic wall homeostasis and response to vascular injury was investigated in inducible <i>Ccn2</i>-deficient mice, with induction of vascular damage by infusion of Ang II (angiotensin II; 15 days), which is known to upregulate CCN2 expression in the aorta., Results: Ang II infusion in CCN2-silenced mice lead to 60% mortality within 10 days due to rapid development and rupture of aortic aneurysms, as evidenced by magnetic resonance imaging, echography, and histological examination. <i>Ccn2</i> deletion decreased systolic blood pressure and caused aortic structural and functional changes, including elastin layer disruption, smooth muscle cell alterations, augmented distensibility, and increased metalloproteinase activity, which were aggravated by Ang II administration. Gene ontology analysis of RNA sequencing data identified aldosterone biosynthesis as one of the most enriched terms in CCN2-deficient aortas. Consistently, treatment with the mineralocorticoid receptor antagonist spironolactone before and during Ang II infusion reduced aneurysm formation and mortality, underscoring the importance of the aldosterone pathway in Ang II-induced aorta pathology., Conclusions: CCN2 is critically involved in the functional and structural homeostasis of the aorta and in maintenance of its integrity under Ang II-induced stress, at least, in part, by disruption of the aldosterone pathway. Thus, this study opens new avenues to future studies in disorders associated to vascular pathologies.

Cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), is considered a fibrotic biomarker and has been suggested as a potential therapeutic target for kidney pathologies. CCN2 is a matricellular protein with four distinct structural modules that can exert a dual function as a matricellular protein and as a growth factor. Previous experiments using surface plasmon resonance and cultured renal cells have demonstrated that the C-terminal module of CCN2 (CCN2(IV)) interacts with the epidermal growth factor receptor (EGFR). Moreover, CCN2(IV) activates proinflammatory and profibrotic responses in the mouse kidney. The aim of this paper was to locate the in vivo cellular CCN2/EGFR binding sites in the kidney. To this aim, the C-terminal module CCN2(IV) was labeled with a fluorophore (Cy5), and two different administration routes were employed. Both intraperitoneal and direct intra-renal injection of Cy5-CCN2(IV) in mice demonstrated that CCN2(IV) preferentially binds to the tubular epithelial cells, while no signal was detected in glomeruli. Moreover, co-localization of Cy5-CCN2(IV) binding and activated EGFR was found in tubules. In cultured tubular epithelial cells, live-cell confocal microscopy experiments showed that EGFR gene silencing blocked Cy5-CCN2(IV) binding to tubuloepithelial cells. These data clearly show the existence of CCN2/EGFR binding sites in the kidney, mainly in tubular epithelial cells. In conclusion, these studies show that circulating CCN2(IV) can directly bind and activate tubular cells, supporting the role of CCN2 as a growth factor involved in kidney damage progression.

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.

Background: CKD leads to vitamin D deficiency. Treatment with vitamin D receptor agonists (VDRAs) may have nephroprotective and anti-inflammatory actions, but their mechanisms of action are poorly understood., Methods: Modulation of the noncanonical NF- κ B2 pathway and its component TNF receptor-associated factor 3 (TRAF3) by the VDRA paricalcitol was studied in PBMCs from patients with ESKD, cytokine-stimulated cells, and preclinical kidney injury models., Results: In PBMCs isolated from patients with ESKD, TRAF3 protein levels were lower than in healthy controls. This finding was associated with evidence of noncanonical NF- κ B2 activation and a proinflammatory state. However, PBMCs from patients with ESKD treated with paricalcitol did not exhibit these features. Experiments in cultured cells confirmed the link between TRAF3 and NF- κ B2/inflammation. Decreased TRAF3 ubiquitination in K48-linked chains and cIAP1-TRAF3 interaction mediated the mechanisms of paricalcitol action.TRAF3 overexpression by CRISPR/Cas9 technology mimicked VDRA's effects. In a preclinical model of kidney injury, paricalcitol inhibited renal NF- κ B2 activation and decreased renal inflammation. In VDR knockout mice with renal injury, paricalcitol prevented TRAF3 downregulation and NF- κ B2-dependent gene upregulation, suggesting a VDR-independent anti-inflammatory effect of paricalcitol., Conclusions: These data suggest the anti-inflammatory actions of paricalcitol depend on TRAF3 modulation and subsequent inhibition of the noncanonical NF- κ B2 pathway, identifying a novel mechanism for VDRA's effects. Circulating TRAF3 levels could be a biomarker of renal damage associated with the inflammatory state., (Copyright © 2020 by the American Society of Nephrology.)

