CKD is highly prevalent worldwide, especially in the elderly.1 Compared with younger counterparts, the elderly have higher rates of CKD, and mortality and renal function decline are worse in older than in younger patients.1,2 Interestingly, CKD-affected kidneys have many characteristics associated with natural aging, such as glomerular sclerosis and interstitial fibrosis,3 tubular atrophy,4 local renin-angiotensin-aldosterone system activation,5 loss of repair capabilities6 in renal tubular cells, and intracellular deposition of lipofuscin, a strong indicator of senescence.7,8 These findings suggest CKD is a clinical manifestation of premature aging or accelerated senescence.9
Cellular senescence, a stress-induced growth arrest, has recently been found in multiple kidney diseases2,7,10,11 and tubulointerstitial nephritis.11 The accumulation of senescent cells has been detected in early clinical nephropathy, when patients have mild proteinuria and normal GFR.4,10 Accelerated senescence may promote more pathologic alterations in kidneys7,8,12 and even reduce the healthy lifespan.13
The key characteristics of cellular senescence are growth arrest and loss of DNA replication, breakdown of DNA double strands,14 and accumulation of senescence-related proteins mainly through p16INK4A-retinoblastoma (Rb) and ARF-p53-p21 pathways.15,16 These pathways collectively halt cell proliferation and accelerate cellular senescence. Senescent cells can be identified by senescence-associated β-galactosidase (SA–β-gal) activity,4,13 and these cells produce components of the senescence-associated secretory phenotype (SASP), including proinflammatory cytokines such as IL-6 and matrix-synthesizing molecules such as TGF-β1.17,18
Cellular senescence can be caused by DNA damage, mitochondria dysfunction, inflammation, oxidative stress, and epigenetic alterations, each of which is a common characteristic feature in CKD.4,13,19,20 Of note, among somatic cells of the kidney, tubular cells are the most likely to transition to the senescent phenotype.4,10,11,21 However, the underlying molecular mechanism of tubular senescence has not been elucidated.
Wnt/β-catenin signaling is an evolutionarily conserved pathway involved in organ development and tissue repair.6 In normal adults, Wnt/β-catenin signaling is silent, but it is reactivated after kidney injury in a wide range of CKD models.5,22,23 Recent publications show the aberrant expression of Wnt/β-catenin is highly associated with progression of renal fibrosis5,23 and tubular atrophy.3 Such pathologic effects may result from increased hyperactivity of local renin-angiotensin-aldosterone system5 and repressed expression of Klotho, an antiaging protein,22 caused by the increase in Wnt/β-catenin signaling. These findings suggest the intimate correlation between Wnt activation and cellular senescence. However, whether Wnt ligands directly promote renal tubular cell senescence and the progression of renal fibrosis remains unknown.
In this study, we examined the role of Wnt9a in CKD-associated renal fibrosis and tubular senescence in vivo and in vitro, which led to the identification of a Wnt9a-induced reciprocal activation loop between senescent tubular cells and activated interstitial fibroblasts. Our results suggest that Wnt9a promotes renal fibrosis through the induction of tubular senescence and crosstalk between tubular cells and interstitial fibroblasts.
Results
Wnt9a Increases in Multiple Types of Clinical Nephropathy and Experimental CKD Models and Associates with Tubular Senescence
To test the clinical relevance of Wnt ligands in the pathogenesis of CKD, we conducted immunostaining in kidney biopsy specimens from 35 patients with various nephropathies, including minimal change disease, IgA nephropathy (IgAN), diabetic nephropathy, membranous nephropathy, lupus nephritis, and others. Most of these diseases are highly associated with accelerated cellular senescence.4,11,12 Compared with the absence of signal in normal human kidneys, the signal for Wnt9a strikingly increased in all biopsy specimens from patients with CKD (Figure 1A). Notably, Wnt9a was predominantly localized in the renal tubular epithelium in human diseased kidneys (Figure 1A, arrows). Furthermore, immunostaining on sequential sections of human IgAN biopsy specimens confirmed the localization of Wnt9a in tubular cells with Ki67-negative nuclei, a feature of senescent cells (Figure 1B, yellow arrows).14 Because p16INK4A is also a typical senescence-related protein marker,13 we examined the colocalization of Wnt9a and p16INK4A expression. As shown in Figure 1C, the expression of Wnt9a nearly completely colocalized with p16INK4A-positive tubules. Furthermore, among 30 patients with sequentially sectioned biopsy specimens available for additional analysis, there was a positive correlation between Wnt9a and p16INK4A expression levels (Figure 1D). Additionally, Wnt9a expression correlated with renal fibrosis and decline of eGFR (Figure 1, E and F). The demographic and clinical data of the patients with CKD are presented in Supplemental Table 1.
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Figure 1.: Wnt9a is induced specifically in renal tubular epithelium in human CKD. (A) Representative micrographs show the expression and localization of Wnt9a in various types of human CKD. Nontumor kidney tissue from the patients who had renal cell carcinoma and underwent nephrectomy was used as normal controls. Arrows indicate Wnt9a-positive tubules. Scale bar, 50 μm. (B) Representative micrographs show Wnt9a localized in those tubules with nuclei negative for expression of Ki67. Sequential paraffin-embedded kidney sections from patients with IgAN were immunostained for Wnt9a and Ki67. Yellow arrows indicate Wnt9a-positive tubules lack Ki67 nuclear expression. Scale bar, 50 μm. (C) Colocalization of Wnt9a and p16INK4A in tubules of patients with IgAN. Sequential paraffin-embedded kidney sections were immunostained for Wnt9a and p16INK4A. Colocalization of Wnt9a and p16INK4A in tubules is indicated by arrows. Scale bar, 50 μm. (D and E) Scatter plots with linear regression show significant correlation between Wnt9a expression levels and (D) p16INK4A expression levels and (E) extent of renal fibrosis. Expression of p16INK4A and fibrosis were quantitatively assessed by analysis of immunostaining and Masson trichrome staining, respectively, by three individuals who were blinded to Wnt9a data. The Spearman correlation coefficient (R) and P value are shown. (F) Linear regression shows an inverse correlation between Wnt9a expression level and eGFR. The Spearman correlation coefficient (R) and P value are shown. LN, lupus nephritis; MCD, minimal change disease; MN, membranous nephropathy.
To further evaluate the role of Wnt9a in tubular senescence and progression of CKD, we assessed the expression of Wnt9a in different CKD models, including mice subjected to ischemia-reperfusion injury (IRI), unilateral ureteral obstruction (UUO), and adriamycin (ADR) nephropathy. As shown in Figure 2, A and B, Wnt9a expression increased as early as 1 day after severe IRI, although this increase was not yet significant. Three days after severe IRI, the key transitional time point at which AKI progresses to CKD through sustained activation of Wnt(s) signaling,24 Wnt9a expression was significantly upregulated, as was the expression of histone γH2AX. γH2AX is a selective marker of DNA double-strand breaks, and an increase in the number of γH2AX foci is an important feature of senescence.25,26 We also assessed the expression of the antiaging protein Klotho, the loss of which derepresses Wnt signaling and serves as a marker of tubular damage and CKD.22 As shown in Figure 2, A and C, as Wnt9a expression increased after IRI, Klotho expression correspondingly decreased. We then checked the expression of Wnt9a in a mouse model of UUO, in which p16INK4A is highly expressed in tubules.27 Our immunostaining results demonstrated that Wnt9a expression was remarkably high in renal tubules of these mice (Figure 2D, arrow). Furthermore, western blot analysis confirmed the upregulation of Wnt9a and γH2AX, as well as the downregulation of Klotho, in UUO mice (Figure 2, E–G). In the ADR nephropathy model, Wnt9a expression was high 3 weeks after ADR injection, and this effect was also accompanied by increased expression of γH2AX and loss of Klotho (Figure 2, H–J). In sequential sections of kidneys from ADR mice, Wnt9a expression largely colocalized with p16INK4A expression (Figure 2K, arrows). In these sections, we observed expression of p16INK4A in the nucleus as well as the tubular cytoplasmic area, which suggests that these cells are subjected to unique disease-specific stresses, as previously reported.11 Wnt9a expression did not correlate with tubular cell apoptosis in sequential sections of ADR-injected kidney (Supplemental Figure 1), consistent with the notion that senescent cells are generally resistant to apoptosis.28 These results suggest the intimate correlation between upregulation of Wnt9a and tubular senescence in kidney injury.
