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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2013 May 30;24(9):1387–1398. doi: 10.1681/ASN.2012101041

Kindlin-2 Mediates Activation of TGF-β/Smad Signaling and Renal Fibrosis

Xiaofan Wei *, Yang Xia †, Feng Li †, Yan Tang †, Jing Nie *, Youhua Liu *, Zhanmei Zhou *, Hongquan Zhang †,✉, Fan Fan Hou *,✉
PMCID: PMC3752947  PMID: 23723426

Abstract

Activation of TGF-β/Smad signaling plays a central role in the pathogenesis of tubulointerstitial fibrosis, but the mechanisms underlying the initial interaction of the TGF-β receptor with Smads, leading to their activation, remain unclear. Here, we found that Kindlin-2, an integrin-binding protein, physically mediated the interaction of the TGF-β type I receptor (TβRI) with Smad3 in human kidney tubular epithelial cells. Kindlin-2 bound to TβRI through its FERM domain and to Smad3 through its N terminus. Overexpression of Kindlin-2 increased TGF-β–induced Smad3 activation. Knockdown of Kindlin-2 significantly suppressed the engagement of TβRI with Smad3 and inhibited TGF-β–induced Smad3 activation, as well as the expression of its target genes. Neither transfection of a Kindlin-2 mutant incapable of binding to β1 integrin nor knockdown of β1 integrin influenced the effect of Kindlin-2 on TGF-β1–induced Smad3 activation, indicating that this effect is independent of integrin. Kindlin-2 expression was markedly increased, predominantly in renal tubular epithelial cells, both in the unilateral ureteral obstruction model of kidney fibrosis and in human tissue exhibiting tubulointerstitial fibrosis. Furthermore, in the unilateral ureteral obstruction model, knocking down Kindlin-2 significantly inhibited activation of TGF-β/Smad signaling, decreased the expression of matrix genes, and ameliorated fibrosis. In summary, Kindlin-2 physically interacts with both TβRI and Smad3, promoting the activation of TGF-β/Smad signaling and contributing to the pathogenesis of tubulointerstitial fibrosis. Blockade of Kindlin-2 might be a rational therapeutic strategy for the treatment of fibrotic kidney diseases.

Progressive renal tubulointerstitial fibrosis (TIF) is the final common pathway of virtually all CKD leading to end stage renal failure.1,2 There is broad agreement that progressive TIF is mediated by multiple mediators including growth factors, metabolic toxins, and stress molecules.3–5 Among them, TGF-β1 has been recognized as a key mediator in the pathogenesis of TIF.6–8

TGF-β1 has a broad spectrum of biologic actions in a wide variety of cell types, including renal tubular epithelial cells (TECs).9,10 TGF-β1 initiates its cellular response by binding with TGF-β type II receptor (TβRII),11,12 which activates TGF-β type I receptor (TβRI), resulting in phosphorylation of Smad2/3, two receptor-associated Smads (R-Smads). The activated R-Smads then translocate into the nucleus, where they regulate the transcription of target genes such as type I collagen (Col I),13 α-smooth muscle actin (α-SMA),14 and Snail15 to exert the biologic functions.

Although activation of R-Smad by TβR is well documented, little is known on the mechanism underlying the interaction of TβR and R-Smad. A previous study reports that SARA16 recruits Smad2 to the TGF-β receptor via controlling the subcellular localization of Smad2, suggesting that interaction of TβR with Smads requires molecules functioned as “adaptors.”17

Kindlins are a group of FERM domain-containing adaptor proteins and have recently attracted attention for their ability to bind to and activate integrins. Moreover, they have also been linked to inherited and acquired human diseases such as Kindler syndrome and cancer.18,19 Kindlin-2, a member of Kindlin protein family, is widely expressed and evolutionarily conserved and emerging as an important regulator of integrin-mediated cell–extracellular matrix interaction.20–22 A recent study23 reports that Kindlin-2 is expressed in glomerular podocytes and plays a role in integrin-dependent regulation of podocyte-matrix adhesion.23 Knockdown of Kindlin-2 significantly decreases TGF-β1–induced extracellular matrix synthesis, suggesting that Kindlin-2 may be involved in TGF-β–induced renal injury.

This study was conducted to test the hypothesis that Kindlin-2, acting as an adaptor protein, mediates activation of TGF-β/Smad signaling and contributes to the pathogenesis of TIF. In this study, we demonstrated that Kindlin-2 promoted activation of TGF-β/Smad signaling in TECs by recruiting Smad3 to TβRI. More importantly, we found that Kindlin-2 expression was upregulated in experimental and human TIF. Depletion of Kindlin-2 in vivo repressed matrix deposition and ameliorated TIF. These findings suggest that Kindlin-2 is an important component of the intracellular machinery that regulates TGF-β/Smad signaling in TECs and shed new light on the mechanism underlying the pathogenesis of TIF.

