Effective Treatment Of Renal Fibrosis: Thioredoxin Domain Containing 5 (TXNDC5)

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Part Ⅱ：Endoplasmic reticulum protein TXNDC5 promotes renal fibrosis by enforcing TGF-β signaling in kidney fibroblasts

Yen-Ting Chen, Pei-Yu Jhao & et al.

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Renal fibrosis, a common pathological manifestation of virtually all types of chronic kidney disease (CKD), often results in diffuse kidney scarring and predisposes to end-stage renal disease. Currently, there is no effective therapy against renal fibrosis. Recently, our laboratory identified an ER-resident protein, thioredoxin domain containing 5 (TXNDC5), as a critical mediator of cardiac fibrosis. Transcriptome analyses of renal biopsy specimens from patients with CKD revealed marked TXNDC5 (thioredoxin domain containing 5) upregulation in fibrotic kidneys, suggesting a potential role of TXNDC5 (thioredoxin domain containing 5) in renal fibrosis. Employing multiple fluorescence reporter mouse lines, we showed that TXNDC5 (thioredoxin domain containing 5) was specifically upregulated in collagen-secreting fibroblasts in fibrotic mouse kidneys. In addition, we showed that TXNDC5 (thioredoxin domain containing 5) was required for TGF-β1–induced fibrogenic responses in human kidney fibroblasts (HKFs), whereas TXNDC5 (thioredoxin domain containing 5) overexpression was sufficient to promote HKF activation, proliferation, and collagen production. Mechanistically, we showed that TXNDC5 (thioredoxin domain containing 5), transcriptionally controlled by the ATF6-dependent ER stress pathway, mediated its profibrogenic effects by enforcing TGF-β signaling activity through posttranslational stabilization and upregulation of type I TGF-β receptor in kidney fibroblasts. Using a tamoxifen-inducible, fibroblast-specific Txndc5 knockout mouse line, we demonstrated that deletion of Txndc5 in kidney fibroblasts mitigated the progression of established kidney fibrosis, suggesting the therapeutic potential of TXNDC5 (thioredoxin domain containing 5) targeting for renal fibrosis and CKD.

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In this study, we demonstrate that TXNDC5 (thioredoxin domain containing 5), an ER-resident protein disulfide isomerase enriched in renal fibroblasts, contributes critically to the pathogenesis of renal fibrosis. TXNDC5 (thioredoxin domain containing 5) was upregulated both in the kidneys from patients with CKD and from mouse models of renal fibrosis induced by UUO, uIRI, or FA treatment. Mechanistic investigations showed that TXNDC5 (thioredoxin domain containing 5) promotes renal fibrogenesis by enhancing profibrotic TGF-β signaling through increasing the protein folding and stability of TGFBR1, leading to the activation and proliferation of renal fibroblasts that produce excessive ECM proteins. In addition, TGF-β1 induces TXNDC5 (thioredoxin domain containing 5) expression through increased ER stress level and ATF6-dependent transcriptional control in renal fibroblasts. Consistent with the profibrogenic effects of TXNDC5 (thioredoxin domain containing 5) observed in kidney fibroblasts, global deletion of Txndc5 (thioredoxin domain containing 5) protects against renal fibrosis induced by UUO, uIRI, or FA. Targeted deletion of Txndc5 (thioredoxin domain containing 5) in collagen-secreting renal fibroblasts, but not in TECs, endothelial cells, and podocytes, could mitigate the establishment and lessen the progression of renal fibrosis in response to injury. Taken together, these results reveal a novel causal role of TXNDC5 (thioredoxin domain containing 5) in the development of renal fibrosis through the regulation of TGFBR1 and TGF-β1 signaling. These data also suggest that targeting TXNDC5 (thioredoxin domain containing 5) is a potentially powerful therapeutic approach to treat or prevent renal fibrosis and CKD. A schematic in Figure 11D illustrates the regulation and fibrogenic function of TXNDC5 (thioredoxin domain containing 5) in kidney fibroblasts.