Figure 2.: Wnt9a is commonly induced and accompanied by increasing expression of γH2AX and loss of Klotho expression in various animal models of CKD. (A) Representative western blots showing the renal expression of Wnt9a, γH2AX, and Klotho in sham and injured kidneys 1 or 3 days after IRI. (B and C) Graphical representations of (B) Wnt9a, γH2AX, and (C) Klotho expression levels in three groups, as indicated. *P<0.05 versus sham controls (n=5–6). (D) Representative staining showing induction of Wnt9a protein expression in tubules in the UUO group. Scale bar, 50 μm. (E) Representative western blots showing the renal expression of Wnt9a, γH2AX, and Klotho in sham and obstructed kidneys 7 days after UUO. (F and G) Graphical representations of (F) Wnt9a, γH2AX, and (G) Klotho protein expression levels in two groups, as indicated. *P<0.05 versus sham controls (n=5–6). (H) Representative western blots showing renal Wnt9a, γH2AX, and Klotho expression in ADR nephropathy 3 weeks after ADR injection. CTL, control. (I and J) Graphical representations of (I) Wnt9a and γH2AX and (J) Klotho protein expression levels in two groups, as indicated. *P<0.05 versus normal controls (n=5–6). (K) Colocalization of Wnt9a and p16INK4A in ADR nephropathy tubules. Sequential paraffin-embedded kidney sections were immunostained for Wnt9a and p16INK4A. Colocalization of Wnt9a and p16INK4A in tubules is indicated by arrows. Scale bar, 50 μm.
We also detected the expression of Wnt9a in a developmental senescence model, consisting of mouse embryonic day 14.5 (E 14.5) mesonephric tubules29 and kidneys from 2-, 12-, and 24-month-old mice (Supplemental Figure 2). Although we detected p16INK4A expression in the E 14.5 mesonephric tubules, we did not detect the expression of Wnt9a. However, the expression of Wnt9a increased in association with the progression of aging-associated kidney fibrosis in adults.
In Vivo Expression of Wnt9a Augments Kidney Injury after IRI
To establish the role of Wnt9a in renal fibrosis, we delivered a Flag-tagged Wnt9a-expression vector (pFlag-Wnt9a) to mice by hydrodynamic-based gene delivery, an approach that is routinely used in our laboratory for in vivo expression of a variety of genes.22,24 As shown in Supplemental Figure 3, Wnt9a was significantly induced in kidneys 24 hours after intravenous injection of naked pFlag-Wnt9a plasmid. We then investigated the effect of exogenous Wnt9a in a mouse model of unilateral IRI (UIRI). As shown in Figure 3A, pFlag-Wnt9a or empty vector (pcDNA3) was administered intravenously by the hydrodynamic-based gene transfer technique22 4 days after UIRI. Western blot analyses revealed that Wnt9a expression was induced at 7 days after a single injection of the Wnt9a expression plasmid (11 days after UIRI) (Figure 3, B and C). We further detected the pathologic morphology by periodic acid–Schiff (PAS) staining, a method that allows discernment of injured tubules through the detection of glycogen content. As shown in Figure 3, D and E, in vivo expression of exogenous Wnt9a significantly exacerbated the morphologic injury induced by IRI alone. This injury was characterized by tubular dilation, hyaline casts, and tubular atrophy with thickened basement membranes, as well as detached epithelial cells in tubular lumens. We then examined urinary N-acetyl-β-D-glucosaminidase (NAG), a well known marker of tubular damage.30 As shown in Figure 3F, urinary NAG level was significantly elevated by IRI injury but further increased by expression of exogenous Wnt9a. Consistent with this result, levels of serum creatinine (Scr) (Figure 3G) and urinary albumin (Ualb) (Figure 3H) were further increased by in vivo expression of exogenous Wnt9a after IRI. These data indicate that the aberrant expression of Wnt9a accelerates pathologic kidney damage and functional decline.
Figure 3.: Overexpression of Wnt9a promotes tubular injury and impairs renal function in IRI. (A) Experimental design. Green arrow indicates the injection of pcDNA3 or pFlag-Wnt9a plasmid. Red arrows indicate the timing of renal IRI surgery. (B) Representative western blots showing renal expression of Wnt9a in three groups, as indicated. (C) Graphical representation of Wnt9a protein expression levels in three groups. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (D) Representative micrographs show kidney morphology after IRI injury in different groups of mice, as indicated. Images of PAS staining are shown. Boxed areas are enlarged in the bottom panels. Arrows indicate injured tubules. Scale bar, 50 μm. (E) Quantitative analyses of injured tubules in three groups, as indicated. Kidney sections were subjected to PAS staining. At least 20 randomly selected fields were evaluated under ×400 magnification and results were averaged for each kidney. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (F) Urinary NAG level, expressed as international units per gram creatinine, in different groups. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (G and H) Ectopic expression of Wnt9a increased Scr and Ualb levels in IRI mice. Quantitative analysis of (G) Scr and (H) Ualb levels in three groups, as indicated. Scr was expressed as milligrams per deciliter, and Ualb was expressed as micrograms per milligram urinary creatinine. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). Ucr, urinary creatinine; UNx, unilateral nephrectomy.
In Vivo Expression of Wnt9a Aggravates Renal Fibrosis and Activates β-Catenin Signaling after IRI
We further investigated the effects of exogenous Wnt9a on matrix gene expression and renal fibrotic lesions after IRI. Analysis of Masson trichrome staining indicated that in vivo expression of exogenous Wnt9a aggravated renal fibrotic lesions (Figure 4, A and B) and further induced fibronectin and α–smooth muscle actin (α-SMA) protein expression in IRI mice (Figure 4, C–E). Consistently, exogenous Wnt9a further upregulated collagen I mRNA levels in kidneys after IRI (Figure 4F). Similar results were observed when kidney sections were immunostained with antibodies against fibronectin and collagen I (Figure 4G). Furthermore, as shown in Figure 4G, exogenous Wnt9a strikingly increased the expression of fibroblast-specific protein–1 (FSP-1), a target gene of β-catenin that is also known as S100A4 protein and is a strong indicator of activated myofibroblasts in diseased kidneys.5,23 These data indicate that activation of Wnt9a is a major driver of kidney fibrogenesis.
Figure 4.: Ectopic expression of Wnt9a aggravates renal fibrosis in IRI mice. (A) Representative micrographs show that Wnt9a exacerbated renal interstitial fibrosis. Kidney sections were subjected to Masson trichrome staining. Arrow indicates positive staining. Scale bar, 50 μm. (B) Graphical representation of kidney fibrotic lesion area in different groups after quantitative determination. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (C) Representative western blots show renal expression of fibronectin and α-SMA in three groups, as indicated. (D and E) Graphical representations of (D) fibronectin and (E) α-SMA expression levels in different groups. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (F) Graphical representation of the relative abundance of collagen I mRNA in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (G) Representative immunostaining micrographs show fibronectin, collagen I, and Fsp-1 expression in different groups. Boxed areas are enlarged and presented in the right column. Arrows indicate positive staining. Scale bar, 50 μm.