Results

Expression and Localization of Kindlin-2 in Human Kidney TECs

The Kindlin protein family consists of three members. Only Kindlin-1 and -2 were detected in kidney tissues.24 We found that Kindlin-2 was highly expressed in human kidney TECs (HKCs), but not in colonic carcinoma cell HCT-116 (negative control) (Figure 1A). By contrast, an extremely low level of Kindlin-1 was detected in HKCs, whereas abundant Kindlin-1 was expressed in HaCaT keratinocytes (positive control) (Figure 1B), suggesting that Kindlin-2 is the major member of Kindlin family expressed in HKCs. Immunofluorescence staining showed that Kindlin-2 was detected in the cytosol in HKCs (Figure 1, C–E).

Figure 1.

Figure 1.

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Expression and localization of Kindlin-2 in HKCs. (A) Cell lysates (20 μg proteins/lane) of HKCs (lane 1) and HCT-116 (lane 2) are analyzed by Western blot with antibody recognizing Kindlin-2. Equal loading is confirmed by probing the membrane with anti-β-actin antibody. (B) Lysates (20 μg protein per lane) of HKCs (lane 1) and HaCaT cell lysates (lane 2) are analyzed by Western blot with antibody recognizing Kindlin-1. β-actin is used to verify equivalent loading. (C–E) HKCs are dually stained with mouse anti-Kindlin-2 mAb (C) and FITC-conjugated phalloidin (D). The image is merged in panel E. Scale bars, 10 μm.

Kindlin-2 Interacts with Smad3 and TβRI in HKCs

We next investigated the effect of TGF-β1 on Kindlin-2 expression. As shown in Figure 2, TGF-β1 upregulated Kindlin-2 expression in a dose-dependent (Figure 2, A and C) and time-dependent (Figure 2, B and D) manner at both mRNA and protein levels.

Figure 2.

Figure 2.

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TGF-β1 upregulates Kindlin-2 expression in HKCs. HKCs are treated with indicated concentrations of TGF-β1 for 48 hours (A and C), or 5 ng/ml of TGF-β1 for the indicated time period (B and D). Expression of Kindlin-2 is determined by quantitative RT-PCR (A and B) and Western blot (C and D). β-actin is used to verify equivalent loading. Values are mean ± SD of three independent experiments. *P<0.05 versus control.

The critical initial step of TGF-β signaling activation requires interaction of TβR with Smads. Therefore, coimmunoprecipitation was performed to examine whether Kindlin-2 interacted with the major components of TGF-β signaling. As shown in Figure 3A, exogenous Kindlin-2 physically bound to Smad3 and TβRI in HKCs. The interaction of endogenous Kindlin-2 with TβRI and Smad3 was detected in the absence of TGF-β1 and enhanced by TGF-β1 stimulation (Figure 3B). Kindlin-2 also weakly interacted with Smad2, Smad4, and TβRII in the presence of TGF-β1 (Figure 3B). Immunofluorescence staining further demonstrated that Kindlin-2 was colocalized with TβRI and Smad3 at the cellular membrane even in the absence of TGF-β1. TGF-β1 augmented the complex formation among Kindlin-2, Smad3, and TβRI (Figure 3C).

Figure 3.

Figure 3.

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Kindlin-2 effectively interacts with Smad3 and TβRI. (A) HKCs are transfected with Flag-tagged-Kindlin-2 expression vector. Forty-eight hours after transfection, cell lysates are immunoprecipitated with anti-Flag antibody or normal IgG followed by immunoblotting using indicated antibodies. (B) HKCs are treated with or without TGF-β1 (5 ng/ml) for 30 minutes and then cell lysates are immunoprecipitated with indicated antibodies followed by immunoblotting using anti-Kindlin-2 mAb. (C) HKCs are transfected with pFlag-Smad2, pFlag-Smad3, pFlag-Smad4, pHA-TβRI, or pHA-TβRII. Forty-eight hours after transfection, cells were treated with or without TGF-β1 (5 ng/ml) for 30 minutes. Expression of Kindlin-2, Smad2, Smad3, Smad4, TβRI, and TβRII is analyzed by immunofluorescence staining. Nuclei are visualized with DAPI. (D) HKCs are transfected with wild-type Smad3, Smad3 mutant Smad3D407E, or empty vector. Cell lysates are immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Kindlin-2 mAb. (E) HKCs are treated with or without TGF-β1 (5 ng/ml) for 30 minutes. Cell lysates are immunoprecipitated using anti-p-Smad3 antibody followed by immunoblotting using anti-Kindlin-2 mAb. (F) HKCs are transfected with the indicated pFlag-Kindlin-2 (amino acids 1–239, amino acids 240–569, or amino acids 570–680). Cell lysates are immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Smad3 or TβRI antibody. DAPI, 4',6-diamidino-2-phenylindole. Scale bar, 10 μm in C.