TGF-β1 signaling plays an essential role in the development of renal fibrosis (32-34). Following insults to the kidneys, TGF-β1 is released from injured TECs and triggers the activation of TGF-β1 signaling in surrounding kidney fibroblasts(4).TGF-β1 binds to TGFBR2, which recruits, phosphorylates, and activates TGF-BR1. Phosphorylated TGFBR1 then activates SMAD3-dependent canonical TGF-β1 signaling, as well as Ras and TAK1, constituents of SMAD-independent, noncanonical TGF-β1 signaling (35). Pharmacological inhibition of TGF-β signaling using neutralizing antibodies (36-38)or inhibitors (39,40)has shown protective effects against renal fibrosis in preclinical models. In particular, pentoxifylline, a nonspecific phosphodiesterase inhibitor clinically proven to slow down the decline of glomerular filtration rate and to improve proteinuria (16,41), was found to attenuate renal fibrosis by blocking the Smad3/4-mediated fibrogenic effects(14). Although TGF-β1 pathway inhibition appears to be an attractive strategy to treat renal fibrosis, most, if not all, pharmacological agents targeting TGF-β1 or TGF-β receptors directly failed in clinical trials(42). Because TGF-β signaling plays essential physiological roles in development, cell differentiation, tissue homeostasis, and immune response(43), direct inhibition of TGF-β signaling could lead to impaired physiological functions and adverse reactions including liver (44) and cardiac toxicity (45, 46). The results presented here reveal a novel positive feedback loop of the TGF-β1/ATF6/TXNDC5/TGFBR1 signaling axis in kidney fibroblasts, where TGF-β1 induces the upregulation of TXNDC5 (thioredoxin domain containing 5) through ER stress and ATF6-mediated transcriptional control. Increased TXNDC5 (thioredoxin domain containing 5) level further enhances the folding and stability of TGFBR1, leading to amplified TGF-β1 signaling and subsequent fibrogenic response. Targeting TXNDC5 (thioredoxin domain containing 5), therefore, could repress fibrogenic TGF-β1 signaling by breaking this positive feedback loop and hence attenuating renal fibrogenesis. The fibroblast-restricted nature of TXNDC5 also makes it a favor-able drug target to avoid the risk of disturbing TGF-β-dependent physiological functions in nonfibroblast cells.

Exploiting a global Txndc5 (thioredoxin domain containing 5) deletion mouse line, we demonstrated that loss of TXNDC5 (thioredoxin domain containing 5) could prevent renal fibrogenesis in response to kidney injuries. However, it is more clinically pertinent to determine if targeting TXNDC5 (thioredoxin domain containing 5) could deter or even resolve existing kidney fibrosis. To address this question, a tamoxifen-inducible, fibroblast-specific Txndc5 (thioredoxin domain containing 5) conditional knockout mouse line (Txnd5ko) was utilized, and it showed that the induction of Txndc5 (thioredoxin domain containing 5) deletion in kidney fibroblasts 10 days after UUO, a time point when renal fibrosis was established (Figure 11B), significantly eased the progression and expansion of renal fibrosis than in control mice. These data strongly suggest the potential of targeting TXNDC5 (thioredoxin domain containing 5) as a novel therapeutic approach to halt the progression of renal fibrosis in patients with CKD. Of note, serum levels of blood urea nitrogen(BUN) and creatinine were not measured in the mouse models used in the current study owing to the fact that BUN/creatinine levels are unaffected in UUO and ulRI models and are somewhat variable in the folic acid nephropathy model(47-49). Further studies are required to determine the impact of Txndc5 (thioredoxin domain containing 5) deletion on renal function using CKD mouse models induced by the approach to treat or prevent renal fibrosis and CKD. A schematic in Figure 11D illustrates the regulation and fibrogenic function of TXNDC5 (thioredoxin domain containing 5) in kidney fibroblasts.

