José M. Muñoz-Félix

TGF-β/BMP proteins as therapeutic targets in renal fibrosis. Where have we arrived after 25 years of trials and tribulations?

The understanding of renal fibrosis in chronic kidney disease (CKD) remains as a challenge. More than 10% of the population of developed countries suffer from CKD. Proliferation and activation of myofibroblasts and accumulation of extracellular matrix proteins are the main features of kidney fibrosis, a process in which a large number of cytokines are involved. Targeting cytokines responsible for kidney fibrosis development might be an important strategy to face the problem of CKD. The increasing knowledge of the signaling pathway network of the transforming growth factor beta (TGF-β) superfamily members, such as the profibrotic cytokine TGF-β1 or the bone morphogenetic proteins (BMPs), and their involvement in the regulation of kidney fibrosis, has stimulated numerous research teams to look for potential strategies to inhibit profibrotic cytokines or to enhance the anti-fibrotic actions of other cytokines. The consequence of all these studies is a better understanding of all these canonical (Smad-mediated) and non-canonical signaling pathways. In addition, the different receptors involved for signaling of each cytokine, the different combinations of type I-type II receptors, and the presence and function of co-receptors that can influence the biological response have been also described. However, are these studies leading to suitable strategies to block the appearance and progression of kidney fibrosis? In this review, we offer a critical perspective analyzing the achievements using the most important strategies developed up till now: TGF-β antibodies, chemical inhibitors of TGF-β receptors, miRNAs and signaling pathways and BMP agonists with a potential role as therapeutic molecules against kidney fibrosis.

The ALK-1/Smad1 pathway in cardiovascular physiopathology. A new target for therapy?

Activin receptor-like kinase-1 or ALK-1 is a type I cell surface receptor for the transforming growth factor-β (TGF-β) family of proteins. The role of ALK-1 in endothelial cells biology and in angiogenesis has been thoroughly studied by many authors. However, it has been recently suggested a possible role of ALK-1 in cardiovascular homeostasis. ALK-1 is not only expressed in endothelial cells but also in smooth muscle cells, myofibroblast, hepatic stellate cells, chondrocytes, monocytes, myoblasts, macrophages or fibroblasts, but its role in these cells have not been deeply analyzed. Due to the function of ALK-1 in these cells, this receptor plays a role in several cardiovascular diseases. Animals with ALK-1 haploinsufficiency and patients with mutations in Acvrl1 (the gene that codifies for ALK-1) develop type-2 Hereditary Hemorrhagic Telangiectasia. Moreover, ALK-1 heterozygous mice develop pulmonary hypertension. Higher levels of ALK-1 have been observed in atherosclerotic plaques, suggesting a possible protector role of this receptor. ALK-1 deficiency is also related to the development of arteriovenous malformations (AVMs). Besides, due to the ability of ALK-1 to regulate cell proliferation and migration, and to modulate extracellular matrix (ECM) protein expression in several cell types, ALK-1 has been now demonstrated to play an important role in cardiovascular remodeling. In this review, we would like to offer a complete vision of the role of ALK-1 in many process related to cardiovascular homeostasis, and the involvement of this protein in the development of cardiovascular diseases, suggesting the possibility of using the ALK-1/smad-1 pathway as a powerful therapeutic target.

ALK1-Smad1/5 signaling pathway in fibrosis development: Friend or foe?

Fibrosis is a common phenomenon associated with several pathologies, characterized by an excessive extracellular matrix deposition that leads to a progressive organ dysfunction. Thus fibrosis has a relevant role in chronic diseases affecting the kidney, the liver, lung, skin (scleroderma) and joints (arthritis), among others. The pathogenesis of fibrosis in different organs share numerous similarities, being one of them the presence of activated fibroblasts, denominated myofibroblast, which act as the main source of extracellular matrix proteins. Transforming growth factor beta-1 (TGF-β1) is a profibrotic cytokine that plays a pivotal role in fibrosis. The TGF-β1/ALK5/Smad3 signaling pathway has been studied in fibrosis extensively. However, an increasing number of studies involving the ALK1/Smad1 pathway in the fibrotic process exist. In this review we offer a perspective of the function of ALK1/Smad1 pathway in renal fibrosis, liver fibrosis, scleroderma and osteoarthritis, suggesting this pathway as a powerful therapeutical target. We also propose several strategies to modulate the activity of this pathway and its consequences in the fibrotic process.

Heterozygous disruption of activin receptor-like kinase 1 is associated with increased renal fibrosis in a mouse model of obstructive nephropathy.