To determine whether the interaction of Kindlin-2 with Smad3 is dictated by the phosphorylation of Smad3, we utilized a Smad3 mutant (Smad3D407E) that binds to TβRI but is neither phosphorylated nor released from the receptor.25 As shown in Figure 3D, Kindlin-2 was also able to interact with Smad3D407E. Consistently, TGF-β1 augmented the association between Kindlin-2 and phosphorylated Smad3 (p-Smad3) (Figure 3E), suggesting that Kindlin-2 could interacted with both phosphorylated and unphosphorylated Smad3. These findings established a novel interaction of Kindlin-2 with TβRI and Smad3 in physiologically relevant settings.

To further characterize the region of Kindlin-2 that is responsible for interaction with Smad3 and TβRI, we prepared deletion mutants of Kindlin-2,26 including N terminal (1–239 aa), FERM domain (240–569 aa), and C terminal (570–680 aa), and transfected them into HKCs, respectively. The coimmunoprecipitation was performed. N terminal of Kindlin-2 strongly interacted with Smad3, whereas FERM domain interacted with TβRI (Figure 3F).

Kindlin-2 is Required for the Engagement of TβRI with Smad3 in HKCs

To determine the involvement of Kindlin-2 in the engagement of TβRI with Smad3, we first examined the physical interaction among Kindlin-2, Smad3, and TβRI under the stimulation of TGF-β1. As shown in Figure 4A, both Smad3 and TβRI were detectable in the immunocomplexes precipitated by anti-Kindlin-2. In the reciprocal experiments, both Kindlin-2 and TβRI were present in the complexes precipitated by anti-Smad3. Similarly, TβRI was able to coimmunoprecipitate with Kindlin-2 and Smad3 (Figure 4A). These data suggest that Kindlin-2 was involved in the engagement of TβRI with Smad3 by forming a multi-component protein complex.

Figure 4.

Figure 4.

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Kindlin-2 is required for TGF-β1–induced interaction of TβRI with Smad3. (A) HKCs are treated with TGF-β1 (5ng/ml) for 30 minutes and then the endogenous interaction among Kindlin-2, Smad3, and TβRI is analyzed by coimmunoprecipitation. (B) pHA-TβRI, pFlag-Smad3, control siRNA, or Kindlin-2–specific siRNA is cotransfected in HKCs followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. The interaction of TβRI with Smad3 is analyzed by coimmunoprecipitation. *P<0.05 versus control siRNA. (C) HKCs are treated with control siRNA or Kindlin-2 siRNA followed by TGF-β1 treatment for 30 minutes. Coimmunoprecipitation is performed to analyze the endogenous interaction between TβRI and Smad3. *P<0.05 versus control siRNA without TGF-β1 group; #P<0.05 versus control siRNA with TGF-β1 group. (D) HKCs are transfected with empty vector or pFlag-Kindlin-2 followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. Coimmunoprecipitation is performed to analyze the endogenous interaction between TβRI and Smad3. *P<0.05 versus empty vector with TGF-β1 group.

Further studies showed that silencing Kindlin-2 significantly reduced TGF-β1–induced both exogenous and endogenous interaction between TβRI and Smad3 (Figure 4, B and C). Consistently, overexpression of Kindlin-2 increased TGF-β1–induced interaction of TβRI and Smad3 (Figure 4D). These results indicate that Kindlin-2 is obligatory for the formation of TβRI-Smad3 complex in TGF-β1 signaling.

Kindlin-2 Facilitates TGF-β1–Induced Smad3 Activation in HKCs

TGF-β1 executes its function mainly through activation of the downstream Smads, particularly Smad3.27,28 Because Kindlin-2 is required for TβRI-Smad3 interaction, we next tested whether Kindlin-2 was involved in TGF-β1–induced activation of Smad3. HKCs were transfected with Flag-tagged-Kindlin-2 followed by incubation with TGF-β1 for 30 minutes. TGF-β1 significantly induced phosphorylation of Smad3. Overexpression of Kindlin-2 further increased the level of Smad3 phosphorylation, although exogenous Kindlin-2 per se in the absence of TGF-β1 did not trigger Smad3 phosphorylation (Figure 5A). Consistently, overexpression of Kindlin-2 significantly increased TGF-β1–induced Smad3 nuclear translocation. However, ectopic Kindlin-2 expression alone did not cause Smad3 nuclear translocation in the absence of TGF-β1 (Figure 5B).

Figure 5.

Figure 5.

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Kindlin-2 is required for TGF-β1–induced Smad3 activation. (A and B) HKCs are transfected with empty vector or pFlag-Kindlin-2 followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. P-Smad3 expression in cytosol (A) or in the nuclei (B) was examined by Western blot. Data are expressed as the mean ± SD of three independent experiments, *P<0.05 versus empty vector. (C and D) HKCs are transfected with control siRNA or Kindlin-2 siRNA followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. P-Smad3 expression in cytosol (C) or in the nuclei (D) is examined by Western blot. *P<0.05 versus control siRNA group. (E) HKCs are cotransfected with Kindlin-2 siRNA and Kindlin-2 siRNA-resistant plasmid followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. The expression of p-Smad3 and Kindlin-2 is analyzed by Western blot. *P<0.05 versus Kindlin-2 siRNA with empty vector group.

