Targeting inflammation to treat diabetic kidney disease: the road to 2030.

Diabetic kidney disease (DKD) is one of the fastest growing causes of chronic kidney disease and associated morbidity and mortality. Preclinical research has demonstrated the involvement of inflammation in its pathogenesis and in the progression of kidney damage, supporting clinical trials designed to explore anti-inflammatory strategies. However, the recent success of sodium-glucose cotransporter-2 inhibitors and the nonsteroidal mineralocorticoid receptor antagonist finerenone has changed both guidelines and standard of care, rendering obsolete older studies directly targeting inflammatory mediators and the clinical development was discontinued for most anti-inflammatory drugs undergoing clinical trials for DKD in 2016. Given the contribution of inflammation to the pathogenesis of DKD, we review the impact on kidney inflammation of the current standard of care, therapies undergoing clinical trials, or repositioned drugs for DKD. Moreover, we review recent advances in the molecular regulation of inflammation in DKD and discuss potential novel therapeutic strategies with clinical relevance. Finally, we provide a road map for future research aimed at integrating the growing knowledge on inflammation and DKD into clinical practice to foster improvement of patient outcomes.

Cellular communication network 2 (connective tissue growth factor) aggravates acute DNA damage and subsequent DNA damage response-senescence-fibrosis following kidney ischemia reperfusion injury.

Chronic allograft dysfunction with progressive fibrosis of unknown cause remains a major issue after kidney transplantation, characterized by ischemia-reperfusion injury (IRI). One hypothesis to account for this is that spontaneous progressive tubulointerstitial fibrosis following IRI is driven by cellular senescence evolving from a prolonged, unresolved DNA damage response (DDR). Since cellular communication network factor 2 ((CCN2), formerly called connective tissue growth factor), an established mediator of kidney fibrosis, is also involved in senescence-associated pathways, we investigated the relation between CCN2 and cellular senescence following kidney transplantation. Tubular CCN2 overexpression was found to be associated with DDR, loss of kidney function and tubulointerstitial fibrosis in both the early and the late phase in human kidney allograft biopsies. Consistently, CCN2 deficient mice developed reduced senescence and tubulointerstitial fibrosis in the late phase; six weeks after experimental IRI. Moreover, tubular DDR markers and plasma urea were less elevated in CCN2 knockout than in wild-type mice. Finally, CCN2 administration or overexpression in epithelial cells induced upregulation of tubular senescence-associated genes including p21, while silencing of CCN2 alleviated DDR induced by anoxia-reoxygenation injury in cultured proximal tubule epithelial cells. Thus, our observations indicate that inhibition of CCN2 can mitigate IRI-induced acute kidney injury, DNA damage, and the subsequent DDR-senescence-fibrosis sequence. Hence, targeting CCN2 might help to protect the kidney from transplantation-associated post-IRI chronic kidney dysfunction.

Deletion of delta-like 1 homologue accelerates renal inflammation by modulating the Th17 immune response.

Preclinical studies have demonstrated that activation of the NOTCH pathway plays a key role in the pathogenesis of kidney damage. There is currently no information on the role of the Delta-like homologue 1 (DLK1), a NOTCH inhibitor, in the regulation of renal damage. Here, we investigated the contribution of DLK1 to experimental renal damage and the underlying molecular mechanisms. Using a Dlk1-null mouse model in the experimental renal damage of unilateral ureteral obstruction, we found activation of NOTCH, as shown by increased nuclear translocation of the NOTCH1 intracellular domain, and upregulation of Dlk2/hey-1 expression compared to wild-type (WT) littermates. NOTCH1 over-activation in Dlk1-null injured kidneys was associated with a higher inflammatory response, characterized by infiltration of inflammatory cells, mainly CD4/IL17A + lymphocytes, and activation of the Th17 immune response. Furthermore, pharmacological NOTCH blockade inhibited the transcription factors controlling Th17 differentiation and gene expression of the Th17 effector cytokine IL-17A and other related-inflammatory factors, linked to a diminution of inflammation in the injured kidneys. We propose that the non-canonical NOTCH ligand DLK1 acts as a NOTCH antagonist in renal injury regulating the Th17-mediated inflammatory response.