We next analyzed the expression of β-catenin signaling, which is activated by Wnt ligand(s) and highly associated with the progression of renal fibrosis.23,31 As shown in Figure 5, A and B, immunostaining and quantitative real-time PCR results showed that in vivo expression of the Wnt9a gene in mice further aggravated the expression of β-catenin and MMP-7, a transcriptional target of canonic Wnt/β-catenin signaling and a pathogenic mediator of kidney fibrosis.3,32 Similarly, quantitative real-time PCR results showed exogenous Wnt9a expression further upregulated the expression of PAI-1, another transcriptional target of β-catenin (Figure 5C).33 Furthermore, as shown in Figure 5, D–G, western blot analyses of whole-kidney lysates revealed that expression of exogenous Wnt9a promoted the expression of β-catenin, MMP-7, and PAI-1 proteins. In addition, exogenous Wnt9a induced the expression of Snail 1, a key transcription factor of β-catenin signaling that is regarded as a “master gene” in the control of renal fibrogenesis (Figure 5, D and H).34
Figure 5.: In vivo expression of exogenous Wnt9a promotes the expressions of β-catenin and targeted genes. (A) Immunohistochemical staining showing the renal induction of β-catenin and MMP-7 after injection of Wnt9a expression plasmid. Arrows indicate positive staining. (B and C) Graphical representations of the relative abundance of (B) MMP-7 and (C) PAI-1 mRNA in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (D) Representative western blots show renal expression of β-catenin, MMP-7, PAI-1, and Snail 1 in three groups, as indicated. (E–H) Graphical representations show expression of (E) β-catenin, (F) MMP-7, (G) PAI-1, and (H) Snail 1 in three groups. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6).
In Vivo Expression of Wnt9a Accelerates Tubular Senescence after IRI
To further clarify the mechanism by which Wnt9a promotes renal fibrosis, we analyzed tubular senescence. As shown in Figure 6A, quantitative real-time PCR results indicated that IRI induced the renal expression of p16INK4A mRNA, and in vivo expression of exogenous Wnt9a dramatically aggravated this effect. Moreover, in vivo expression of exogenous Wnt9a further inhibited the expression of Klotho after IRI (Figure 6, B and C). We next assessed the protein expression levels of p16INK4A and TGF-β1, which is secreted by tubular cells that have adopted the SASP.17,18 Immunostaining results indicated that in vivo expression of exogenous Wnt9a significantly aggravated the IRI-induced increase in p16INK4A and TGF-β1 expression and SA–β-gal activity in tubules (Figure 6D). These findings strongly suggest that Wnt9a-induced tubular senescence could be a mediator of renal fibrosis (Figure 6D).
Figure 6.: Overexpression of Wnt9a after IRI accelerates cellular senescence in renal tubules. (A) Expression of p16INK4A mRNA in different groups was assessed by real-time PCR. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (B) Representative western blots show that Klotho expression was further downregulated after injection of Wnt9a expression plasmid. (C) Graphical representations of Klotho protein expression levels in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (D) Representative staining micrographs show p16INK4A and TGF-β1 expression and SA–β-gal activity in different groups. Paraffin sections were immunostained with antibodies against p16INK4A and TGF-β1. Frozen kidney sections were stained for SA–β-gal activity, which appears as bright-blue granular staining in the cytoplasm of tubular epithelial cells. Boxed areas are enlarged and presented in the right column. Arrows indicate positive staining. Scale bar, 50 μm.
To further investigate the role of Wnt9a in tubular senescence, we assessed tubular senescence and renal fibrosis in 9-month-old mice subjected to exogenous Wnt9a expression without additional renal damage. As shown in Supplemental Figure 4, overexpression of Wnt9a in the absence of disease was sufficient to accelerate tubular senescence and renal fibrosis. These data suggest the important role of Wnt9a in tubular senescence and development of renal fibrosis.
Knockdown of Wnt9a Protects Renal Function and Kidney Fibrogenesis after IRI
To further confirm the role of Wnt9a in kidney fibrogenesis, mice were intravenously injected with an shRNA vector encoding the interference sequence for Wnt9a (pLVX-shWnt9a) through a hydrodynamic-based gene delivery approach (Figure 7A). As shown in Figure 7, B and C, renal expression of Wnt9a was almost completely abolished in IRI mice by intravenous injection of Wnt9a shRNA plasmid. The IRI-induced increase in Scr and Ualb levels was also significantly blocked by knockdown of Wnt9a expression (Figure 7, D and E). Immunofluorescence revealed that knockdown of Wnt9a also reduced the deposition of major interstitial matrix proteins, including fibronectin and collagen I (Figure 7F), and western blot analysis showed that knockdown of Wnt9a largely inhibited the expression of fibronectin and α-SMA in IRI kidneys (Figure 7, G–I). Consistently, Masson trichrome staining revealed that knockdown of Wnt9a attenuated the extent of renal fibrotic lesions in these mice (Figure 7, J and K).
Figure 7.: Knockdown of Wnt9a ameliorates renal injury after IRI. (A) Experimental design. Green arrow indicates the injection of pLVX-shRNA (Ctrl-shR) or pLVX-shWnt9a (Wnt9a-shR) plasmids. Red arrows indicate the timing of renal IRI surgery. (B) Representative western blots show expression of Wnt9a was nearly completely abolished after injection with Wnt9a-shR plasmid. (C) Graphical representation of Wnt9a protein expression levels in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (D and E) Knockdown of Wnt9a significantly decreased Scr and secretion of Ualb. Quantification of (D) Scr and (E) Ualb levels in three groups, as indicated. Ualb level was expressed as micrograms per milligram urinary creatinine. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (F) Representative immunofluorescence micrographs show collagen I and fibronectin expression in three groups. Arrows indicate positive staining. Scale bar, 50 μm. (G) Representative western blots show fibronectin and α-SMA expression in three groups, as indicated. (H and I) Graphical representations of (H) fibronectin and (I) α-SMA protein expression levels in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (J) Representative micrographs show collagen deposition in different groups, as indicated. Paraffin sections were used for Masson trichrome staining. Arrow indicates positive staining. Scale bar, 50 μm. (K) Graphical representation of the extent of fibrosis. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). Ucr, urinary creatinine; UNx, unilateral nephrectomy.
Knockdown of Wnt9a Inhibits Tubular Senescence and Epithelial Injury after IRI
We next investigated the relevance of Wnt9a knockdown in tubular senescence and cellular injury. As shown in Figure 8A, staining analyses revealed that knockdown of Wnt9a after IRI largely blocked the IRI-induced increase in p16INK4A expression and SA–β-gal activity in tubules. Conversely, the tubular expression of Klotho was remarkably preserved by knockdown of Wnt9a after IRI (Figure 8A). As shown in Figure 8, B–E, knockdown of Wnt9a significantly inhibited IRI-induced expression of p16INK4A and TGF-β1 and reversed the expression of Klotho in kidneys. To further confirm the role of Wnt9a in tubular damage, we examined the expression of E-cadherin, an epithelial marker that maintains normal epithelial integrity.35 As shown in Figure 8F, knockdown of Wnt9a after IRI largely preserved the expression of E-cadherin in tubules. These data further suggest that Wnt9a has an important role in tubular senescence and cellular injury.
Figure 8.: Knockdown of Wnt9a inhibits tubular cell senescence in IRI model. (A) Representative micrographs show staining for p16INK4A expression, SA–β-gal activity, and Klotho expression in different groups. Paraffin sections were immunostained with antibodies against p16INK4A and Klotho. Frozen kidney sections were stained for SA–β-gal activity. Arrows indicate positive staining. Scale bar, 50 μm. (B) Western blot analyses show p16INK4A, TGF-β1, and Klotho expression in three groups, as indicated. (C–E) Graphical representations of (C) p16INK4A, (D) TGF-β1, and (E) Klotho protein expression levels in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6). (F) Representative micrographs show knockdown of Wnt9a reversed expression of E-cadherin after IRI, as indicated. Frozen sections were immunostained with antibody against E-cadherin. Scale bar, 50 μm. Ctrl-shR, injection of pLVXshRNA; Wnt9a-shR, injection of pLVX-shWnt9a.