To further confirm the role of Kindlin-2 on TGF-β1–induced Smad3 activation, Kindlin-2 was silenced by small interfering RNA (siRNA) in HKCs. Compared with scramble siRNA controls, knockdown of Kindlin-2 significantly inhibited TGF-β1–induced Smad3 phosphorylation (Figure 5C) and its nuclear translocation (Figure 5D). This effect was specific, because transfection of a mutant plasmid that is resistant to Kindlin-2 siRNA effectively restored TGF-β1–induced Smad3 phosphorylation (Figure 5E). Overexpression or knockdown of Kindlin-2 did not affect TGF-β1–induced Smad3 activation in fibroblasts (NRK-49F) (Supplemental Figure 1). These results indicate that Kindlin-2 is required for TGF-β1–induced Smad3 activation in tubular cells.

The Role of Kindlin-2 in TGF-β/Smad Signaling is Independent of β1 Integrin

It is well established that, among the members of integrin family, β1 integrin is the most critical one and is expressed ubiquitously in different tissues including renal proximal tubular cells.29 Kindlin-2 could bind and activate β1 integrin.20,30 To address whether the promoting effect of Kindlin-2 on TGF-β/Smad signaling is dependent on β1 integrin, we first examined whether β1 integrin is involved in the interaction of Kindlin-2 with Smad3 and TβRI. HKCs were transfected with wild-type Kindlin-2 (Flag-Kindlin-2 WT) or Flag-Kindlin-2 QW Mutant, which is not able to interact with integrin, and coimmunoprecipitation was performed. As shown in Figure 6A, Kindlin-2 QW mutant binds to both Smad3 and TβRI as that of Kindlin-2 WT. To further confirm this result, HKCs were cotransfected with Flag-Kindlin-2 and β1 integrin siRNA. Knocking down β1 integrin did not affect the interaction of Kindlin-2 with Smad3 and TβRI, suggesting that interaction of Kindlin-2 with Smad3 and TβRI is independent of β1 integrin (Figure 6B).

Figure 6.

Figure 6.

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The role of Kindlin-2 in TGF-β/Smad signaling is independent of β1 integrin. (A) A Flag–Kindlin-2 QW mutant deficient in binding to β1 integrin is generated. Flag, Flag–Kindlin-2 WT, or QW mutant is transfected into HKCs. Cell lysates are immunoprecipitated with anti-Flag antibody by immunoblotting using indicated antibodies. (B) HKCs are cotransfected with indicated siRNA and Flag–Kindlin-2, cell lysates are immunoprecipitated with anti-Flag antibody by immunoblotting using indicated antibodies. (C) Flag, Flag–Kindlin-2 WT, or QW mutant is transfected into HKCs followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. P-Smad3 expression is examined by Western blot. *P<0.05 versus Flag with TGF-β1 group. (D) HKCs are cotransfected with indicated siRNA and Flag–Kindlin-2 followed by TGF-β1 (5 ng/ml) treatment for 30 minutes. The expression of p-Smad3 is analyzed by Western blot. *P<0.05 versus Flag with TGF-β1 group. (E) HKCs are treated with 5 ng/ml of TGF-β1 for the indicated time period. β1 integrin expression is determined by Western blot. *P<0.05 versus control. (F and G) HKCs are transfected with control siRNA, Smad3 siRNA, or Kindlin-2 siRNA followed by TGF-β1 (5 ng/ml) treatment for 24 hours. The expression of β1 integrin is determined by Western blot. * P<0.05 versus control siRNA with TGF-β1 group.

Next, we tested whether the promoting effect of Kindlin-2 on TGF-β1–induced Smad3 phosphorylation requires β1 integrin. As shown in Figure 6C, like Flag-Kindlin-2 WT, QW Mutant also increased the level of Smad3 phosphorylation. To further verify this result, HKCs were cotransfected with Flag-Kindlin-2 and β1 integrin siRNA followed by treatment with TGF-β1. The results showed that knocking down β1 integrin did not influence the role of Kindlin-2 in promoting TGF-β1–induced Smad3 phosphorylation (Figure 6D).

Consistent with a previous study,29 we found that TGF-β1 upregulated β1 integrin expression in a time-dependent manner (Figure 6E). Knocking down Smad3 significantly suppressed TGF-β1–induced β1 integrin expression (Figure 6F), suggesting that this effect of TGF-β1 is dependent on Smad3. Furthermore, knockdown of Kindlin-2 remarkably decreased β1 integrin expression after TGF-β1 stimulation, indicating that Kindlin-2-augmented Smad3 activation is the upstream event of TGF-β1–induced β1 integrin expression.

Kindlin-2 Promotes the Expression of TGF-β1 Target Genes in HKCs

We next investigated the effect of Kindlin-2 on the expression of TGF-β signaling target genes. HKCs were transfected with Kindlin-2 or control siRNA, followed by stimulation of TGF-β1. The results showed that knockdown of Kindlin-2 significantly inhibited TGF-β1–induced upregulation of Col I, α-SMA, and Snail at both mRNA (Figure 7B) and protein levels (Figure 7C).