Introduction: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease worldwide. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway participates in the development and progression of DN. Among the different mechanisms involved in JAK/STAT negative regulation, the family of suppressor of cytokine signaling (SOCS) proteins has been proposed as a new target for DN. Our aim was to evaluate the effect of SOCS1 mimetic peptide in a mouse model of obesity and type 2 diabetes (T2D) with progressive DN., Research Design and Methods: Six-week-old BTBR (black and tan brachyuric) mice with the ob/ob (obese/obese) leptin-deficiency mutation were treated for 7 weeks with two different doses of active SOCS1 peptide (MiS1 2 and 4 µg/g body weight), using inactive mutant peptide (Mut 4 µg) and vehicle as control groups. At the end of the study, the animals were sacrificed to obtain blood, urine and kidney tissue for further analysis., Results: Treatment of diabetic mice with active peptide significantly decreased urine albumin to creatinine ratio by up to 50%, reduced renal weight, glomerular and tubulointerstitial damage, and restored podocyte numbers. Kidneys from treated mice exhibited lower inflammatory infiltrate, proinflammatory gene expression and STAT activation. Concomitantly, active peptide administration modulated redox balance markers and reduced lipid peroxidation and cholesterol transporter gene expression in diabetic kidneys., Conclusion: Targeting SOCS proteins by mimetic peptides to control JAK/STAT signaling pathway ameliorates albuminuria, morphological renal lesions, inflammation, oxidative stress and lipotoxicity, and could be a therapeutic approach to T2D kidney disease., Competing Interests: Competing interests: JE and CG-G are inventors on a patent application regarding clinical utility of SOCS peptide., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Uraemic cardiomyopathy, a process mainly associated with increased myocardial fibrosis, is the leading cause of death in chronic kidney disease patients and can be prevented by vitamin D receptor activators (VDRAs). Since some microRNAs (miRNAs) have emerged as regulators of the fibrotic process, we aimed to analyse the role of specific miRNAs in VDRA prevention of myocardial fibrosis as well as their potential use as biomarkers., Methods: Wistar rats were nephrectomized and treated intraperitoneally with equivalent doses of two VDRAs: calcitriol and paricalcitol. Biochemical parameters, cardiac fibrosis, miRNA (miR-29b, miR-30c and miR-133b) levels in the heart and serum and expression of their target genes collagen I (COL1A1), matrix metalloproteinase 2 (MMP-2) and connective tissue growth factor (CTGF) in the heart were evaluated., Results: Both VDRAs attenuated cardiac fibrosis, achieving a statistically significant difference in the paricalcitol-treated group. Increases in RNA and protein levels of COL1A1, MMP-2 and CTGF and reduced expression of miR-29b and miR-30c, known regulators of these pro-fibrotic genes, were observed in the heart of chronic renal failure (CRF) rats and were attenuated by both VDRAs. In serum, significant increases in miR-29b, miR-30c and miR-133b levels were observed in CRF rats, which were prevented by VDRA use. Moreover, vitamin D response elements were identified in the three miRNA promoters., Conclusions: VDRAs, particularly paricalcitol, attenuated cardiac fibrosis acting on COL1A1, MMP-2 and CTGF expression, partly through regulation of miR-29b and miR-30c. These miRNAs and miR-133b could be useful serum biomarkers for cardiac fibrosis and also potential new therapeutic targets., (© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

The classical view of the immune system has changed by the discovery of novel T-helper (Th) subsets, including Th17 (IL-17A-producing cells). IL-17A participates in immune-mediated glomerulonephritis and more recently in inflammatory pathologies, including experimental renal injury. Peritoneal dialysis patients present chronic inflammation and Th1/Th2 imbalance, but the role of the Th17 response in peritoneal membrane damage has not been investigated. In peritoneal biopsies from dialyzed patients, IL-17A immunostaining was found mainly in inflammatory areas and was absent in the healthy peritoneum. IL-17A-expressing cells included lymphocytes (CD4+ and γδ), neutrophils, and mast cells. Elevated IL-17A effluent concentrations were found in long-term peritoneal dialysis patients. Studies in mice showed that repeated exposure to recombinant IL-17A caused peritoneal inflammation and fibrosis. Moreover, chronic exposure to dialysis fluids resulted in a peritoneal Th17 response, including elevated IL-17A gene and protein production, submesothelial cell infiltration of IL-17A-expressing cells, and upregulation of Th17 differentiation factors and cytokines. IL-17A neutralization diminished experimental peritoneal inflammation and fibrosis caused by chronic exposure to dialysis fluids in mice. Thus, IL-17A is a key player of peritoneum damage and it may be a good candidate for therapeutic intervention in peritoneal dialysis patients.

Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR-binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-β-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein susceptible to proteolytic degradation. CCN2 levels have been suggested as a potential risk biomarker in several chronic diseases. In body fluids, CCN2 full-length and its degradation fragments can be found; however, their in vivo effects are far from being elucidated. CCN2 was described as a profibrotic mediator, but this concept is changing to a proinflammatory cytokine. In vitro, CCN2 full-length and its C-terminal module IV (CCN2(IV)) exert proinflammatory properties. Emerging evidence suggest that Th17 cells, and its effector cytokine IL-17A, participate in chronic inflammatory diseases. Our aim was to explore whether CCN2(IV) could regulate the Th17 response. In vitro, stimulation of human naive CD4+ T lymphocytes with CCN2(IV) resulted in differentiation to Th17 phenotype. The in vivo effects of CCN2(IV) were studied in C57BL/6 mice. Intraperitoneal administration of recombinant CCN2(IV) did not change serum IL-17A levels, but caused an activation of the Th17 response in the kidney, characterized by interstitial infiltration of Th17 (IL17A+/CD4+) cells and upregulation of proinflammatory mediators. In CCN2(IV)-injected mice, elevated renal levels of Th17-related factors (IL-17A, IL-6, STAT3 and RORγt) were found, whereas Th1/Th2 cytokines or Treg-related factors (TGF-β and Foxp-3) were not modified. Treatment with an anti-IL-17A neutralizing antibody diminished CCN2(IV)-induced renal inflammation. Our findings unveil that the C-terminal module of CCN2 induces the Th17 differentiation of human Th17 cells and causes a renal Th17 inflammatory response. Furthermore, these data bear out that IL-17A targeting is a promising tool for chronic inflammatory diseases, including renal pathologies.

Angiotensin II (Ang II) modulates vasomotor tone, cell growth, and extracellular matrix deposition. This study analyzed the effect of atorvastatin in the possible alterations induced by Ang II on structure and mechanics of mesenteric resistance arteries and the signaling mechanisms involved. Wistar rats were infused with Ang II (100 ng/kg per day, SC minipumps, 2 weeks) with or without atorvastatin (5 mg/kg per day). Ang II increased blood pressure and plasmatic malondialdehyde levels. Compared with controls, mesenteric resistance arteries from Ang II-treated rats showed the following: (1) decreased lumen diameter; (2) increased wall/lumen; (3) decreased number of adventitial, smooth muscle, and endothelial cells; (4) increased stiffness; (5) increased collagen deposition; and (6) diminished fenestrae area and number in the internal elastic lamina. Atorvastatin did not alter blood pressure but reversed all of the structural and mechanical alterations of mesenteric arteries, including collagen and elastin alterations. In mesenteric resistance arteries, Ang II increased vascular O(2)(.-) production and diminished endothelial NO synthase and CuZn/superoxide dismutase but did not modify extracellular-superoxide dismutase expression. Atorvastatin improved plasmatic and vascular oxidative stress, normalized endothelial NO synthase and CuZn/superoxide dismutase expression, and increased extracellular-superoxide dismutase expression, showing antioxidant properties. Atorvastatin also diminished extracellular signal-regulated kinase 1/2 activation caused by Ang II in these vessels, indicating an interaction with Ang II-induced intracellular responses. In vascular smooth muscle cells, collagen type I release mediated by Ang II was reduced by different antioxidants and statins. Moreover, atorvastatin downregulated the Ang II-induced NADPH oxidase subunit, Nox1, expression. Our results suggest that statins might exert beneficial effects on hypertension-induced vascular remodeling by improving vascular structure, extracellular matrix alterations, and vascular stiffness. These effects might be mediated by their antioxidant properties.

Connective tissue growth factor (CTGF) is increased in several pathologies associated with fibrosis, including multiple renal diseases. CTGF is involved in biological processes such as cell cycle regulation, migration, adhesion and angiogenesis. Its expression is regulated by various factors involved in renal damage, such as transforming growth factor- , Angiotensin II, high concentrations of glucose and cellular stress. CTGF is involved in the initiation and progression of renal damage to be able to induce an inflammatory response and promote fibrosis, identified as a potential therapeutic target in the treatment of kidney diseases. In this paper we review the main actions of CTGF in renal disease, the intracellular action mechanisms and therapeutic strategies for its blocking.