Knockdown of p16INK4A Inhibits Wnt9a-Induced Renal Fibrosis in IRI Mice
To establish a causative link between Wnt9a-induced renal fibrosis and senescence, we examined shRNA-mediated in vivo silencing of p16INK4A in the model of UIRI mice injected with pFLAG-Wnt9a (Figure 9A). The silencing efficacy of renal p16INK4A was confirmed by western blotting. Exogenous Wnt9a-induced expression of p16INK4A in IRI mice was mostly abolished by intravenous injection of p16INK4A shRNA plasmid (Figure 9, B and C). As shown in Figure 9D, knockdown of p16INK4A tended to prevent the increase in Scr induced by IRI and aggravated by exogenous Wnt9a, but this finding was not statistically significant (P=0.07; n=5–6). Knockdown of p16INK4A also reduced the renal fibrosis observed after IRI and Wnt9a overexpression (Figure 9, E and F) and significantly attenuated Wnt9a-induced expression of fibronectin and α-SMA (Figure 9, G–J). These data suggest that Wnt9a could induce tubular senescence, which has a causative link to development of renal fibrosis.
Figure 9.: Knockdown of p16INK4A inhibits Wnt9a-induced renal fibrosis in IRI mice. (A) Experimental design. Red arrow indicates the injection of pcDNA3, pFlag-Wnt9a, or pFlag-Wnt9a with pLVX-shp16INK4A (p16-shR) plasmid. Black arrows indicate the timing of renal IRI surgery. (B) Representative western blots show expression of p16INK4A was largely abolished after injection with p16-shR plasmid. (C) Graphical representation of p16INK4A protein expression levels in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6); # P<0.05 versus IRI injected with pFlag-Wnt9a (n=5–6). (D) Knockdown of p16INK4A prevented the increase in Scr levels induced by Wnt9a in IRI mice. Quantitative assessment of Scr level (mg/dl) in four groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6); # P<0.05 versus IRI injected with pFlag-Wnt9a (n=5–6). (E) Representative micrographs show collagen deposition in different groups, as indicated. Paraffin sections were used for Masson trichrome staining. Arrow indicates positive staining. Scale bar, 50 μm. (F) Graphical representation of the extent of fibrosis after quantitative determination. (G) Representative western blots show fibronectin and α-SMA expression in four groups, as indicated. (H and I) Graphical representations of (H) fibronectin and (I) α-SMA protein expression levels in different groups, as indicated. *P<0.05 versus sham controls (n=5–6); †P<0.05 versus IRI alone (n=5–6); # P<0.05 versus IRI injected with pFlag-Wnt9a (n=5–6). (J) Representative immunostaining micrographs show fibronectin expression in different groups. Arrow indicates positive staining. Scale bar, 50 μm. UNx, unilateral nephrectomy.
Wnt9a Induces Cellular Senescence in Tubular Cells In Vitro
We further examined the cellular senescence in a cultured tubular epithelial cell line (HK-2). Quantitative real-time PCR results indicated that transfection of the Wnt9a expression plasmid significantly induced the mRNA expression of p16INK4A (Figure 10A). Moreover, Wnt9a induced a significant increase in γH2AX nuclear foci in these cells (Figure 10B). We next examined other senescence-related proteins p14ARF; p53; p21, a cell-cycle inhibitor and a target of the p53 gene that is often expressed by senescent cells; and hypophosphorylated Rb, which represses E2F-targeted DNA replication-related enzymes.36,37 As shown in Figure 10, C–G, overexpression of Wnt9a significantly induced the expression of p14ARF, p53, and p21, and reduced the level of Rb phosphorylation at S807/811. These results were also confirmed by treatment with recombinant human Wnt9a protein (data not shown). As shown in Supplemental Figure 5, the addition of recombinant Wnt9a to the standard growth media significantly inhibited the growth of HK-2 cells from passage 3 (P3) through P6.
Figure 10.: Wnt9a induces cellular senescence in cultured proximal tubular epithelial cells. (A) Expression of p16INK4A mRNA in different groups was assessed by real-time PCR in HK-2 cells transfected with empty vector (pcDNA3) or Wnt9a expression plasmid (pFlag-Wnt9a) for 24 hours. *P<0.05 versus pcDNA3 group (n=3). (B) Representative micrographs of HK-2 cells immunostained for γH2AX (red) and counterstained with DAPI (blue) after transfection with pcDNA3 or pFlag-Wnt9a. Arrow indicates positive staining for γH2AX. Scale bar, 10 μm. (C) Representative western blots show Wnt9a transfection decreases the phosphorylation of Rb and promotes expression of p14ARF, p53, and p21. (D–G) Graphical representations of the levels of phosphorylated (D) Rb and (E) p14ARF, (F) p53, and (G) p21 protein expression in two groups, as indicated. *P<0.05 versus pcDNA3 group (n=3). (H) Representative western blots show incubation with recombinant Wnt9a for 24 hours dose-dependently promotes secretion of TGF-β1. (I) Graphical representations of TGF-β1 protein expression levels in four groups, as indicated. *P<0.05 versus medium alone (n=3). (J) Representative western blots show ICG-001inhibits Wnt9a-induced p53 and TGF-β1 expression. HK-2 cells were preincubated with ICG-001 (5 μM) or neutralizing antibody against type II receptor of TGF-β1 (5 μg/ml), then treated with Wnt9a (50 ng/ml) for 24 hours. (K and L) Graphical representations of (K) p53 and (L) TGF-β1 protein expression levels in four groups, as indicated. *P<0.05 versus medium alone (n=3); †P<0.05 versus Wnt9a alone (n=3). (M) Representative western blots show interference of p16INK4A blocks Wnt9a-induced increase in α-SMA and TGF-β1 expression. HK-2 cells were transfected with control (CTL) or p16INK4A-specific siRNA, then treated with Wnt9a (50 ng/ml) for 24 hours. (N–P) Graphical representations of (N) p16INK4A, (O) α-SMA, and (P) TGF-β1 protein expression levels in three groups, as indicated. *P<0.05 versus CTL siRNA alone (n=3); †P<0.05 versus Wnt9a alone (n=3). siRNA, small interfering RNA.
Administration of recombinant Wnt9a protein also significantly induced the expression of TGF-β1 in a dose-dependent manner (Figure 10, H and I). To further assess the downstream targets of Wnt9a, we pretreated HK-2 cells with ICG-001, a small molecule that inhibits β-catenin–mediated gene transcription,5 or a neutralizing antibody against TGF-β receptor II. As shown in Figure 10, J–L, administration of ICG-001 significantly inhibited the Wnt9a-induced expression of p53 and TGF-β1, but administration of the neutralizing antibody against TGF-β receptor II did not. Similarly, ICG-001 repressed the expression of p21 (Supplemental Figure 6).15 These results suggest Wnt9a induces tubular senescence through activation of β-catenin but not TGF-β1.
We then examined the effect on fibrogenic responses of siRNA-mediated inhibition of p16INK4A in the Wnt9a-treated tubular cell line. As shown in Figure 10, M–P, the knockdown of p16INK4A expression significantly blocked the Wnt9a-induced expression of α-SMA and TGF-β1. These data further confirm that tubular senescence has an important role in the Wnt9a-induced fibrogenic response.