The CCN2 Polymorphism rs12526196 Is a Risk Factor for Ascending Thoracic Aortic Aneurysm.

Cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a downstream mediator of other profibrotic factors including transforming growth factor (TGF)-ß 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.

Interplay between extracellular matrix components and cellular and molecular mechanisms in kidney fibrosis.

Chronic kidney disease (CKD) is characterized by pathological accumulation of extracellular matrix (ECM) proteins in renal structures. Tubulointerstitial fibrosis is observed in glomerular diseases as well as in the regeneration failure of acute kidney injury (AKI). Therefore, finding antifibrotic therapies comprises an intensive research field in Nephrology. Nowadays, ECM is not only considered as a cellular scaffold, but also exerts important cellular functions. In this review, we describe the cellular and molecular mechanisms involved in kidney fibrosis, paying particular attention to ECM components, profibrotic factors and cell-matrix interactions. In response to kidney damage, activation of glomerular and/or tubular cells may induce aberrant phenotypes characterized by overproduction of proinflammatory and profibrotic factors, and thus contribute to CKD progression. Among ECM components, matricellular proteins can regulate cell-ECM interactions, as well as cellular phenotype changes. Regarding kidney fibrosis, one of the most studied matricellular proteins is cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), currently considered as a fibrotic marker and a potential therapeutic target. Integrins connect the ECM proteins to the actin cytoskeleton and several downstream signaling pathways that enable cells to respond to external stimuli in a coordinated manner and maintain optimal tissue stiffness. In kidney fibrosis, there is an increase in ECM deposition, lower ECM degradation and ECM proteins cross-linking, leading to an alteration in the tissue mechanical properties and their responses to injurious stimuli. A better understanding of these complex cellular and molecular events could help us to improve the antifibrotic therapies for CKD.

TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease.

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-κB2 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-κB2 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-κB2/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-κB2 activation and decreased renal inflammation. In VDR knockout mice with renal injury, paricalcitol prevented TRAF3 downregulation and NF-κB2-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-κB2 pathway, identifying a novel mechanism for VDRA's effects. Circulating TRAF3 levels could be a biomarker of renal damage associated with the inflammatory state.

CCN2 Increases TGF-ß Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-ß Pathway Regulation.

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 ß (TGF-ß). 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-ß 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-ß receptors (TßR) in the TGF-ß pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TßRI and TßRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TßRI levels, an increase in TßRII 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 TßRI inhibitor did not modify TßRII increment induced by CCN2(VI), demonstrating a TGF-ß-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TßRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TßRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-ß pathway activation by increasing TßRII expression in an EGFR-SMAD dependent manner activation.

Interleukin-17A induces vascular remodeling of small arteries and blood pressure elevation.

An important link exists between hypertension and inflammation. Hypertensive patients present elevated circulating levels of proinflammatory cytokines, including interleukin-17A (IL-17A). This cytokine participates in host defense, autoimmune and chronic inflammatory pathologies, and cardiovascular diseases, mainly through the regulation of proinflammatory factors. Emerging evidence also suggests that IL-17A could play a role in regulating blood pressure and end-organ damage. Here, our preclinical studies in a murine model of systemic IL-17A administration showed that increased levels of circulating IL-17A raised blood pressure induced inward remodeling of small mesenteric arteries (SMAs) and arterial stiffness. In IL-17A-infused mice, treatment with hydralazine and hydrochlorothiazide diminished blood pressure elevation, without modifying mechanical and structural properties of SMA, suggesting a direct vascular effect of IL-17A. The mechanisms of IL-17A seem to involve an induction of vascular smooth muscle cell (VSMC) hypertrophy and phenotype changes, in the absence of extracellular matrix (ECM) proteins accumulation. Accordingly, treatment with an IL-17A neutralizing antibody diminished SMA remodeling in a model of angiotensin II (Ang II) infusion. Moreover, in vitro studies in VSMCs reported here, provide further evidence of the direct effects of IL-17A on cell growth responses. Our experimental data suggest that IL-17A is a key mediator of vascular remodeling of the small arteries, which might contribute, at least in part, to blood pressure elevation.