TGF-β1 signaling plays an essential role in the development of renal fibrosis (32-34). Following insults to the kidneys, TGF-β1 is released from injured TECs and triggers the activation of TGF-β1 signaling in surrounding kidney fibroblasts(4).TGF-β1 binds to TGFBR2, which recruits, phosphorylates, and activates TGF-BR1. Phosphorylated TGFBR1 then activates SMAD3-dependent canonical TGF-β1 signaling, as well as Ras and TAK1, constituents of SMAD-independent, noncanonical TGF-β1 signaling (35). Pharmacological inhibition of TGF-β signaling using neutralizing antibodies (36-38)or inhibitors (39,40)has shown protective effects against renal fibrosis in preclinical models. In particular, pentoxifylline, a nonspecific phosphodiesterase inhibitor clinically proven to slow down the decline of glomerular filtration rate and to improve proteinuria (16,41), was found to attenuate renal fibrosis by blocking the Smad3/4-mediated fibrogenic effects(14). Although TGF-β1 pathway inhibition appears to be an attractive strategy to treat renal fibrosis, most, if not all, pharmacological agents targeting TGF-β1 or TGF-β receptors directly failed in clinical trials(42). Because TGF-β signaling plays essential physiological roles in development, cell differentiation, tissue homeostasis, and immune response(43), direct inhibition of TGF-β signaling could lead to impaired physiological functions and adverse reactions including liver (44) and cardiac toxicity (45, 46). The results presented here reveal a novel positive feedback loop of the TGF-β1/ATF6/TXNDC5/TGFBR1 signaling axis in kidney fibroblasts, where TGF-β1 induces the upregulation of TXNDC5 (thioredoxin domain containing 5) through ER stress and ATF6-mediated transcriptional control. Increased TXNDC5 (thioredoxin domain containing 5) level further enhances the folding and stability of TGFBR1, leading to amplified TGF-β1 signaling and subsequent fibrogenic response. Targeting TXNDC5 (thioredoxin domain containing 5), therefore, could repress fibrogenic TGF-β1 signaling by breaking this positive feedback loop and hence attenuating renal fibrogenesis. The fibroblast-restricted nature of TXNDC5 (thioredoxin domain containing 5) also makes it a favor-able drug target to avoid the risk of disturbing TGF-β-dependent physiological functions in nonfibroblast cells.

Exploiting a global Txndc5 (thioredoxin domain containing 5) deletion mouse line, we demonstrated that loss of TXNDC5 (thioredoxin domain containing 5) could prevent renal fibrogenesis in response to kidney injuries. However, it is more clinically pertinent to determine if targeting TXNDC5 (thioredoxin domain containing 5) could deter or even resolve existing kidney fibrosis. To address this question, a tamoxifen-inducible, fibroblast-specific Txndc5 (thioredoxin domain containing 5) conditional knockout mouse line (Txnd5ko) was utilized, and it showed that the induction of Txndc5 deletion in kidney fibroblasts 10 days after UUO, a time point when renal fibrosis was established (Figure 11B), significantly eased the progression and expansion of renal fibrosis than in control mice. These data strongly suggest the potential of targeting TXNDC5 (thioredoxin domain containing 5) as a novel therapeutic approach to halt the progression of renal fibrosis in patients with CKD. Of note, serum levels of blood urea nitrogen(BUN) and creatinine were not measured in the mouse models used in the current study owing to the fact that BUN/creatinine levels are unaffected in UUO and ulRI models and are somewhat variable in the folic acid nephropathy model(47-49). Further studies are required to determine the impact of Txndc5 (thioredoxin domain containing 5) deletion on renal function using CKD mouse models induced by 5/6 nephrectomy and/or cyclosporin/cisplatin treatment, models with apparent changes in BUN and creatinine levels(48).

The magnitude of fibrotic area reduction observed in Tendeseko mice was relatively lower than that observed in Txndc 57mice. This could be owing to the incomplete deletion of Txndc5 (thioredoxin domain containing 5) in renal fibroblasts related to the efficiency of Cre-mediated recombination. Although residential renal fibroblasts are considered the major source of scar-forming myofibroblasts in fibrogenic kidney diseases (50), epithelial-to-mesenchymal transition(EMT, refs. 51, 52) and endothelial-to-mesenchymal transition(EndoMT, ref.53) have been shown to contribute to the development of renal fibrosis. Because TXNDC5 (thioredoxin domain containing 5) was also expressed in a fraction of TECs and endothelial cells, we could not exclude the possibility that TXNDC5 could also contribute to kidney fibrogenesis through promoting EMTor EndoMTin these cells. Further studies are required to test this hypothesis directly. The tamoxifen-inducible, fibroblast-specific conditional knockout mouse line used in the present study, however, allows deletion of Txndc5in collagen-producing active myofibroblasts regardless of their origins (i.e., from TECs or endothelial cells). The notion that fibroblast-specific deletion of Txndc5 (thioredoxin domain containing 5) effectively eased the development and progression of postinjury renal fibrosis in mice, therefore, remains unchanged whether TXNDC5 (thioredoxin domain containing 5) plays any role in EMT-or EndoMT-mediated kidney fibrogenesis.

Although TGF-β signaling is generally considered a central mediator of renal fibrosis, TGF-β signaling per se can be essential to maintain the homeostasis and health of kidney tissue. Conditional deletion of Tbgbr2 in renal tubular cells, for example, leads to increased NF-kB signaling and renal inflammation (54). Deletion of Tbgbr2 in collecting ducts exacerbates renal fibrosis in response to UUO, possibly by enhancing paracrine TGF-β signaling between epithelial and interstitial cells (55).In a study by Neelisetty et al, selective deletion of Tbgbr2 in matrix-producing interstitial cells using Colla2-Cre/ERT2*Tgfbr2 and Tenascin C-Cre/ERT*Tgfbr2M mice did not show protection against renal fibrosis induced by UUO or aristolochic acid (56), suggesting that abrogating TGF-β signaling in renal fibroblasts may not be sufficient to reduce postinjury renal fibrosis. Here, we showed that fibroblast-specific Txndc5 (thioredoxin domain containing 5) deletion (using Colla2-Cre/ERT2*Txndc5) (thioredoxin domain containing 5) results in reduced TGFBR1, TGF-βsignalingactivityand fibrosis in the kidneys. The observation that deletion of Txndc5 (thioredoxin domain containing 5), but not Tgfbr2, in renal fibroblasts/interstitial cells reduces renal fibrosis suggests that deletion of Txndc5 (thioredoxin domain containing 5) may have additional anti-fibrotic effects that are independent of TGF-β signaling. Indeed, we have previously shown that in cardiac fibroblasts, TXNDC5 (thioredoxin domain containing 5), as a PDI, also promotes fibrogenesis by facilitating the folding and production of ECM proteins (17). It is also possible that Txndc5 (thioredoxin domain containing 5) deletion could result in additional inhibition of fibrogenic signal-ing pathways independent of TGF-β receptors such as PDGF, CCN2, Hedgehog, and HIF-1α signaling (57), thereby potentiating its antifibrotic effects in the kidneys. Further experiments will be required to test these hypotheses directly.

In conclusion, the present study revealed an essential and previously unrecognized role of renal fibroblast-enriched ER protein TXNDC5 (thioredoxin domain containing 5) in the pathogenesis of renal fibrosis. Experimental evidence suggests that TXNDC5 (thioredoxin domain containing 5) promotes renal fibrosis by augmenting TGF-β signaling activity by enhancing the folding and stability of TGFBR1 in renal fibroblasts, leading to excessive myofibroblast transdifferentiation, proliferation, and ECM production. Targeted deletion of Txndc5 (thioredoxin domain containing 5) in renal fibroblasts protects against the development and progression of postinjury renal fibrosis. These results suggest that targeting TXNDC5 (thioredoxin domain containing 5) could be a novel and powerful approach to treat or prevent renal fibrosis and CKD.

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