Wnt9a Leads to Cell Senescence in Primary Cultured Tubular Cells
In primary cultured mouse renal tubular cells (Figure 11A), incubation with recombinant Wnt9a triggered the upregulation of SA–β-gal activity (Figure 11B). Consistently, recombinant Wnt9a induced the accumulation of nuclear γH2AX foci and repressed the expression of E-cadherin (Figure 11C). As shown in Figure 11, D–F, treatment with recombinant Wnt9a significantly induced the expression of p16INK4A and p19ARF and inhibited the phosphorylation of Rb and the expression of proliferating cell nuclear antigen (PCNA). These data provide striking evidence that Wnt9a induces renal tubular cell senescence.
Figure 11.: Wnt9a induces cellular senescence in primary cultured tubular cells. (A) Representative micrographs show freshly isolated tubules and cultured primary tubular cells. Renal tubules were isolated from mouse kidneys and cultivated for use as primary tubular cells. (B) Representative micrographs show staining of SA–β-gal activity induced by Wnt9a. The primary cultured tubular cells were treated with Wnt9a (50 ng/ml) for 8 days before staining. Arrow indicates positive staining. (C) Representative micrographs of cells immunostained for γH2AX (red) and E-cadherin (green) and counterstained with DAPI (blue). Primary tubular cells were treated with Wnt9a or medium alone for 8 days. Arrows indicate positive staining. Scale bar, 10 μm. (D) Representative western blots show p16INK4A, p19ARF, phosphorylated Rb, and PCNA expression levels in two groups, as indicated. (E and F) Graphical representation of (E) p16INK4A and p19ARF, and (F) phosphorylated Rb and PCNA expression levels in two groups, as indicated. The primary cultured tubular cells were treated with Wnt9a (50 ng/ml) for 4 days before assessment. *P<0.05 versus medium alone (n=3). Ctrl, control.
Wnt9a Induces Cell Communication between Senescent Tubular Cells and Activated Fibroblasts
We next examined the role of Wnt9a in cell-cell communication between tubular cells and fibroblasts. As shown in Figure 12, A–D, treatment of a cultured fibroblast cell line (NRK-49F) with recombinant Wnt9a induced protein expression of the proliferation-related genes PCNA and cyclin D1 and dose-dependently induced cell proliferation.
Figure 12.: Wnt9a induces communication between senescent tubules and activated fibroblasts. (A) Representative western blots showing expression of PCNA and cyclin D1 after NRK-49F cells were treated with or without Wnt9a recombinant protein (50 ng/ml) for 24 hours. (B and C) Graphical representation of (B) PCNA and (C) cyclin D1 protein expression levels in two groups, as indicated. *P<0.05 versus medium alone (n=3). (D) Graphical representation shows that Wnt9a promoted NRK-49F cell proliferation as assessed by a colorimetric MTT assay. *P<0.05 versus medium alone (n=3). (E) Flow chart shows the experimental design and procedures. HK-2 cells were transfected with the Wnt9a expression plasmid for 24 hours to induce cellular senescence or with empty vector as controls. The supernatant was collected (Wnt9a-TCM or CTL-TCM) and used to stimulate NRK-49F cells. (F) Representative micrographs of NRK-49F cells immunostained for fibronectin (red) and counterstained with DAPI (blue). NRK-49F cells were plated on coverslips and preincubated with neutralizing antibody against TGF-β1 receptor II (5 μg/ml) for 1 hour, then treated with Wnt9a-TCM or CTL-TCM for 24 hours before immunostaining. Scale bar, 20 μm. (G) Representative western blots show PCNA and α-SMA expression in three groups, as indicated. (H and I) Graphical representations of (H) PCNA and (I) α-SMA protein expression levels in different groups, as indicated. *P<0.05 versus CTL-TCM group (n=4); †P<0.05 versus Wnt9a-TCM alone (n=4). (J) Representative western blots show TGF-β1 promoted Wnt9a expression in cultured fibroblasts. NRK-49F cells were stimulated with TGF-β1 (5 ng/ml) for 24 hours. Whole-cell kidney lysates were analyzed for Wnt9a. (K) Graphical representation of Wnt9a levels in (J). *P<0.05 versus control (n=3). (L) Schematic presentation of reciprocal activation loop between senescent tubular cells and activated fibroblasts. In pathologic conditions, Wnt9a is dramatically upregulated. Wnt9a induces tubular cell senescence and promotes fibroblast activation. Tubular cells exhibiting the SASP secrete TGF-β1, which aggravates fibroblast proliferation and facilitates matrix protein production and deposition. The activated fibroblasts also produce Wnt9a, which further promotes tubular cell senescence. All of these lead to the pathogenesis of renal fibrotic foci. CTL-TCM, the supernatant collected from tubular cells with empty vector transfection; ECM, excellular matrix.
To further confirm the role of Wnt9a in communication between tubular cells and fibroblasts, we transfected HK-2 cells with the Wnt9a expression plasmid and collected the supernatant as conditioned medium (Wnt9a-TCM; Figure 12E). Treatment of NRK-49F cells with Wnt9a-TCM induced the secretion of fibronectin, as detected by immunofluorescence. Similarly, Wnt9a-TCM enhanced the expression of PCNA, as well as α-SMA, which suggests fibroblast-to-myofibroblast transdifferentiation (Figure 12, G–I).38 Of note, TGF-β receptor II blockade inhibited the Wnt9a-TCM–induced secretion of fibronectin and expression of PCNA and α-SMA (Figure 12, F–I). Interestingly, treatment of NRK-49F cells with TGF-β1 induced the expression of Wnt9a in these cells (Figure 12, J and K). These findings suggest that Wnt9a has different functions in tubular cells and fibroblasts. Furthermore, the Wnt9a–TGF-β pathway may regulate a reciprocal activation loop between senescent tubular cells and activated fibroblasts that accelerates the pathogenesis of renal fibrosis (Figure 12L).
Discussion
CKD exhibits characteristics of a premature senescence syndrome,9,11 including many features of aging9 such as increased oxidative stress, persistent inflammation, and loss of Klotho.5,22,39 Accelerated senescence has been observed in patients with advanced clinical impairment, and even in proteinuric patients with normal GFR,4 suggesting that cell senescence is an early event in CKD. In multiple types of CKD, an accelerated senescence phenotype has been observed in defined cell types, mainly tubular cells.4,7
Cellular senescence is an irreversible fate of damaged cells that features growth arrest, DNA damage, and the enhanced expression of senescence-related proteins such as p16INK4A and p21. Emerging evidence suggests that accumulation of chronic senescent cells could promote pathologies and shorten healthy lifespan.4,13 Senescent cells not only lose growth and repair ability, but also secrete SASP components, including proinflammatory cytokines and growth factors, that attract inflammatory cells and affect the neighboring cells. Thus, understanding the mechanism that triggers cellular senescence is of great importance. Manipulating senescence by clearance of p16INK4A-positive cells and calorie restriction can significantly reduce renal fibrosis and tubular atrophy and increase survival.10,13,14 However, the underlying mechanism that promotes cellular senescence has not been clarified. The results of this study demonstrate that Wnt9a is involved in premature senescence in tubular cells and in promoting renal fibrosis.
Wnt/β-catenin signaling is silent in normal adult kidneys but reactivated in a wide variety of nephropathies.24,40,41 Of note, cellular senescence has been shown to have an important role in the pathologic features of these conditions.4,14 In this study, we showed that Wnt9a has a decisive role in driving tubular senescence and renal fibrosis.