Figure 7.

Figure 7.

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Knockdown of Kindlin-2 inhibits the expression of TGF-β1–induced downstream target genes in HKCs. (A) Western blot analysis of Kindlin-2 expression in HKCs transfected with either control siRNA or Kindlin-2 siRNA. (B and C) HKCs are transfected with control siRNA or Kindlin-2 siRNA followed by TGF-β1 (5n g/ml) treatment for 48 hours. The expression of Col I, α-SMA, and Snail was determined by quantitative RT-PCR (B) or Western blot (C). *P<0.05 versus control siRNA without TGF-β1 group; #P<0.05 versus control siRNA with TGF-β1 group.

Depletion of Kindlin-2 Attenuates TIF in UUO Mice

To explore the role of Kindlin-2 in the pathogenesis of TIF in vivo, we examined the expression of Kindlin-2 in UUO mice, a well characterized model of TIF.31 Immunohistochemistry staining showed remarkably upregulation of Kindlin-2 in the renal tissues of UUO mice. Kindlin-2 expression was detected predominantly in TECs (Figure 8C and Supplemental Figure 1). This finding was further confirmed by real-time PCR (Figure 8D) and Western blot (Figure 8F) in renal homogenate. The induction of Kindlin-2 was accompanied by increased expression of TGF-β1 and p-Smad3 (Figure 8, C, E, G, and H). Knocking down Kindlin-2 in vivo significantly reduced the renal expression of TGF-β1, p-Smad3, and integrin β1 in the kidneys with ureteral ligation (Figure 8, C, E, G, and H), but did not in the contralateral kidneys (Supplemental Figure 2). Likewise, knockdown of Kindlin-2 significantly decreased the renal expression of Col I, α-SMA, and Snail at both mRNA and protein levels (Figure 8, D–G). Most importantly, depletion of Kindlin-2 significantly attenuated TIF (Figure 8B), suggesting that Kindlin-2 plays a critical role in the pathogenesis of TIF.

Figure 8.

Figure 8.

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Depletion of Kindlin-2 ameliorates renal tubulointerstitial fibrosis. (A) Western blot analysis of Kindlin-2 expression in mice injected with control siRNA or Kindlin-2 siRNA (twice a week). *P<0.05 versus control siRNA (B) Representative micrographs demonstrate kidney injury at 7 days after UUO in different groups as indicated. Kidney sections are subjected to Masson trichrome staining. *P<0.05 versus sham; #P<0.05 versus UUO with control siRNA (n=5 for each group). (C) Representative micrographs show the abundance and distribution of Kindlin-2, p-Smad3, and TGF-β1 in the kidney of different groups of mice as indicated 7 days after UUO, respectively. (D–H) The expression of Kindlin-2, α-SMA, FN, Col I, Snail, β1 integrin, TGF-β1, or p-Smad3 in the kidney of different groups of mice 7 days after UUO is determined by quantitative RT-PCR (D and E) or Western blot (F–H). *P<0.05 versus sham; #P<0.05 versus UUO with control siRNA (n=5 for each group). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Scale bar, 50 μm in B and C.

Kindlin-2 is Upregulated in Human TIF

We examined the expression of Kindlin-2, TGF-β1, and p-Smad3 in renal biopsies samples from eight patients with chronic TIF. Normal kidney tissues adjacent to the tumor (n=3) served as controls. Immunohistochemical staining revealed that the expression of Kindlin-2 was upregulated in fibrotic renal tissue, predominantly in proximal and distal tubules. Kindlin-2 was also weakly expressed in fibroblast and glomerular cells such as podocytes and parietal epithelial cells (Supplemental Figure 3). Overexpression of Kindlin-2 was associated with upregulation of TGF-β1 and p-Smad3 (Figure 9), suggesting that dysregulated Kindlin-2 implicates in the pathogenesis of human TIF as well. However, we were unable to analyze the correlation of Kindlin-2 staining level with the severity of fibrosis due to the relatively equal degree of fibrosis in eight patients (six of eight patients had an interstitial fibrosis score of 2).

Figure 9.

Figure 9.

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Increased expression of Kindlin-2 in human fibrotic kidney. (A1) Representative micrographs show the collagen deposition (blue) detected by Masson trichrome staining (a and b) and Kindlin-2 (c and d), TGF-β1 (e and f), and p-Smad3 (g and h) expression detected by immunohistochemical staining in human normal kidneys or fibrotic kidneys from patients with renal tubulointerstitial fibrosis. (A2) Control studies using nonimmuno-IgG or PBS instead of primary antibody (anti-Kindlin-2) showed negative staining. (B) Relative amount of collagen deposition in human normal kidneys (n=3) and human fibrotic kidneys (n=8). (C) Relative expression of Kindlin-2, TGF-β1, and p-Smad3 in human normal kidneys (n=3) and human fibrotic kidneys (n=8). *P<0.05 versus normal kidney. Scale bar in A1, 50 μm.

Discussion

It is well established that activation of TGF-β/Smad signaling plays a critical role in the pathogenesis of TIF. Although phosphorylation of R-Smad by TβR has been documented as a key initial event in activation of TGF-β/Smad signaling, there remains a gap in understanding the mechanism underlying the interaction of TβR with R-Smad. This study established Kindlin-2 as a previously unrecognized adaptor in TGF-β signaling by building a molecular platform for the engagement of TβRI and Smad3. Depletion of Kindlin-2 in vitro and in vivo significantly suppressed TGF-β–triggered Smad3 activation and the target gene expression. Importantly, we found that Kindlin-2 expression was markedly induced in vivo, predominantly in TECs, in both UUO and human TIF. Knocking down Kindlin-2 in UUO remarkably ameliorated TIF. These results demonstrated for the first time that Kindlin-2 played a crucial role in activation of TGF-β/Smad signaling and might be involved in the pathogenesis of TIF.

The most novel finding in this study is that Kindlin-2 regulates the activation of TGF-β/Smad signaling in TECs. TGF-β/Smad signaling is a central intracellular signaling involved in a multitude of biologic processes including tissue fibrogenesis.8,9 The conclusion is supported by several lines of evidence. First, Kindlin-2 was mainly expressed in renal TECs. In cultured HKCs, Kindlin-2 interacted with TβRI through its FERM domain and bound with Smad3 via its N terminal. Knockdown of Kindlin-2 largely prevented the interaction of TβRI and Smad3, suggesting that Kindlin-2 acts as a “bridge” for engagement of TβRI with Smad3. Second, overexpression of Kindlin-2 enhanced TGF-β1–induced Smad3 activation. Vice versa, depletion of Kindlin-2 significantly inhibited TGF-β1–induced Smad3 activation both in vitro and in vivo. Third, knockdown of Kindlin-2 suppressed the expression of TGF-β1 target genes in both TGF-β1–stimulated HKCs and in UUO kidney. Although renal fibroblasts respond strongly to TGF-β1,32 our data showed that Kindlin-2 was not required for TGF-β1–induced Smad3 activation in fibroblasts, probably because of the weak expression of Kindlin-2 in this type of cells (Supplemental Figure 1). Taken together, our data demonstrated for the first time that Kindlin-2 plays a critical role in activation of TGF-β/Smad signaling in renal TECs.

Previous studies have shown that Kindlin-2 binds to β1 integrin and functions as an adaptor protein in integrins activation.20,30 In this study, we demonstrated that the role of Kindlin-2 in TGF-β/Smad signaling is independent of β1 integrin: first, knockdown of β1 integrin did not affect the interaction of Kindlin-2 with TβRI and Smad3. In addition, β1 integrin siRNA did not influence the promoting effect of Kindlin-2 on TGF-β–induced Smad3 phosphorylation. Meanwhile, Kindlin-2 mutant not binding to integrin could mimic the effect of wild-type Kindlin-2. Furthermore, depletion of Kindlin-2 or Smad3 remarkably inhibited TGF-β1–induced β1 integrin expression, suggesting that Kindlin-2-augemented Smad3 activation is the upstream event of TGF-β1–induced β1 integrin expression. Consistent with our study, a previous study showed that TGF-β1–induced upregulation of β1 integrin is Smad3 dependent.29 Finally, renal expression of β1 integrin was significantly upregulated in UUO mice, a model with significantly increased expression of TGF-β1. Depletion of Kindlin-2 in this model significantly decreased β1 integrin expression in renal tissues. Given that Kindlin-2 plays critical role in initiating of TGF-β/Smad signaling, the pathway regulates a variety of proinflammatory and profibrotic downstream events and not only integrin activation.

Supporting our findings, several other adaptors have been suggested to regulate TGF-β/Smad signaling. SARA regulates the TGF-β pathway via bringing Smad2 to TβR.16 ELF affects TGF-β–dependent transcriptional response by controlling the localization of Smad3 and Smad4.33 These reports, together with our findings, suggest that formation of the TβR/Smads complex requires various adaptor proteins that positively or negatively modulate the activity of TGF-β/Smad signaling. Therefore, identifying individual adaptor protein and its function would be an important step for understanding the mechanisms underlying the activation of the signaling.

TIF is considered as a central event in the progression of CKD.34 Extensive studies have revealed that the impairment of renal function correlates better with the extent of tubulointerstitial damage than with the degree of glomerular injury.35 Although the exact mechanisms underlying TIF have not completely understood, dysregulation of TGF-β signaling is believed to play a pivotal role.36 Our in vivo data implicated Kindlin-2 in the development and progression of TIF primarily by modulating TGF-β signaling. In UUO mice, Kindlin-2 was significantly upregulated, predominantly in renal TECs. Induction of Kindlin-2 was accompanied by enhanced phosphorylation of Smad3 and upregulation of the fibrosis-related genes such as Col I, α-SMA, and Snail.37 Furthermore, in vivo depletion of Kindlin-2 inhibited Smad3 activation, suppressed matrix genes expression, decreased renal β1 integrin expression, and altogether resulted in attenuated TIF in UUO mice. Supporting the observations, Kindlin-2 was significantly upregulated in kidney specimens from patients with TIF, which was associated with overexpression of TGF-β1 and activation of Smad3. Altogether, these data have demonstrated that Kindlin-2 is a crucial contributor to the development of TIF.

