Cardiotrophin-1 therapy reduces disease severity in a murine model of glomerular disease.

Cardiotrophin-1 (CT-1), a member of the interleukin (IL)-6 cytokine family, has renoprotective effects in mouse models of acute kidney disease and tubulointerstitial fibrosis, but its role in glomerular disease is unknown. To address this, we used the mouse model of nephrotoxic nephritis to test the hypothesis that CT-1 also has a protective role in immune-mediated glomerular disease. Using immunohistochemistry and analysis of single-cell RNA-sequencing data of isolated glomeruli, we demonstrate that CT-1 is expressed in the glomerulus in male mice, predominantly in parietal epithelial cells and is downregulated in mice with nephrotoxic nephritis. Furthermore, analysis of data from patients revealed that human glomerular disease is also associated with reduced glomerular CT-1 transcript levels. In male mice with nephrotoxic nephritis and established proteinuria, administration of CT-1 resulted in reduced albuminuria, prevented podocyte loss, and sustained plasma creatinine, compared with mice administered saline. CT-1 treatment also reduced fibrosis in the kidney cortex, peri-glomerular macrophage accumulation and the kidney levels of the pro-inflammatory mediator complement component 5a. In conclusion, CT-1 intervention therapy delays the progression of glomerular disease in mice by preserving kidney function and inhibiting renal inflammation and fibrosis.

Association of Alk1 and Endoglin Polymorphisms with Cardiovascular Damage.

Cardiovascular diseases are associated to risk factors as obesity, hypertension and diabetes. The transforming growth factor-ß1 receptors ALK1 and endoglin regulate blood pressure and vascular homeostasis. However, no studies relate the association of ALK1 and endoglin polymorphisms with cardiovascular risk factors. We analysed the predictive value of the ALK1 and endoglin polymorphisms on cardiovascular target organ damage in hypertensive and diabetic patients in 379 subjects with or without hypertension and diabetes in a Primary Care setting. The ALK1 rs2071219 polymorphism (AA genotype) is associated with a lower presence of diabetic retinopathy and with the absence of altered basal glycaemia. Being carrier of the ALK1 rs3847859 polymorphism (G allele) is associated with lower basal heart rate and with higher LDL-cholesterol levels. The endoglin rs3739817 polymorphism (AA genotype) is associated with higher levels of LDL-cholesterol, and being carrier of the endoglin rs10987759 polymorphism (C allele) is associated with higher haemoglobin levels and with an increased heart rate. Summarizing, several ALK1 and endoglin gene polymorphisms increase the risk of cardiovascular events. The analysis of these polymorphisms in populations at risk, in combination with the determination of other parameters and biomarkers, could implement the diagnosis and prognosis of susceptibility to cardiovascular damage.

Endothelial-epithelial communication in polycystic kidney disease: Role of vascular endothelial growth factor signalling.

Whereas targeting the cyst epithelium and its molecular machinery has been the prevailing clinical strategy for polycystic kidney disease, the endothelium, including blood vasculature and lymphatics, is emerging as an important player in this disorder. In this Review, we provide an overview of the structural and functional alterations to blood vasculature and lymphatic vessels in the polycystic kidney. We also discuss evidence for vascular endothelial growth factor signalling, otherwise critical for endothelial cell development and maintenance, as being a fundamental molecular pathway in polycystic kidney disease and a potential therapeutic target for modulating cyst expansion.

Generating Mutant Renal Cell Lines Using CRISPR Technologies.

Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles ("indels") at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.

Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease.

Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis; a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease.

Cardiotrophin-1 opposes renal fibrosis in mice: Potential prevention of chronic kidney disease.