Although barely detectable in normal adult kidneys, Wnt9a expression is clearly induced, predominantly in tubular epithelial cells, in humans and mice after kidney injury and in aging-associated kidney fibrosis. In addition, Wnt9a colocalizes with p16INK4A-positive tubules. Here, we observed the expression of p16INK4A not only in nuclei but also in the tubular cytoplasmic area. This is consistent with results from other groups, suggesting the dysregulation of the nuclear transport mechanism or mislocalization owing to overexpression.11 We also found the expression of Wnt9a positively correlates with p16INK4A expression, renal fibrosis, and decline of eGFR, and is accompanied by an increase of γH2AX foci and loss of Klotho, a marker of tubular damage and CKD,22 suggesting the intimate correlation between activation of Wnt9a and cellular senescence in kidney injury. Our additional in vivo and in vitro studies showed that renal expression of Wnt9a induces β-catenin signaling activation and promotes renal fibrosis and accelerates tubular senescence. Notably, these effects require p16INK4A expression. These data undoubtedly show a causative role of Wnt9a in controlling tubular senescence and renal fibrosis in vivo and suggest that Wnt9a gene deletion could be a plausible strategy for therapeutic intervention of cellular senescence and renal fibrosis.
Supporting our findings, it was previously reported that Wnt/β-catenin signaling could accelerate senescence in bone and skin,42 and cellular senescence of bone marrow–mesenchymal stem cells and hematopoietic stem/progenitor cells.43
It is noteworthy that Wnt9a has different functions in tubular cells and fibroblasts. Although we demonstrated that Wnt9a induces senescence in tubular cells, it has a proliferative role in fibroblasts, suggesting Wnt9a functions through differential mechanisms in these cells to jointly reinforce the pathogenesis of renal fibrosis. The other interesting observation in this study is that Wnt9a evokes cell communication between senescent tubular cells and interstitial fibroblasts. Consistent with other studies,18 we observed that Wnt9a induced senescent tubular cells to secrete the SASP component TGF-β1, which led to interstitial fibroblast proliferation and transition to myofibroblasts.
Of note, TGF-β1 reinforces paracrine senescence in normal cells by regulating p15INK4B and p21,18 and has a potent mitogenic role in fibroblasts and induces myofibroblast transition.44 In our study, we found TGF-β1 also induces Wnt9a in fibroblasts, thereby creating a vicious cycle of tubular senescence, fibroblast activation, and renal fibrosis. The evidence further suggests that signaling crosstalk between tubular epithelial cells and interstitial fibroblasts is an important mechanism in accelerating progressive disease.45
In summary, we have shown that aberrantly expressed Wnt9a is critically involved in mediating tubular senescence and renal fibrosis. Wnt9a also directly induces fibroblast proliferation and activation. Importantly, TGF-β1 and Wnt signaling set up a vicious activation loop that promotes renal fibrosis. Although more studies are needed, these results provide proof of principle that targeted inhibition of tubular Wnt9a protects against fibrosis through breakdown of the reciprocal activation loop involving senescent tubular cells and activated interstitial fibroblasts.
Concise Methods
Animal Models
Male C57BL/6 mice, weighing approximately 20–22 g, were purchased from Southern Medical University Animal Center (Guangzhou, China) and housed in a standard environment on a regular light/dark cycle with free access to water and chow. Renal IRI was established as described.24 Briefly, bilateral renal pedicles were clipped for 32 minutes using microaneurysm clamps. During the ischemic period, body temperature was maintained between 37°C and 38°C using a temperature-controlled heating system. After removal of the clamps, reperfusion of the kidneys was visually confirmed. Mice were euthanized 7 days after IRI, and kidney tissues were collected for various analyses.
The male C57BL/6 mouse model of UUO was established by double-ligating the left ureter using 4–0 silk after a midline abdominal incision. Sham-operated mice had their ureters exposed and manipulated but not ligated. Mice were euthanized 7 days after UUO. For the ADR nephropathy model, male BALB/c mice were administered a single intravenous injection of ADR (doxorubicin hydrochloride; Sigma, St. Louis, MO) at 10.5 mg/kg body weight. Mice were euthanized 3 weeks after ADR injection. For the UIRI model, male C57BL/6 mice were subjected to unilateral renal IRI by an established protocol, as described previously.24 Briefly, left renal pedicles were clipped for 35 minutes using microaneurysm clamps for IRI injury. After removal of the clamps, reperfusion of the kidneys was visually confirmed. Ten days later, the intact right kidney was removed via a right flank incision. For studying the effects of Wnt9a, four sets of experiments were performed. The detailed experimental designs are presented in Figures 3A, 7A, and 9A and Supplemental Figure 4A. In vivo expression or knockdown of Wnt9a in mice was carried out by a hydrodynamic-based gene delivery approach, as described previously.22 Briefly, the human Wnt9a and p16INK4A siRNA sequences (5′-GCTTCAAGGAGACTGCCTT-3′ and 5′-CACCAGAGGCAGUAACCAUTT-3′, respectively) were ligated into an shRNA expression plasmid (pLVX-shRNA). Groups of mice were administered human Wnt9a expression plasmid (pFlag-Wnt9a) or shRNA expression plasmid (pLVX-shWnt9a or pLVX-shp16INK4A) by rapid injection of a large volume of DNA solution through the tail vein.
Determination of SCr, Ualb, and NAG Level
Scr level was determined by an automatic chemistry analyzer (AU480; Beckman Coulter, Pasadena, CA). Ualb was measured by using a mouse Albumin ELISA Quantitation kit, according to the manufacturer's protocol (Bethyl Laboratories, Inc., Montgomery, TX). Urine creatinine was determined by a routine procedure, as described previously.22 Ualb was standardized to urine creatinine and expressed as μg/mg urinary creatinine. Urinary NAG level was analyzed by a commercial kit (CSB-E07444m; CUSABIO Life Science, Wuhan, China) and expressed as IU/g urinary creatinine.
Cell Culture and Treatment
Human proximal tubular epithelial cells (HK-2) and normal rat kidney interstitial fibroblast cell lines (NRK-49F) were obtained from the American Type Culture Collection (Manassas, VA) and maintained per routine protocol. HK-2 cells or NRK-49F cells were treated with human recombinant Wnt9a (R & D Systems, Minneapolis, MN) at the indicated concentration or transfected with Wnt9a expression plasmid (pFlag-Wnt9a). Whole-cell lysates were prepared and subjected to Western blot analyses. Some cells were also detected by immunofluorescence.
Primary mouse tubular cells were isolated and cultivated as previously described.46 Briefly, the kidneys were peeled off and minced, then digested in 0.75 mg/ml collagenase for 40 minutes at 37°C, after which the mashed tissue was sieved in PBS. The tubular tissues were isolated using 31% Percoll gradients, resuspended, and washed twice with PBS. Finally, tubules were suspended in DMEM supplemented with 10% bovine calf serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and hormone mix (5 mg/ml insulin, 1.25 ng/ml prostaglandin PG E1, 34 pg/ml triiodothyronine, 5 mg/ml transferrin, 1.73 ng/ml sodium selenite, 18 ng/ml hydrocortisone, and 25 ng/ml EGF). Cells were grown in cell culture dishes for 4–8 days until they reached 60%–80% confluency. Medium was changed on days 2 and 5, then every 3 days.
Western Blot Analysis
Protein expression was analyzed by Western blot analysis as described previously.5 The primary antibodies used were as follows: anti-p16INK4A (ab189034; Abcam), anti-p21 (sc-397; Santa Cruz Biotechnology), anti-PCNA (sc-56; Santa Cruz Biotechnology), anti-cyclinD1 (sc-753; Santa Cruz Biotechnology), anti-p14ARF (sc-8613; Santa Cruz Biotechnology), anti–p-Rb (sc-16670; Santa Cruz Biotechnology), anti-p19ARF (ab202225; Abcam), anti-p53 (sc-126; Santa Cruz Biotechnology), anti–Flag-tag (KM3002; Sungene Biotech Co.), anti–TGF-β1 (sc-146; Santa Cruz Biotechnology), anti–β-catenin (610154; BD Biosciences), anti-fibronectin (F3648; Sigma-Aldrich), anti–α-SMA (ab5694; Abcam), anti–MMP-7 (GTX104658; GeneTex, Inc.), anti-snail1 (ab180714; Abcam), anti–PAI-1 (AF1179; R&D Systems), anti-Klotho (AF1819; R&D Systems), anti-Wnt9a (ab125957; Abcam), anti–γ-H2AX (ab26350; Abcam), anti–α-tubulin (RM2007; Ray Antibody Biotech, Beijing, China), anti–β-actin (RM001; Ray Antibody Biotech), and anti-GAPDH (RM2002; Ray Antibody Biotech).