In summary, we demonstrated that Kindlin-2 facilitated activation of TGF-β/Smad signaling in renal TECs via recruiting Smad3 to TβRI and contributed to the pathogenesis of TIF. In view of the central role of TGF-β/Smads in fibrosis and other human diseases, our findings offer novel insights into the mechanism underlying activation of TGF-β/Smad signaling and development of TIF. These studies are also instrumental for designing rational strategies for the treatment of fibrotic kidney diseases.

Concise Methods

Plasmids

The construction of plasmid pFlag-Kindlin-2 was previously reported.38 A Smad3 mutant (Smad3D407E) was constructed (Sunbiotech Co., Beijing, China) as described previously.25 Expression plasmids for TβRI and TβRII were provided by Dr. Chen YG (Tsinghua University, Beijing, China). Kindlin-2 QW mutant is an integrin-binding deficient Kindlin-2 Q614A/W615A mutant.39 Kindlin-2 siRNA-resistant mutant is a siRNA-resistant Kindlin 2 G147C/G150A mutant.26

Cell Culture and Transfection

HKCs were cultured as described.40 HKCs were grown in DMEM/F12 medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA). NRK-49F cells were cultured in DMEM medium supplemented with 10% FCS. The cells were seeded in complete medium. When the cells reached at approximately 70% confluence, they were serum-starved overnight and then treated with recombinant TGF-β1 (R&D Systems, Minneapolis, MN) for various time periods as indicated.

For transient transfection, 50%–80% confluent cells were transfected with indicated plasmids or siRNA using the Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen).

siRNA Inhibition of Kindlin-2

Specific siRNA targeting human Kindlin-2 (Kindlin-2 siRNA) was designed according to the human Kindlin-2 cDNA sequence and synthesized by Qiagen (Hamburg, Germany). The sense targeting sequence was as follows41: AAGCUGGUGGAGAAACUCG. Integrin β1 and Smad3 siRNA was designed and synthesized by RiboBio Co. (Guangzhou, China). The sense targeting sequence was as follows: Integrin β1, GGTAGAAAGTCGGGACAAA; and Smad3, TGGTGCGAGAAGGCGGTCA. Three siRNAs targeting rat Kindlin-2 were synthesized by RiboBio. The sense targeting sequence was as follows: (1) CCTGGATCAGGAAGCATAT; (2) CCGGTAACATCACCAGAAA; and (3) GCCTCAAGCTCTTCTTGAT. An irrelevant dsRNA with the sense sequence CGAGUGGUCUAGUUGAGAA was used as the control.

Real-Time PCR

Total RNA from HKCs or mice kidney tissue homogenate was extracted by Trizol (Invitrogen). Two micrograms of RNA was reverse transcribed using MMLV RT (Promega, Sunnyvale, CA). Real-time PCR was set up using SYBR Green mix (Applied Biosystems, Foster City, CA) with the following PCR condition: 95°C for 3 minutes; 90°C for 20 seconds, and 60°C for 1 minute, for 40 cycles. The sequences of the primer pairs are given in Supplemental Table. Expression of various genes was determined by the comparative CT method (2-ΔΔCT).

Western Blot Analyses

Cell or tissue lysates were prepared using PBS-TDS lysis buffer containing 1× cocktail inhibitor (Boehringer Mannheim, Mannheim, Germany). Samples were heated at 95°C for 5 minutes and then separated on SDS-PAGE gels. Transfer membranes were immunoblotted with primary antibodies against Kindlin-2 (Millipore, Billerica, MA), Kindlin-1 (Millipore), Col I (Bioworld, Minneapolis, MN), α-SMA (Abcam, Cambridge, MA), Snail (Millipore), fibronectin (Abcam), p-Smad3 (Abcam), Smad2 (Epitomics, Burlingame, CA), Smad3 (Epitomics), Smad4 (Epitomics), β1 integrin (Epitomics), TβRI (Santa Cruz Biotechnology, Santa Cruz, CA), TβRII (Santa Cruz), Actin (Santa Cruz), YY-1 (Santa Cruz), or Flag (Sigma-Aldrich, St. Louis, MO) overnight at 4°C. After extensive washing in TBS buffer, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody or for 1 hour at room temperature. Immobilized antibodies were then detected by enhanced chemiluminescence (Amersham Biosciences, Sunnyvale, CA). Quantification was performed by measurement of the intensity of the bands using ImageJ analysis software (National Institutes of Health, Bethesda, MD).