Molecular Regulation of Notch Signaling by Gremlin.

Gremlin is a member of the TGF-ß superfamily that can act as a BMP antagonist, and recently, has been described as a ligand of the vascular endothelial growth factor receptor 2 (VEGFR2). Gremlin shares properties with the Notch signaling pathway. Both participate in embryonic development and are reactivated in pathological conditions. Gremlin is emerging as a potential therapeutic target and biomarker of renal diseases. Here we review the role of the Gremlin-VEGFR2 axis in renal damage and downstream signaling mechanisms, such as Notch pathway.

Increased miR-7641 Levels in Peritoneal Hyalinizing Vasculopathy in Long-Term Peritoneal Dialysis Patients.

Peritoneal hyalinizing vasculopathy (PHV) represents the cornerstone of long-term peritoneal dialysis (PD), and especially characterizes patients associated with encapsulating peritoneal sclerosis. However, the mechanisms of PHV development remain unknown. A cross sectional study was performed in 100 non-selected peritoneal biopsies of PD patients. Clinical data were collected and lesions were evaluated by immunohistochemistry. In selected biopsies a microRNA (miRNA)-sequencing analysis was performed. Only fifteen patients (15%) showed PHV at different degrees. PHV prevalence was significantly lower among patients using PD fluids containing low glucose degradation products (GDP) (5.9% vs. 24.5%), angiotensin converting enzyme inhibitors (ACEIs) (7.5% vs. 23.4%), statins (6.5% vs. 22.6%) or presenting residual renal function, suggesting the existence of several PHV protective factors. Peritoneal biopsies from PHV samples showed loss of endothelial markers and induction of mesenchymal proteins, associated with collagen IV accumulation and wide reduplication of the basement membrane. Moreover, co-expression of endothelial and mesenchymal markers, as well as TGF-ß1/Smad3 signaling activation were found in PHV biopsies. These findings suggest that an endothelial-to-mesenchymal transition (EndMT) process was taking place. Additionally, significantly higher levels of miR-7641 were observed in severe PHV compared to non-PHV peritoneal biopsies. Peritoneal damage by GDPs induce miRNA deregulation and an EndMT process in submesothelial vessels, which could contribute to collagen IV accumulation and PHV.

Connective tissue growth factor induces renal fibrosis via epidermal growth factor receptor activation.

Connective tissue growth factor (CCN2/CTGF) is a matricellular protein that is overexpressed in progressive human renal diseases, mainly in fibrotic areas. In vitro studies have demonstrated that CCN2 regulates the production of extracellular matrix (ECM) proteins and epithelial-mesenchymal transition (EMT), and could therefore contribute to renal fibrosis. CCN2 blockade ameliorates experimental renal damage, including diminution of ECM accumulation. We have reported that CCN2 and its C-terminal degradation product CCN2(IV) bind to epidermal growth factor receptor (EGFR) to modulate renal inflammation. However, the receptor involved in CCN2 profibrotic actions has not been described so far. Using a murine model of systemic administration of CCN2(IV), we have unveiled a fibrotic response in the kidney that was diminished by EGFR blockade. Additionally, in conditional CCN2 knockout mice, renal fibrosis elicited by folic acid-induced renal damage was prevented, and this was linked to inhibition of EGFR pathway activation. Our in vitro studies demonstrated a direct effect of CCN2 via the EGFR pathway on ECM production by fibroblasts and the induction of EMT in tubular epithelial cells. Our studies clearly show that the EGFR regulates CCN2 fibrotic signalling in the kidney, and suggest that EGFR pathway blockade could be a potential therapeutic option to block CCN2-mediated profibrotic effects in renal diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Branched-chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation.

Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids implicated in glucose metabolism and maintenance of correct brain function. Elevated BCAA levels can promote an inflammatory response in peripheral blood mononuclear cells. However, there are no studies analysing the direct effects of BCAA on endothelial cells (ECs) and its possible modulation of vascular function. In vitro and ex vivo studies were performed in human ECs and aorta from male C57BL/6J mice, respectively. In ECs, BCAA (6 mmol/L) increased eNOS expression, reactive oxygen species production by mitochondria and NADPH oxidases, peroxynitrite formation and nitrotyrosine expression. Moreover, BCAA induced pro-inflammatory responses through the transcription factor NF-κB that resulted in the release of intracellular adhesion molecule-1 and E-selectin conferring endothelial activation and adhesion capacity to inflammatory cells. Pharmacological inhibition of mTORC1 intracellular signalling pathway decreased BCAA-induced pro-oxidant and pro-inflammatory effects in ECs. In isolated murine aorta, BCAA elicited vasoconstrictor responses, particularly in pre-contracted vessels and after NO synthase blockade, and triggered endothelial dysfunction, effects that were inhibited by different antioxidants, further demonstrating the potential of BCAA to induce oxidative stress with functional impact. In summary, we demonstrate that elevated BCAA levels generate inflammation and oxidative stress in ECs, thereby facilitating inflammatory cells adhesion and endothelial dysfunction. This might contribute to the increased cardiovascular risk observed in patients with elevated BCAA blood levels.

Gremlin activates the Notch pathway linked to renal inflammation.

Preclinical studies suggest that Gremlin participates in renal damage and could be a potential therapeutic target for human chronic kidney diseases. Inflammation is a common characteristic of progressive renal disease, and therefore novel anti-inflammatory therapeutic targets should be investigated. The Notch signaling pathway is involved in kidney development and is activated in human chronic kidney disease, but whether Gremlin regulates the Notch pathway has not been investigated. In cultured tubular cells, Gremlin up-regulated gene expression of several Notch pathway components, increased the production of the canonical ligand Jagged-1, and caused the nuclear translocation of active Notch-1 (N1ICD). In vivo administration of Gremlin into murine kidneys elicited Jagged-1 production, increased N1ICD nuclear levels, and up-regulated the gene expression of the Notch effectors hes-1 and hey-1 All these data clearly demonstrate that Gremlin activates the Notch pathway in the kidney. Notch inhibition using the γ-secretase inhibitor DAPT impaired renal inflammatory cell infiltration and proinflammatory cytokines overexpression in Gremlin-injected mice and in experimental models of renal injury. Moreover, Notch inhibition blocked Gremlin-induced activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathway, identifying an important mechanism involved in the anti-inflammatory actions of Notch inhibition. In conclusion, Gremlin activates the Notch pathway in the kidney and this is linked to NF-κB-mediated inflammation, supporting the hypothesis that Notch inhibition could be a potential anti-inflammatory strategy for renal diseases.

Role of Epidermal Growth Factor Receptor (EGFR) and Its Ligands in Kidney Inflammation and Damage.

Chronic kidney disease (CKD) is characterized by persistent inflammation and progressive fibrosis, ultimately leading to end-stage renal disease. Although many studies have investigated the factors involved in the progressive deterioration of renal function, current therapeutic strategies only delay disease progression, leaving an unmet need for effective therapeutic interventions that target the cause behind the inflammatory process and could slow down or reverse the development and progression of CKD. Epidermal growth factor receptor (EGFR) (ERBB1), a membrane tyrosine kinase receptor expressed in the kidney, is activated after renal damage, and preclinical studies have evidenced its potential as a therapeutic target in CKD therapy. To date, seven official EGFR ligands have been described, including epidermal growth factor (EGF) (canonical ligand), transforming growth factor-α, heparin-binding epidermal growth factor, amphiregulin, betacellulin, epiregulin, and epigen. Recently, the connective tissue growth factor (CTGF/CCN2) has been described as a novel EGFR ligand. The direct activation of EGFR by its ligands can exert different cellular responses, depending on the specific ligand, tissue, and pathological condition. Among all EGFR ligands, CTGF/CCN2 is of special relevance in CKD. This growth factor, by binding to EGFR and downstream signaling pathway activation, regulates renal inflammation, cell growth, and fibrosis. EGFR can also be "transactivated" by extracellular stimuli, including several key factors involved in renal disease, such as angiotensin II, transforming growth factor beta (TGFB), and other cytokines, including members of the tumor necrosis factor superfamily, showing another important mechanism involved in renal pathology. The aim of this review is to summarize the contribution of EGFR pathway activation in experimental kidney damage, with special attention to the regulation of the inflammatory response and the role of some EGFR ligands in this process. Better insights in EGFR signaling in renal disease could improve our current knowledge of renal pathology contributing to therapeutic strategies for CKD development and progression.

