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.
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.
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.
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.
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.
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
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).
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).
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.
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.
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.
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.
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.
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).
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.
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).
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.
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.
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).
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