Immunoprecipitation

Immunoprecipitation was performed according to a previously described method.42 Lysates were prepared in RIPA buffer (1× PBS, pH 7.4, 0.5% Sodium deoxycholate, 1% Triton X-100, 0.1% SDS) with protease inhibitor cocktail, followed by centrifugation to remove cell debris. Protein complexes were obtained by incubating precleared lysates with indicated antibodies or normal IgG (as controls) overnight at 4°C, respectively. Immune complexes were washed three times with RIPA buffer and separated by SDS-PAGE gels. Transfer membranes were probed with indicated primary antibodies and horseradish peroxidase–conjugated antibody TrueBlot (eBioscience, San Diego, CA) as the secondary antibody.43 The membranes were detected by enhanced chemiluminescene, as mentioned above.

Immunofluorescence Staining

Immunofluorescence staining was performed as previously described .44 Briefly, the cells cultured on coverslips were washed with cold PBS twice, fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and stained with the specific primary antibodies against Flag, HA (Sigma-Aldrich), or Kindlin-2 for 14 hours at 4°C, followed by incubation with secondary antibody conjugated with Alexa Fluor 488 or 588 (Invitrogen). Cells were also stained with 4',6-diamidino-2-phenylindole to visualize the nuclei. Intracellular localization was visualized using a confocal microscopy (Leica Microsystems, Wetzlar, Germany).

Animal Model and Kindlin-2 siRNA Treatment

Male ICR mice weighing 20–25 g were obtained from and housed in a Beijing University animal facility. The mice were divided into three groups (n=5 in each group): (1) sham-operated mice receiving control siRNA, (2) UUO mice receiving control siRNA, and (3) UUO mice receiving Kindlin-2 siRNA. UUO was performed using an established procedure as described.45,46 Administration of Kindlin-2 siRNA or control siRNA into mice was performed as described previously.47–49 Briefly, synthetic Kindlin-2 or control siRNA (50 μg in 1 ml of PBS) was rapidly injected (within 10 seconds) into one of the tail side veins of the mice (twice a week). Mice were euthanized at 7 days after surgery. All experiments were approved by the Beijing University Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments of Beijing University.

Partially 2′-O-methyl modified siRNA against Kindlin-2 and the negative control siRNA were synthesized by RiboBio. The target sequence used for knockdown of Kindlin-2 in this study was 5′-AAG TTG GTG GAA AAA CTC GAT-3′.

Immunohistochemistry

Immunohistochemical staining for specific protein expression was performed on kidney sections using methods previously described .50 Briefly, sections (4 μm thickness) were deparaffinized with xylene, followed by rehydration in ethanol. Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. Sections were incubated overnight at 4°C with primary antibodies against Kindlin-2 (Abcam), TGF-β1 (Santa Cruz), p-Smad3 (Abcam), AQP-1 (Abcam), NCC (Millipore), and FSP-1 (Epitomics). After extensive washing in PBS buffer, sections were then incubated for 30 minutes with secondary antibodies (Dako, Carpinteria, CA). Control experiments included omission of the primary antibodies and substitution of the primary antibodies with nonimmune rabbit or mouse IgG. The immunostaining was examined by an Olympus BX51 microscope (Olympus, Tokyo, Japan). Positive stains were quantified using image analysis software50 (Image Pro-Plus, Media Cybernetics, Silver Spring, MD).

Masson Trichrome Staining

Selected renal sections were stained using the Masson Trichrome Stain Kit (Richard-Allan Scientific, Kalamazoo, MI) according to the manufacturer’s protocols.51

Human Kidney Specimens

Kidney biopsies samples were obtained from eight patients with chronic renal tubulointerstitial fibrosis (three with IgA nephropathy, two with membranous nephropathy, one with membranoproliferative nephritis, one with chronic tubulointerstitial nephropathy, and one with crescentic GN). Interstitial fibrosis scores of the patients were calculated as previously described.52 The mean (±SD) of interstitial fibrosis score was 2.3±0.4. Normal kidney tissues adjacent to renal carcinoma (n=3) obtained from nephrectomies were used as controls. The study was approved by Medical Ethics committee of Southern Medical University. Written informed consent was obtained from all patients.

Statistical Analyses

Data are presented as the mean ± SD. Comparisons between two groups were made using two-tailed t test. Differences among more than two groups were compared using one-way ANOVA. Pairwise comparisons were evaluated by the Student–Newman–Keuls procedure or Dunnett’s T3 procedure when the assumption of equal variances did not hold. A P value <0.05 was considered statistically significant.

Disclosures

None.

Acknowledgments

This work was supported by the Major State Basic Research Development Program of China (973 program) (grants 2012CB517700 to F.F.H. and 2010CB912203 to H.Z.). This work was also partially supported by the 973 program (grant 2011CB504005 to F.F.H.), the National Natural Science Foundation of China (key program grants U0932002 to F.F.H. and 30830048 to H.Z.), and a Leading Academic Discipline Project of Beijing Education Bureau (to H.Z.).

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

See related editorial, “Kindlin-2: A New Player in Renal Fibrogenesis,” on pages 1339–1340.

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