Role of αv Integrins in TGF-β–Mediated Renal Fibrosis

CKD is a significant public health concern affecting about 14% of adults globally, and its prevalence has been increasing over the past 30 years. In the United States alone, it is estimated that one in seven adults, about 35.5 million people, have CKD (data from US Centers for Disease Control and Prevention). Regardless of the initial cause of CKD, renal fibrosis is a final common pathway of tissue injury, resulting in irreversible loss of kidney function. Renal fibrosis is a complex process involving fibroblast activation, recruitment of inflammatory cells, and epithelia-to-mesenchymal transition (EMT), leading to excessive deposition of extracellular matrix (ECM) and widespread tissue scarring.1 Fibroblasts are key effectors of fibrosis, and TGF-β is crucial in fibroblast activation.2 This is supported by data from murine models where transgenic overexpression of TGF-β causes extensive lung fibrosis.3 Furthermore, TGF-β1 blocking antibodies prevent fibrosis in skin, liver, lung, and kidneys.4 Single-cell sequencing has identified a subset of fibroblasts expressing signature genes encoding components of pathologic ECM that are upregulated by TGF-β.5,6 TGF-β also contributes to increased ECM stiffness, further facilitating fibroblast activation through mechanotransduction pathways.1 As such, TGF-β induces fibroblast proliferation, recruitment, and transformation into myofibroblasts that, in turn, produce ECM proteins such as collagens and fibronectin. In addition to mediating fibroblast activation, TGF-β contributes to organ fibrosis in many other ways. It inhibits proteases that degrade ECM, such as matrix metalloproteinases, while promoting inhibitors such as tissue inhibitor of matrix metalloproteinase 3 and plasminogen activator inhibitor 1 (also known as serpin E1). The end result is accumulation of excessive ECM. TGF-β also stimulates the expression of fibrogenic factors in epithelial cells and perpetuates excessive cell senescence and a persistent senescence-associated secretory phenotype, creating an inflammatory microenvironment and eventual progression to tissue fibrosis.2 Finally, TGF-β has been well established as the main trigger of EMT, an important process during tissue fibrosis where renal tubular epithelial cells lose epithelial characteristics such as polarity and specialized cell-cell junction and acquire migratory behavior and features of fibroblasts. The TGF-β family of proteins consist of three isoforms, TGF-β1, TGF-β2, and TGF-β3 (together referred to as TGF-β), that are widely expressed by many cell types. All three isoforms are produced as a disulfide-linked dimeric precursor, which is cleaved by the endoprotease furin in the Golgi to form the latency-associated peptide (LAP) in the N-terminal portion and mature TGF-β in the C-terminal portion. However, on secretion into the extracellular space, the mature TGF-β remains in a latent form that is noncovalently associated with LAP. The latent TGF-β/LAP complex binds to latent TGF-β binding proteins and is tethered to the ECM. Activation of latent TGF-β relies heavily on αv integrins. Binding of LAP to αv integrins on adjacent cells, namely, αvβ1, αvβ6, and αvβ8, leads to a conformational change of LAP that releases the captive TGF-β to allow its binding to receptors. TGF-β binding to its receptors in fibroblasts triggers downstream SMAD (comprise a family of structurally similar proteins that are the main signal transducers for receptors of the TGF-β superfamily) signaling pathways that upregulate the transcription of an array of profibrotic genes including collagen α1 and α2.2 The net effect is increased accumulation and decreased degradation of fibrillar collagen. In addition, TGF-β interacts with other signaling pathways, such as Wnt-related integration site–β-catenin, growth factors such as connective tissue growth factor, and bone morphogenetic proteins to amplify fibrotic responses, regulate tissue plasticity, and facilitate EMT. As mentioned earlier, αv integrins are important activators of latent TGF-β. They primarily bind to the arginine-glycine-aspartic acid which is a peptide sequence recognized by LAP (arginine-glycine-aspartic acid) sequence of LAPs, causing a conformational change that facilitates the release of active TGF-β. Integrins are transmembrane receptors with a α and a β subunit. There are five αv integrins, αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8. Studies using a platelet derived growth factor receptorβ promoter–driven Cre system to delete αv integrins in activated fibroblasts identified these integrins as core regulators of fibroblast activity across solid organs including the kidneys.7 The profibrotic effect of αv integrins is most likely contributed by αvβ1, αvβ6, and αvβ8 integrins because global deletion of αvβ3 or αvβ5 did not protect mice against liver fibrosis.7αvβ1 integrin is the main αv integrin expressed in the activated fibroblasts. Small-molecule inhibitors against αvβ1 successfully decreased TGF-β activation and attenuated bleomycin-induced lung fibrosis, carbon tetrachloride–induced liver fibrosis, unilateral ureteral obstruction operation–induced renal fibrosis, and bleomycin-induced renal injury in mouse models.8,9αvβ6 expression is restricted to the epithelial cells, and its expression increases significantly during tissue injury. Evidence supports a feedforward circuit between TGF-β and αvβ6 where TGF-β amplifies its signal by enhancing αvβ6 expression in the epithelium that, in turn, binds to latent TGF-β to release active TGF-β for its action. Integrin β6 knockout mice develop exaggerated inflammation in the lungs and skin but are protected from fibrosis in multiple organs including the kidneys.2 Integrin αvβ8 is expressed widely in neuroepithelial cells, astrocytes, myeloid cells, T cells, epithelial cells, and fibroblasts. Together with αvβ6, it is crucial for TGF-β–mediated embryonic development and immune homeostasis.2 However, the role of αvβ8 in organ fibrosis remains unclear. αvβ8 expression has been identified in fibroblasts of the lung and liver but not yet in renal fibroblasts.8 In addition, fibroblast-specific deletion of αvβ8 (mice with global β8 deletion die in utero) did not protect mice against liver fibrosis.7 In this issue, Marin et al. explored the role of αvβ8 integrin in renal fibrosis and its potential as a therapeutic target in CKD.10 Using human CKD datasets (European Renal cDNA Bank CKD data set and Clinical Phenotyping Resource and Biobank Core), they demonstrated that αvβ8 integrin is upregulated primarily in the tubulointerstitium of CKD kidneys, particularly in diabetic kidney disease (DKD), and is associated with TGF-β activation and kidney function decline. Interstitial Itgb8 gene expression correlates inversely with eGFR and positively with TGF-β activation score (derived from analysis of 30 TGF-β downstream genes). These suggest involvement of αvβ8 integrin in TGF-β–mediated renal fibrosis. Next, they examined the efficacy of monoclonal anti-αvβ8 antibody MEDI8367. It appears to specifically bind to human (not mouse) integrin β8 in vitro, reducing its affinity for LAP, thus preventing β8-mediated TGF-β activation, without affecting its cell adhesion function. MEDI8367 was then tested in ureteral obstruction operation (a severe fibrosis model) where MEDI8367 inhibited TGF-β–mediated renal fibrosis as evidenced by decreased activation of TGF-β and its downstream phospho-SMAD2/3 and/or collagen deposition. The effects are similar to mAb against TGF-β. It also decreased albuminuria in five/six nephrectomy model, a surrogate for renal function decline. The authors also tested the αvβ8 integrin blockade in the db/db uninephrectomized meaning one kidney is surgically removed DKD model. Because MEDI8367 was specific to human β8 integrin, murine antibodies 3G9 and RAD2640 were used. Both antibodies target murine αvβ6 integrin, but RAD2640 also has cross-reactivity with murine αvβ8. RAD2640, but not 3G9, reduced albuminuria in db/db uninephrectomized meaning one kidney is surgically removed DKD mice. This suggests that αvβ8, either alone or together with αvβ6, is important for development of kidney injury in diabetic kidney mouse models. However, the degree of kidney fibrosis was not quantified in the DKD mouse model. Overall, Marin et al. demonstrated that αvβ8 integrin is involved in TGF-β–mediated renal fibrosis supported by data from both human CKD kidney tissues and mouse models of renal fibrosis. Single-cell RNA sequencing data from the study using a CKD cohort showed Itgb8 expression in various kidney cell types, with the highest expression in the renal tubular epithelium. However, whether renal fibroblasts express β8 remains unclear because it was not assessed. It also remains to be elucidated if αvβ8 exerts its effect primarily from its expression in the epithelial cells or from various renal cell types. Future research on cell-type–specific expression and activity of Itgb8 during renal fibrosis is necessary. Moreover, αvβ8 signaling likely varies among different types of CKD including DKD, hypertensive nephrosclerosis, and glomerular diseases. A comprehensive study using disease-specific preclinical models is required to better understand the specific αvβ8 signaling pathway and help generate meaningful therapeutic targets. Finally, data from mouse models appear to support monoclonal anti-αvβ8 antibody MEDI8367 as a promising treatment for renal fibrosis. Additional study of its effect on αvβ8 activity through TGF-β–dependent and non-TGF-β–dependent pathways will help better characterize its mechanism of action and aid in future clinical trial design (phase 1 clinical trial of MEDI8367 in healthy participants and those with CKD was conducted with 12 participants enrolled). Currently, there are at least two drugs targeting TGF-β–activating integrins in clinical trials for fibrotic diseases: a dual small-molecule inhibitor of αvβ1 and αvβ6 in phase 2 clinical trials for treatment of pulmonary fibrosis and primary sclerosing cholangitis and a small-molecule inhibitor of αvβ1 in a phase 1 trial for nonalcoholic liver fibrosis.2 Currently, no effective agent exists that can directly halt renal fibrosis in CKD. Development of therapies against renal fibrosis is much needed.