Tubulointerstitial fibrosis is characterized by an accumulation of extracellular matrix in the renal interstitium, myofibroblast activation, cell infiltration, and tubular cell apoptosis, leading to chronic renal failure. Activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β1 type I receptor with a pivotal role in endothelial proliferation and migration, but its role in the development of renal fibrosis is unknown. To assess this we used the unilateral ureteral obstruction model of tubulointerstitial fibrosis in ALK1 haploinsufficient (ALK1(+/-)) and wild-type mice. After 15 days, there was an increase in extracellular matrix protein expression in the obstructed kidneys from both ALK1(+/+) and ALK1(+/-) mice, but obstructed kidneys from ALK1(+/-) mice showed significantly higher expression of type I collagen than those from wild-type mice. Ureteral obstruction increased kidney myofibroblasts markers (α-smooth muscle actin and S100A4), without differences between mouse genotypes. ALK1 expression was increased after ureteral obstruction, and this increased expression was located in myofibroblasts. Moreover, cultured renal fibroblasts from ALK1(+/-) mice expressed more collagen type I and fibronectin than fibroblasts derived from wild-type mice. Thus, ALK1 modulates obstruction-induced renal fibrosis by increased extracellular matrix synthesis in myofibroblasts, but without differences in myofibroblast number.

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-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β 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-β1 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-β/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.

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-β 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-β1 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.

L-Endoglin Overexpression Increases Renal Fibrosis after Unilateral Ureteral Obstruction

Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG+) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG+ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG+ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG+ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG+ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG+ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development.

Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

The involvement of Ras‐GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H‐ and N‐Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K‐Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K‐Ras knockout (KO) mouse embryonic fibroblasts (K‐ras−/−) stimulated with transforming growth factor‐β1 (TGF‐β1) exhibited reduced proliferation and impaired mobility than wild‐type fibroblasts. Moreover, an increase on ECM production was observed in K‐Ras KO fibroblasts in basal conditions. The absence of K‐Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF‐β1‐induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K‐ras but reduced migration and ECM proteins expression only in wild‐type fibroblasts, while the PI3K‐AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K‐Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K‐Ras may be a crucial mediator in TGF‐β1‐mediated effects in this cell type. J. Cell. Physiol. 231: 2224–2235, 2016.

Overexpression of the short endoglin isoform reduces renal fibrosis and inflammation after unilateral ureteral obstruction.

Transforming growth factor beta 1 (TGF-β1) is one of the most studied cytokines involved in renal tubulo-interstitial fibrosis, which is characterized by myofibroblast abundance and proliferation, and high buildup of extracellular matrix in the tubular interstitium leading to organ failure. Endoglin (Eng) is a 180-kDa homodimeric transmembrane protein that regulates a great number of TGF-β1 actions in different biological processes, including ECM synthesis. High levels of Eng have been observed in experimental models of renal fibrosis or in biopsies from patients with chronic kidney disease. In humans and mice, two Eng isoforms are generated by alternative splicing, L-Eng and S-Eng that differ in the length and composition of their cytoplasmic domains. We have previously described that L-Eng overexpression promotes renal fibrosis after unilateral ureteral obstruction (UUO). However, the role of S-Eng in renal fibrosis is unknown and its study would let us analyze the possible function of the cytoplasmic domain of Eng in this process. For this purpose, we have generated a mice strain that overexpresses S-Eng (S-ENG(+)) and we have performed an UUO in S-ENG(+) and their wild type (WT) control mice. Our results indicate that obstructed kidney of S-ENG(+) mice shows lower levels of tubulo-interstitial fibrosis, less inflammation and less interstitial cell proliferation than WT littermates. Moreover, S-ENG(+) mice show less activation of Smad1 and Smad2/3 pathways. Thus, S-Eng overexpression reduces UUO-induced renal fibrosis and some associated mechanisms. As L-Eng overexpression provokes renal fibrosis we conclude that Eng-mediated induction of renal fibrosis in this model is dependent on its cytoplasmic domain.

Functional specific roles of H‐ras and N‐ras. A proteomic approach using knockout cell lines

Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H‐, N‐ and K‐Ras. To improve the understanding of H‐ and N‐Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H‐ras and/or N‐ras, using proteomics tools combining 2DE, semi‐quantitative image analysis, in‐gel trypsin digestion and mass spectrometry. There are four up‐regulated proteins due to the loss of expression of H‐Ras (including cyclin‐dependent kinase inhibitor 2A) and eight down‐regulated (including stress‐70 protein, dihydropyrimidinase‐related‐protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP‐dissociation inhibitor 1) and six up‐regulated proteins (e.g. leukocyte elastase inhibitor A, L‐lactate dehydrogenase B chain, c‐Myc‐responsive protein Rcl, interleukin‐1 receptor antagonist protein) due to the loss of expression of both N‐ and H‐Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H‐ and N‐Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets.