Kidney Fibrosis: Fundamental Questions, Challenges, and Perspectives
Youhua Liu
Abstract
Kidney fibrosis is characterized by excessive deposition of extracellular matrix (ECM) leading to tissue scarring in renal parenchyma and represents the common outcome of a wide variety of chronic kidney disease (CKD).[1] As CKD is associated with high morbidity and mortality, it imposes an enormous healthcare burden worldwide. CKD is typically progressive, with detrimental impacts on kidney structure and function that lead to the development of end-stage renal disease, which requires dialysis or kidney transplantation for the survival of afflicted patients.[2] Studies have shown that a diverse array of cell types and signaling pathways orchestrate the fibrotic process in CKD, which greatly complicates the identification of relevant biomarkers and druggable therapeutic targets.[3,4] As a result, there is still no specific treatment available in clinical practice for kidney fibrosis. There is a need for a better understanding of the mechanisms underlying kidney fibrosis to enable the development of effective therapeutic strategies to prevent or reverse the onset and progression of kidney fibrosis and improve the long-term outcomes of patients with CKD. Towards this end, three fundamental questions relating to kidney fibrosis need to be addressed, as outlined below. HOW DOES KIDNEY FIBROSIS INITIATE? Fibrosis after organ injury is an evolutionarily conserved response, which, at least initially, is reparative and protective. We previously showed that fibroblast activation after ischemic-reperfusion injury was an extremely early event, occurring as early as 1 h post-injury and long before the occurrence of other major cellular events, such as renal infiltration of inflammatory cells and tubular cell death.[5] This early wave of fibroblast activation was characterized by the expression of vimentin but not α-smooth muscle actin. Fibroblast proliferation after organ injury is protective, as blockade of such proliferation would exacerbate kidney damage and inhibit tubular repair and regeneration. Activated fibroblasts promote wound healing via numerous mechanisms, including encircling the injurious site, restoring tissue integrity, and secreting pro-regenerative factors, such as hepatocyte growth factor. However, perfect wound healing is not always possible, especially when the injury is severe or recurring. In these circumstances, activated fibroblasts do not resolve by apoptosis or returning to quiescent state. Instead, they become α-smooth muscle actin-positive, contractile myofibroblasts, and produce a large amount of ECM proteins, resulting in the formation of tissue fibrosis or scars. Kidney fibrosis is usually not homogeneous across renal parenchyma. Rather, it occurs at particular focal sites, suggesting that the local tissue microenvironment may play a role in renal fibrogenesis. We recently proposed the concept of the fibrogenic niche, a hypothesis put forward to describe the specialized microenvironment that promotes the activation of fibroblasts in discrete locations.[6] Proteomic profiling has revealed that the backbone of the fibrogenic niche is composed of a specialized ECM network, consisting of structurally unrelated matricellular proteins, including tenascin-C, fibrillin-1, and vitronectin, to name a few.[7] As a functional unit, the fibrogenic niche spontaneously induces or facilitates fibroblast activation and proliferation, tubular cell phenotypic changes, macrophage activation, and endothelial cell apoptosis. In essence, the fibrogenic niche induces pathological features that recapitulate key events in the pathogenesis of CKD. How the fibrogenic niche promotes kidney fibrosis is an intriguing question. Several potential mechanisms may account for its actions in fibrogenesis.[6] Matricellular proteins, such as tenascin-C, are the major components of the fibrogenic niche.[8] Many matricellular proteins can recruit various soluble factors, including Wnt, hedgehog ligands, and transforming growth factor-β (TGF-β), from the extracellular milieu, thereby creating a microenvironment with high local levels of profibrotic factors.[9] In addition, matricellular proteins elicit their cellular activities via receptor-mediated outside-in signaling, as they can bind to and activate a range of cell membrane receptors, such as integrins, Toll-like receptor 4, and low-density lipoprotein receptor-related protein 6.[6,10] Importantly, matricellular proteins typically bind to the ECM and become part of the ECM network, and they are insoluble and non-diffusional. These characteristic features of matricellular proteins suggest that they can operate in a spatially confined manner, a scenario consistent with the concept of the fibrogenic niche.[11] The fibrogenic niche hypothesis potentially provides a rational explanation for how kidney fibrosis develops in a particular locality and then spreads to form fibrotic foci. This hypothesis represents a paradigm shift in our thinking of the patho-mechanisms underlying the onset and progression of kidney fibrosis. Future studies are needed to dissect the molecular makeup of the fibrogenic niche and its mechanism of action. It is conceivable that disrupting the formation of the fibrogenic niche could be an effective therapeutic strategy for fibrotic CKD. WHAT ARE THE KEY REGULATORS CONTROLLING KIDNEY FIBROSIS? Another fundamental question in the field of kidney fibrosis is the identity of the key regulators that control its onset and progression. Over the past several decades, much effort has focused on discovering these key regulators to enable the design of specific strategies to target them and the relevant pathways, thereby halting or reversing renal fibrogenesis.[1,3,4] Among the many factors studied, TGF-β is undoubtedly one of the most well investigated and potent driver of kidney fibrosis.[12] TGF-β is a multifunctional cytokine that regulates various cellular processes, including cell proliferation, differentiation, senescence, and ECM synthesis.[12] It is upregulated in different cell types in kidney disease and stimulates the activation and expansion of fibroblasts and myofibroblasts. In addition, TGF-β promotes the epithelial-to-mesenchymal transition, tubular cell cycle arrest and senescence, and immune cell recruitment, further contributing to the fibrotic process. Thus, TGF-β is an attractive target for therapeutic interventions in CKD, and countless therapeutic approaches targeting TGF-β signaling in kidney fibrosis have been explored, including TGF-β receptor inhibitors, anti-TGF-β antibodies, Smad pathway modulators, and target-specific gene therapies.[12] These approaches have been used to inhibit the profibrotic actions of TGF-β and attenuate kidney fibrosis in various animal models of CKD. Apart from TGF-β, the roles of other profibrotic factors, such as fibroblast growth factor-2, platelet-derived growth factor, and epidermal growth factor receptor (EGFR) ligands, in kidney fibrogenesis have been investigated. Not surprisingly, various intracellular signaling cascades, including phosphatidylinositol 3ʹ-kinase/Akt kinase, Janus kinase/signal transduction and transcription activation, and the Src family of non-receptor tyrosine kinase and mitogen-activated protein kinase pathways, triggered by these factors or other cytokines are involved, either directly or indirectly, in the pathogenesis of kidney fibrosis.[13,14] Several developmental signaling pathways, including Wnt/β-catenin, hedgehog, and Notch, are also implicated in kidney fibrosis after organ injury.[15,16] Wnt proteins are a family of secreted glycoproteins that regulate numerous cellular processes such as cell proliferation, differentiation, tissue patterning, and organ development. Canonical Wnt signaling involves binding of Wnt ligands to cell surface receptors, resulting in stabilization and nuclear translocation of β-catenin.[14,17] Once in the nucleus, β-catenin interacts with transcription factors of the T-cell factor/Lymphoid enhancer factor family, leading to transcriptional activation of fibrosis-related genes, such as fibronectin, Snail1, matrix metalloproteinase-7, plasminogen activator inhibitor-1, and all components of the renin-angiotensin-aldosterone system (RAAS).[17,18] In fibrotic CKD, dysregulation of Wnt signaling contributes to fibroblast activation, excessive production of ECM components, tubular epithelial-to-mesenchymal transition, cellular senescence, activation and polarization of macrophages, and disruption of the normal renal architecture.[18,19] Wnt/β-catenin activation occurs via different regulatory mechanisms, including increased expression of Wnt ligands, alterations in the levels or activity of Wnt receptors, modification of N6-methyladenosine-mediated stabilization of β-catenin mRNA,[20] or EGFR-mediated transactivation of β-catenin protein.[21] Targeting Wnt signaling in different regulatory levels has emerged as a potential therapeutic strategy for kidney fibrosis.[17] Potential interventional approaches include blocking Wnt ligand-receptor interactions, inhibiting β-catenin stabilization, or interfering with the transcriptional activity of β-catenin/T-cell factor complexes. Further investigations and clinical trials are urgently needed to evaluate the safety and efficacy of these interventions in patients with CKD. Angiotensin II (Ang II), the main effector of the RAAS, is strongly implicated in the development and progression of kidney fibrosis.[22,23] Ang II contributes to kidney fibrosis through multiple mechanisms. It activates resident fibroblasts within the kidneys, causing them to transform into matrix-producing myofibroblasts. Furthermore, Ang II stimulates the production of various profibrotic factors, such as TGF-β and platelet-derived growth factors. These factors promote the proliferation and activation of fibroblasts, further driving the fibrotic process. In addition, Ang II promotes renal inflammation by stimulating the release of inflammatory cytokines and chemokines. Ang II also induces vasoconstriction of renal blood vessels, reducing blood flow and oxygen delivery to the kidneys. This leads to tissue hypoxia, which triggers the release of hypoxia-inducible factors and generates reactive oxygen species within the kidneys, leading to oxidative stress. Oxidative stress, in turn, promotes inflammation, fibroblast activation, and the production of ECM proteins, contributing to kidney fibrosis.[23] In the vast majority of CKD cases, multiple profibrotic signaling pathways are activated simultaneously. Importantly, many of these signaling pathways stimulate each other and engage in crosstalk. Together, they constitute a core signaling network in renal fibrogenesis. As shown in Figure 1, TGF-β induces the expression of many Wnt ligands, and the activation of Wnt/β-catenin promotes the induction of components of the RAAS,[22]leading to an increase in Ang II generation. Ang II is known to induce TGF-β expression. As such, TGF-β, Wnt and Ang II establish a vicious feed-forward cycle, leading to profound amplification of profibrotic signaling. We propose that this TGF-β/Wnt/Ang II (TWA) cycle is the core signaling network that drives kidney fibrosis in CKD [Figure 1].Figure 1.: Core signal network that drives kidney fibrosis. TWA, transforming growth factor-β (TGF-β)/Wnt/angiotensin II; Shh, sonic hedgehog; HGF, hepatocyte growth factor. CAN KIDNEY FIBROSIS BE STOPPED OR REVERSED? The third fundamental question is whether and how kidney fibrosis can be halted or even reversed. Much research has focused on developing treatments to prevent or slow the progression of kidney fibrosis.[3,14] This is an important goal, and there has been some limited success, with several approaches now used in the treatment of patients with fibrotic CKD.[3] Due to its profibrotic effects, targeting Ang II, a crucial element of the TWA cycle [Figure 1], has become the mainstay of treatments for kidney fibrosis. Agents that block the effects of Ang II, such as angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, have been shown to reduce kidney fibrosis and slow the progression of CKD.[24] These agents attenuate inflammation, decrease profibrotic factor production, and improve renal blood flow.[24] This leads to a reduction in fibroblast activation and ECM deposition, promoting the preservation of kidney structure and function.[25] Other major elements of the TWA cycle, including TGF-β and Wnt, could be attractive targets for therapeutic intervention of kidney fibrosis. Indeed, numerous animal studies have shown that inhibition of either TGF-β or Wnt/β-catenin signaling inhibits the activation of myofibroblasts, reduces inflammation, and ameliorates fibrotic lesions. However, in clinical trials, inhibition of TGF-β failed to reduce kidney fibrosis in CKD patients.[26] Although targeting the Wnt/β-catenin pathway via various approaches has been shown to prevent or halt fibrosis in experimental animal models,[17] the safety and efficacy of blocking this pathway in fibrotic CKD in humans remain to be tested in clinical trials. Given the complexity of kidney fibrosis, combination therapies that target multiple pathways or processes may be more effective than single-target approaches. Investigators are exploring the potential synergistic effects of combining different therapeutic strategies to improve outcomes. Several new therapeutic options, in addition to conventional RAAS inhibition, have been reported to be effective in ameliorating fibrotic CKD, including sodium-glucose cotransporter 2 inhibitors and mineralocorticoid receptor antagonists.[27,28] There are many challenges in developing an effective therapy for kidney fibrosis. The logic behind current therapies is to target a so-called key profibrotic pathway, such as Ang II. However, the regulation and function of Ang II is extraordinarily complex and involves numerous players, such as angiotensinogen, renin, angiotensin-converting enzyme, and the type 1 Ang II receptor. Long-term treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers in patients with CKD typically results in compensatory upregulation of renin, a protein that acts not only as an enzyme converting angiotensinogen to angiotensin I but also as a cytokine that binds to the plasma membrane (pro)-renin receptor. The activation of renin/(pro)-renin receptor triggers a cascade of signaling events, leading to ECM production and kidney fibrosis,[29] which offsets the beneficial effects of these anti-RAAS drugs. Another conundrum in developing therapeutics for kidney fibrosis is that some profibrotic mediators, such as TGF-β, are biologically pleotropic, with many diverse functions. The failure of anti-TGF-β therapy in clinical trials is widely believed to be linked to its dysregulation of the immune response and renal inflammation. Finally, many mediators or signaling pathways, such as Wnt/β-catenin, matrix metalloproteinase-7, and EGFR ligands, have different, sometime opposite, effects in CKD versus acute kidney injury (AKI). For example, inhibition of Wnt/β-catenin signaling appears anti-fibrotic in CKD, whereas Wnt/β-catenin activation is beneficial in AKI, as it promotes tubular repair and regeneration.[30] The opposite actions of many signaling pathways in CKD and AKI make them difficult to be valuable therapeutic targets. Of note, several endogenous mediators, such as Klotho, glutathione peroxidase 3, and peroxisome proliferator–activated receptor γ coactivator-1α, are lost or downregulated after kidney injury.[31,32] As such, restoration of these mediators is kidney protective in both CKD and AKI. For example, Klotho is highly expressed in renal tubular epithelium in normal kidney, and Klotho deficiency is strongly associated with the development of both AKI and CKD after injury.[32,33] Interestingly, Klotho or Klotho-derived peptides (KP1 and KP6) directly inhibits TGF-β and Wnt/β-catenin signaling by physically interacting with TGF-β receptor II and Wnt ligands, respectively.[32-35] Furthermore, Klotho inhibits the expression of multiple RAAS components, including angiotensinogen, renin, angiotensin-converting enzyme, and Ang II type 1 receptor.[36] Collectively, Klotho appears to target all elements of the TWA cycle [Figure 1], leading to renal protection after injury. In this context, Klotho or its mimics could be an effective remedy for mitigating kidney fibrosis in CKD. A major question in kidney fibrosis field is whether, once established, fibrosis can be reversed. Studies on other organs, such as liver, indicate that tissue fibrosis, even at an advanced stage, can be reversed with appropriate treatments.[37,38] Furthermore, in diabetic kidney disease, glomerulosclerosis, a form of glomerular fibrosis, can be reversed after diabetes is cured.[39] However, kidneys have only a limited capacity for regeneration after injury. The general consensus is that nephron numbers depleted after injury do not recover, although tubular epithelium repair and regeneration occur.[40] Therefore, it would be extremely difficult, if not impossible, for the kidneys to fully recover when nephrons are destroyed after renal fibrosis. Intriguingly, kidney fibrosis was resolved, and nephron numbers were restored after nephrectomy in a study on the Mexican salamander, commonly known as the axolotl. Hence, it would be useful to investigate differences in the mediators and signaling pathways involved in injury repair and tissue remodeling in humans and axolotls. Information from such studies could eventually be translated into improved therapies for patients with fibrotic CKD.