AIM: Chronic kidney disease is characterized by tubulointerstitial fibrosis involving inflammation, tubular apoptosis, fibroblast proliferation and extracellular matrix accumulation. Cardiotrophin-1, a member of the interleukin-6 family of cytokines, protects several organs from damage by promoting survival and anti-inflammatory effects. However, whether cardiotrophin-1 participates in the response to chronic kidney injury leading to renal fibrosis is unknown. METHODS: We hypothesized and assessed the potential role of cardiotrophin-1 in a mice model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO). RESULTS: Three days after UUO, obstructed kidneys from cardiotrophin-1-/- mice show higher expression of inflammatory markers IL-1ß, Cd68, ICAM-1, COX-2 and iNOs, higher activation of NF-κB, higher amount of myofibroblasts and higher severity of tubular damage and apoptosis, compared with obstructed kidneys from wild-type littermates. In a later stage, obstructed kidneys from cardiotrophin-1-/- mice show higher fibrosis than obstructed kidneys from wild-type mice. Interestingly, administration of exogenous cardiotrophin-1 prevents the increased fibrosis resulting from the genetic knockout of cardiotrophin-1 upon UUO, and supplementation of wild-type mice with exogenous cardiotrophin-1 further reduces the renal fibrosis induced by UUO. In vitro, renal myofibroblasts from cardiotrophin-1-/- mice have higher collagen I and fibronectin expression and higher NF-κB activation than wild-type cells. CONCLUSIONS: Cardiotrophin-1 participates in the endogenous response that opposes renal damage by counteracting the inflammatory, apoptotic and fibrotic processes. And exogenous cardiotrophin-1 is proposed as a candidate for the treatment and prevention of chronic renal fibrosis.

Corrigendum: Association of VAV2 and VAV3 polymorphisms with cardiovascular risk factors.

This corrects the article DOI: 10.1038/srep41875.

Association of VAV2 and VAV3 polymorphisms with cardiovascular risk factors.

Hypertension, diabetes and obesity are cardiovascular risk factors closely associated to the development of renal and cardiovascular target organ damage. VAV2 and VAV3, members of the VAV family proto-oncogenes, are guanosine nucleotide exchange factors for the Rho and Rac GTPase family, which is related with cardiovascular homeostasis. We have analyzed the relationship between the presence of VAV2 rs602990 and VAV3 rs7528153 polymorphisms with cardiovascular risk factors and target organ damage (heart, vessels and kidney) in 411 subjects. Our results show that being carrier of the T allele in VAV2 rs602990 polymorphism is associated with an increased risk of obesity, reduced levels of ankle-brachial index and diastolic blood pressure and reduced retinal artery caliber. In addition, being carrier of T allele is associated with increased risk of target organ damage in males. On the other hand, being carrier of the T allele in VAV3 rs7528153 polymorphism is associated with a decreased susceptibility of developing a pathologic state composed by the presence of hypertension, diabetes, obesity or cardiovascular damage, and with an increased risk of developing altered basal glycaemia. This is the first report showing an association between VAV2 and VAV3 polymorphisms with cardiovascular risk factors and target organ damage.

Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-ß in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-ß1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors.

Relationship between target organ damage and blood pressure, retinal vessel calibre, oxidative stress and polymorphisms in VAV-2 and VAV-3 genes in patients with hypertension: a case-control study protocol (LOD-Hipertension).

INTRODUCTION: Target organ damage (TOD) is associated with increased cardiovascular risk. The study objectives were to analyse the relationship of TOD to blood pressure, size of retinal arteries and veins, oxidative stress and different polymorphisms in the VAV-2 and VAV-3 genes in participants with hypertension. METHODS AND ANALYSIS: A case-control study to analyse the relationship between clinical, biochemical and genetic parameters and presence of cardiac, vascular and renal TOD in 486 patients with hypertension. Participants with TOD will be considered as cases, and those without TOD will be enrolled as controls. This will be a collaborative study conducted by the groups of Primary Care, Cardiovascular and Metabolic and Degenerative Diseases of the Instituto de Investigación Biomédica of Salamanca (IBSAL). Assessment of cardiac, renal and vascular TOD. Measurement of peripheral and central blood pressure, size of eye fundus arteries and veins, and oxidative stress, and polymorphisms in the VAV-2 and VAV-3 genes. ETHICS AND DISSEMINATION: The study will be conducted after approval is obtained from the Ethics Committee of Hospital Clínico Universitario of Salamanca. All study participants will sign an informed consent to agree to participate in the study, and another consent to agree on the genetic study, in compliance with the Declaration of Helsinki and the WHO standards for observational studies. The results of this study will allow for an understanding of the relationship of the different TODs with blood pressure, retinal artery and vein diameters, oxidative stress and polymorphisms in VAV-2 and VAV-3 genes. TRIAL REGISTRATION NUMBER: Clinical Trials. gov Identifier: NCT02022618.

ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-ß1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-ß1 with an important role in angiogenesis whose function in cellular biology and TGF-ß signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-ß1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-ß/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.