Immunofluorescence Staining
Kidney cryosections were fixed with 3.7% paraformalin for 15 minutes at room temperature. Primary cultured mouse tubular cells, HK-2 cells, or NRK-49F cells cultured on coverslips were fixed with cold methanol/acetone (1:1) for 15 minutes at room temperature, then blocked with 10% normal donkey serum in PBS. Slides were incubated with antibodies against fibronectin (F3648; Sigma-Aldrich), E-cadherin (3195s; Cell Signaling Technology), collagen I (BA0325; Boster Biotechnology), or γ-H2AX (ab26350; Abcam). After washing, the slides were incubated with Cy3- or Cy2-conjugated donkey anti-mouse or donkey anti-rabbit IgG (Jackson Immuno-Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (Sigma-Aldrich) according to the manufacturer’s instructions. Images were captured by fluorescence microscopy (Leica DMi8; Leica Microsystems, Buffalo Grove, IL).
RT and Real-Time PCR
Total RNA isolation was carried out using the TRIzol RNA isolation system (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. The first strand of complementary DNA was synthesized using 1 μg of RNA in 20 μl of reaction buffer containing AMV-RT and random primers at 42°C for 60 minutes. Real-time PCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). The sequences of the primer pairs are shown in Supplemental Table 2.
Histology and Immunohistochemistry
Paraffin-embedded mouse kidney sections (4-µm thickness) were prepared by a routine procedure. PAS and Masson trichrome staining were conducted by standard protocol. Immunohistochemical staining was performed using routine protocol. Antibodies used were as follows: anti-Wnt9a (ab189010; Abcam), anti-Ki67 (ab16667; Abcam), anti-p16INK4A (ab189034; Abcam), anti-fibronectin (F3648; Sigma-Aldrich), anti–FSP-1 (S100A4; Merk MILLIPORE), anti–β-catenin (ab15180; Abcam), anti–MMP-7 (3801s; Cell Signaling Technology), anti–TGF-β1 (sc-146; Santa Cruz Biotechnology), and anti-Klotho (AF1819; R&D Systems). Human biopsy sequential sections were obtained from diagnostic renal biopsies performed at the Nanfang Hospital and Huadu District People’s Hospital, and stained with rabbit polyclonal anti-Wnt9a (Abcam) and mouse monoclonal anti-p16INK4A (Abcam). All studies involving human kidney sections were approved by the Institutional Ethics Committee at the Nanfang Hospital and Huadu District People’s Hospital, Southern Medical University.
SA–β-gal and TUNEL Staining
Frozen sections (3 μm) were used for detection of SA–β-gal activity according to the manufacturer’s instructions (9860; Cell Signaling Technology). Paraffin sections (4 μm) were fixed and stained for TUNEL staining by a commercial kit (KGA703; KeyGEN BioTECH, Nanjing, China).
Renal Immunohistochemical Analyses
Semiquantitation of Wnt9a and p16INK4A staining was performed by the method previously reported.47 Briefly, digital images at 2448×1920- pixel resolution were captured at ×400 magnification by the DP 27 CCD camera (Olympus, Japan) coupled to an Olympus BX-51 microscope (Olympus). Images of randomly selected fields were taken and the staining scores of Wnt9a and p16INK4A were calculated by the Image-Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, MD) and expressed as the ratio of integrated optical density (IOD) to observed area (IOD/area), representing the relative expression levels of Wnt9a and p16INK4A.
Semiquantitative Scoring of Renal Fibrosis
Masson trichrome staining was conducted by routine procedures in kidney biopsy specimens from patients with CKD with different causesetiologies. Stained slides were observed under a microscope by the DP 27 CCD camera (Olympus, Japan), and images at 2448×1920- pixel resolution captured at high-powered (×400) fields from randomly selected fields were taken. Each image per section was split into 100 squares. Tissue fibrosis as defined by blue staining was scored by three experienced observers in a blinded fashion. The percentage of blue staining in each image was calculated, and the mean values of the fibrosis scores determined by the three observers were reported.
Statistical Analyses
All data examined were expressed as mean ±SEM. Statistical analyseis of the data were carried out using SPSS 13.0 (SPSS Inc., Chicago, IL). Comparison between groups was made using one-way ANOVA followed by Student–Newman–Keuls test or Dunnett’s T3 procedure. P<0.05 was considered significant.
Disclosures
None.
This work was supported by the National Natural Science Foundaion of China grants 81722011, 81570620, 81521003, and 81370014; Guangdong Science Foundation grant 2014A030312014; Guangzhou Projects grant 201504010001; and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (JQ201401).
References
1. Minutolo R, Borrelli S, De Nicola L: CKD in the elderly: Kidney senescence or blood pressure-related nephropathy? Am J Kidney Dis 66: 184–186, 201526210723
2. Clements ME, Chaber CJ, Ledbetter SR, Zuk A: Increased cellular senescence and vascular rarefaction exacerbate the progression of kidney fibrosis in aged mice following transient ischemic injury. PLoS One 8: e70464, 201323940580
3. Zhou D, Tian Y, Sun L, Zhou L, Xiao L, Tan RJ, et al.: Matrix Metalloproteinase-7 is a urinary biomarker and pathogenic mediator of kidney fibrosis. J Am Soc Nephrol 28: 598–611, 201727624489
4. Verzola D, Gandolfo MT, Gaetani G, Ferraris A, Mangerini R, Ferrario F, et al.: Accelerated senescence in the kidneys of patients with type 2 diabetic nephropathy. Am J Physiol Renal Physiol 295.: F1563–F1573, 200818768588
5. Zhou L, Li Y, Hao S, Zhou D, Tan RJ, Nie J, et al.: Multiple genes of the renin-angiotensin system are novel targets of Wnt/β-catenin signaling. J Am Soc Nephrol 26: 107–120, 201525012166
6. Zhou D, Tan RJ, Fu H, Liu Y: Wnt/β-catenin signaling in kidney injury and repair: A double-edged sword. Lab Invest 96: 156–167, 201626692289
7. Kakoki M, Kizer CM, Yi X, Takahashi N, Kim HS, Bagnell CR, et al.: Senescence-associated phenotypes in Akita diabetic mice are enhanced by absence of bradykinin B2 receptors. J Clin Invest 116: 1302–1309, 200616604193
8. Bobkova NV, Evgen’ev M, Garbuz DG, Kulikov AM, Morozov A, Samokhin A, et al.: Exogenous Hsp70 delays senescence and improves cognitive function in aging mice. Proc Natl Acad Sci U S A 112: 16006–16011, 201526668376
9. Stenvinkel P, Larsson TE: Chronic kidney disease: A clinical model of premature aging. Am J Kidney Dis 62: 339–351, 201323357108
10. Zhu K, Kakehi T, Matsumoto M, Iwata K, Ibi M, Ohshima Y, et al.: NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney. Free Radic Biol Med 83: 21–30, 201525701431
11. Sis B, Tasanarong A, Khoshjou F, Dadras F, Solez K, Halloran PF: Accelerated expression of senescence associated cell cycle inhibitor p16INK4A in kidneys with glomerular disease. Kidney Int 71: 218–226, 200717183247
12. Liu J, Yang JR, He YN, Cai GY, Zhang JG, Lin LR, et al.: Accelerated senescence of renal tubular epithelial cells is associated with disease progression of patients with immunoglobulin A (IgA) nephropathy. Transl Res 159: 454–463, 201222633096
13. Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al.: Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 530: 184–189, 201626840489
14. Braun H, Schmidt BM, Raiss M, Baisantry A, Mircea-Constantin D, Wang S, et al.: Cellular senescence limits regenerative capacity and allograft survival. J Am Soc Nephrol 23: 1467–1473, 201222797186
15. Sturmlechner I, Durik M, Sieben CJ, Baker DJ, van Deursen JM: Cellular senescence in renal ageing and disease. Nat Rev Nephrol 13: 77–89, 201728029153
16. Campisi J, d’Adda di Fagagna F: Cellular senescence: When bad things happen to good cells. Nat Rev Mol Cell Biol 8: 729–740, 200717667954
17. Melk A, Schmidt BM, Takeuchi O, Sawitzki B, Rayner DC, Halloran PF: Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney. Kidney Int 65: 510–520, 200414717921
18. Acosta JC, Banito A, Wuestefeld T, Georgilis A, Janich P, Morton JP, et al.: A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol 15: 978–990, 201323770676
19. Mo H, Wu Q, Miao J, Luo C, Hong X, Wang Y, et al.: C-X-C chemokine receptor type 4 plays a crucial role in mediating oxidative stress-induced podocyte injury. Antioxid Redox Signal 27: 345–362, 201727960539
20. Li SY, Susztak K: The long noncoding RNA Tug1 connects metabolic changes with kidney disease in podocytes. J Clin Invest 126: 4072–4075, 201627760046
21. Satriano J, Mansoury H, Deng A, Sharma K, Vallon V, Blantz RC, et al.: Transition of kidney tubule cells to a senescent phenotype in early experimental diabetes. Am J Physiol Cell Physiol 299: C374–C380, 201020505038
22. Zhou L, Li Y, Zhou D, Tan RJ, Liu Y: Loss of Klotho contributes to kidney injury by derepression of Wnt/β-catenin signaling. J Am Soc Nephrol 24: 771–785, 201323559584
23. He W, Dai C, Li Y, Zeng G, Monga SP, Liu Y: Wnt/beta-catenin signaling promotes renal interstitial fibrosis. J Am Soc Nephrol 20: 765–776, 200919297557
24. Xiao L, Zhou D, Tan RJ, Fu H, Zhou L, Hou FF, et al.: Sustained activation of Wnt/β-catenin signaling drives AKI to CKD progression. J Am Soc Nephrol 27: 1727–1740, 201626453613
25. Cardus A, Uryga AK, Walters G, Erusalimsky JD: SIRT6 protects human endothelial cells from DNA damage, telomere dysfunction, and senescence. Cardiovasc Res 97: 571–579, 201323201774
26. d’Adda di Fagagna F: Living on a break: Cellular senescence as a DNA-damage response. Nat Rev Cancer 8: 512–522, 200818574463
27. Wolstein JM, Lee DH, Michaud J, Buot V, Stefanchik B, Plotkin MD: INK4a knockout mice exhibit increased fibrosis under normal conditions and in response to unilateral ureteral obstruction. Am J Physiol Renal Physiol 299: F1486–F1495, 201020861074
28. Romero Y, Bueno M, Ramirez R, Álvarez D, Sembrat JC, Goncharova EA, et al.: mTORC1 activation decreases autophagy in aging and idiopathic pulmonary fibrosis and contributes to apoptosis resistance in IPF fibroblasts [published online ahead of print August 26, 2016]. Aging Cell doi:10.1111/acel.1251427566137
29. Muñoz-Espín D, Cañamero M, Maraver A, Gómez-López G, Contreras J, Murillo-Cuesta S, et al.: Programmed cell senescence during mammalian embryonic development. Cell 155: 1104–1118, 201324238962
30. Mise K, Hoshino J, Ueno T, Hazue R, Hasegawa J, Sekine A, et al.: Prognostic value of tubulointerstitial lesions, urinary N-acetyl-β-d-glucosaminidase, and urinary β2-microglobulin in patients with type 2 diabetes and biopsy-proven diabetic nephropathy. Clin J Am Soc Nephrol 11: 593–601, 201626801478
31. Zhou L, Li Y, He W, Zhou D, Tan RJ, Nie J, et al.: Mutual antagonism of Wilms’ tumor 1 and β-catenin dictates podocyte health and disease. J Am Soc Nephrol 26: 677–691, 201525071087
32. He W, Tan RJ, Li Y, Wang D, Nie J, Hou FF, et al.: Matrix metalloproteinase-7 as a surrogate marker predicts renal Wnt/β-catenin activity in CKD. J Am Soc Nephrol 23: 294–304, 201222095947
33. He W, Tan R, Dai C, Li Y, Wang D, Hao S, et al.: Plasminogen activator inhibitor-1 is a transcriptional target of the canonical pathway of Wnt/beta-catenin signaling. J Biol Chem 285: 24665–24675, 201020519507
34. Simon-Tillaux N, Hertig A: Snail and kidney fibrosis. Nephrol Dial Transplant 32: 224–233, 201728186539
35. Liu Y: Epithelial to mesenchymal transition in renal fibrogenesis: Pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol 15: 1–12, 200414694152
36. Zhao L, Zhang Y, Gao Y, Geng P, Lu Y, Liu X, et al.: JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein. Cell Death Differ 22: 1630–1640, 201525698448
37. Ding G, Franki N, Kapasi AA, Reddy K, Gibbons N, Singhal PC: Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats. Exp Mol Pathol 70: 43–53, 200111170790
38. LeBleu VS, Taduri G, O’Connell J, Teng Y, Cooke VG, Woda C, et al.: Origin and function of myofibroblasts in kidney fibrosis. Nat Med 19: 1047–1053, 201323817022
39. Zhou LL, Cao W, Xie C, Tian J, Zhou Z, Zhou Q, et al.: The receptor of advanced glycation end products plays a central role in advanced oxidation protein products-induced podocyte apoptosis. Kidney Int 82: 759–770, 201222622498
40. von Toerne C, Schmidt C, Adams J, Kiss E, Bedke J, Porubsky S, et al.: Wnt pathway regulation in chronic renal allograft damage. Am J Transplant 9: 2223–2239, 200919681821
41. Zhou T, He X, Cheng R, Zhang B, Zhang RR, Chen Y, et al.: Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy. Diabetologia 55: 255–266, 201222016045
42. Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, Chen J, et al.: Augmented Wnt signaling in a mammalian model of accelerated aging. Science 317: 803–806, 200717690294
43. Gu Z, Tan W, Feng G, Meng Y, Shen B, Liu H, et al.: Wnt/β-catenin signaling mediates the senescence of bone marrow-mesenchymal stem cells from systemic lupus erythematosus patients through the p53/p21 pathway. Mol Cell Biochem 387: 27–37, 201424130040
44. Meng XM, Tang PM, Li J, Lan HY: TGF-β/Smad signaling in
renal fibrosis. Front Physiol 6: 82, 201525852569
45. Tan RJ, Zhou D, Liu Y: Signaling crosstalk between tubular epithelial cells and interstitial fibroblasts after kidney injury. Kidney Dis (Basel) 2: 136–144, 201627921041
46. Bernhardt A, Fehr A, Brandt S, Jerchel S, Ballhause TM, Philipsen L, et al.: Inflammatory cell infiltration and resolution of kidney inflammation is orchestrated by the cold-shock protein Y-box binding protein-1. Kidney Int 92: 1157–1177, 201728610763
47. Wang J, Liang M, Xu J, Cao W, Wang GB, Zhou ZM, et al.: Renal expression of advanced oxidative protein products predicts progression of
renal fibrosis in patients with IgA nephropathy. Lab Invest 94: 966–977, 201425068662