Epigenetic Modification Mechanisms Involved in Inflammation and Fibrosis in Renal Pathology.

The growing incidence of obesity, hypertension, and diabetes, coupled with the aging of the population, is increasing the prevalence of renal diseases in our society. Chronic kidney disease (CKD) is characterized by persistent inflammation, fibrosis, and loss of renal function leading to end-stage renal disease. Nowadays, CKD treatment has limited effectiveness underscoring the importance of the development of innovative therapeutic options. Recent studies have identified how epigenetic modifications participate in the susceptibility to CKD and have explained how the environment interacts with the renal cell epigenome to contribute to renal damage. Epigenetic mechanisms regulate critical processes involved in gene regulation and downstream cellular responses. The most relevant epigenetic modifications that play a critical role in renal damage include DNA methylation, histone modifications, and changes in miRNA levels. Importantly, these epigenetic modifications are reversible and, therefore, a source of potential therapeutic targets. Here, we will explain how epigenetic mechanisms may regulate essential processes involved in renal pathology and highlight some possible epigenetic therapeutic strategies for CKD treatment.

Inhibition of Bromodomain and Extraterminal Domain Family Proteins Ameliorates Experimental Renal Damage.

Renal inflammation has a key role in the onset and progression of immune- and nonimmune-mediated renal diseases. Therefore, the search for novel anti-inflammatory pharmacologic targets is of great interest in renal pathology. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) proteins, was previously found to preserve renal function in experimental polycystic kidney disease. We report here that JQ1-induced BET inhibition modulated the in vitro expression of genes involved in several biologic processes, including inflammation and immune responses. Gene silencing of BRD4, an important BET protein, and chromatin immunoprecipitation assays showed that JQ1 alters the direct association of BRD4 with acetylated histone-packaged promoters and reduces the transcription of proinflammatory genes (IL-6, CCL-2, and CCL-5). In vivo, JQ1 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, antimembrane basal GN, and infusion of Angiotensin II. Notably, JQ1 downregulated the expression of several genes controlled by the NF-κB pathway, a key inflammatory signaling pathway. The RelA NF-κB subunit is activated by acetylation of lysine 310. In damaged kidneys and cytokine-stimulated renal cells, JQ1 reduced the nuclear levels of RelA NF-κB. Additionally, JQ1 dampened the activation of the Th17 immune response in experimental renal damage. Our results show that inhibition of BET proteins reduces renal inflammation by several mechanisms: chromatin remodeling in promoter regions of specific genes, blockade of NF-κB pathway activation, and modulation of the Th17 immune response. These results suggest that inhibitors of BET proteins could have important therapeutic applications in inflammatory renal diseases.

Regulation of miR-29b and miR-30c by vitamin D receptor activators contributes to attenuate uraemia-induced cardiac fibrosis.

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.

IL-17A is a novel player in dialysis-induced peritoneal damage.

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.

A new procedure to induce aortic aneurysms in mice.

Aortic aneurysms (AAs) are a major public health challenge, featured by a progressive impairs in aortic wall integrity that drives to aortic dilation and, in end stage, to its rupture. Despite important advances in the surgical treatment of aortic aneurysms, there is currently no pharmacological intervention that prevents their development, reduces their expansion, or avoids their rupture. In addition to classic risk factors such age or gender, several heritable connective tissue disorders have been associated with AA developing, highlighting the role of extracellular matrix (ECM) genes alterations in the developing of AA. In this sense, we have recently demonstrated that global deletion of the cellular communicating network factor 2 (CCN2), previously known as connective tissue growth factor (CTGF) due to its role in the extracellular matrix formation, predisposes to early and lethal AAs development after Angiotensin II (Ang II) infusion in mice. Here, we detail the protocol to induce and detect AAs generation in inducible global CCN2 knockout mice after Ang II infusion which allow the characterization of CCN role in AA development and may help to the development of pharmacological target for AA treatment.

BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney-an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery.