Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: Klotho binds to Wnt and blocks Wnt-mediated gene transcription. (A) Co-immunoprecipitation demonstrates that Klotho binds to Wnt1 in tubular epithelial cells. HKC-8 cells were transfected with V5-tagged, full-length, membrane Klotho expression vector (pV5-mKlotho) and HA-tagged Wnt1 expression vector (pHA-Wnt1). Cell lysates were immunoprecipitated with anti-Klotho, followed by immunoblotting with anti-Wnt1 or anti-Klotho, respectively. (B) Klotho also binds to Wnt4. Co-immunoprecipitation shows that full-length, membrane Klotho also binds to Wnt4 in HKC-8 cells. (C) The extracellular KL1 domain of Klotho is sufficient for mediating Wnt/Klotho interaction. HKC-8 cells were transfected with truncated, secreted Klotho expression vector (pV5-sKlotho) and Wnt1 expression vector (pHA-Wnt1). (D) Interaction of Klotho and Wnt1 occurs in the extracellular space. HKC-8 cells were transfected with pV5-mKlotho and pHA-Wnt1. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by immunoblotting with anti-HA or anti-V5, respectively. Cell lysates were immunoblotted with antibodies against Klotho and actin. (E) Klotho inhibits Wnt1-mediated β-catenin gene transcription in a dose-dependent manner. HKC-8 cells were co-transfected with TOP-Flash reporter plasmid and different amounts of full-length Klotho plasmid (pV5-mKlotho) as indicated in the absence or presence of Wnt1 expression vector. *P<0.05 versus pHA-Wnt1 alone (n=3). (F) Klotho does not inhibit activated β-catenin–triggered gene transcription. HKC-8 cells were co-transfected with TOP-Flash reporter plasmid and Klotho plasmid in the absence or presence of the N-terminally truncated, constitutively activated β-catenin expression vector (pDel-β-cat). IB, immunoblotting; IP, immunoprecipitation.

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Western Blot, Cell Culture

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: Klotho inhibits Wnt-mediated β-catenin activation and represses its target genes in vitro. (A) Immunofluorescence staining shows that ectopic expression of Klotho blocked the Wnt1-mediated β-catenin activation and its nuclear translocation. HKC-8 cells were transfected with Wnt1 expression plasmid (pHA-Wnt1) and Klotho expression vector (pV5-mKlotho) as indicated. Cells were then immunostained for β-catenin and DAPI, the nuclear marker. Arrows indicate nuclear staining of β-catenin. (B and C) Klotho inhibits Wnt1-mediated β-catenin nuclear translocation. Nuclear (B) and cytoplasmic (C) protein was prepared from HKC-8 cells co-transfected with various plasmids as indicated and immunoblotted with antibodies against β-catenin, TATA-binding protein (TBP), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). n-β-catenin, nuclear β-catenin; c-β-catenin, cytoplasmic β-catenin. The ratios of nuclear β-catenin to TATA-binding protein and cytoplasmic β-catenin to GAPDH are presented in the bottom panels. (D–G) Klotho dose-dependently inhibits Wnt1-mediated β-catenin activation and its target genes, such as PAI-1 and Snail1. Western blot analyses (D) and quantitative data for active β-catenin (E), PAI-1 (F), and Snail1 (G) are shown. Different doses of pV5-mKlotho (1, 2 and 4 µg/well) were used. *P<0.05 versus pHA-Wnt1 alone.

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Activation Assay, In Vitro, Immunofluorescence, Staining, Expressing, Translocation Assay, Transfection, Plasmid Preparation, Marker, Binding Assay, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: TGF-β1 represses Klotho expression and Klotho inhibits the TGF-β1–mediated induction of various fibrogenic genes in vitro. (A–D) Western blot analyses show that TGF-β1 inhibited Klotho expression in a dose- and time-dependent manner. HKC-8 cells were incubated with different doses of TGF-β1 for 24 hours (A and B) or the same concentration of TGF-β1 (2 ng/ml) for various periods of time (C and D) as indicated. Representative Western blots (A and C) and quantitative data (B and D) are presented. *P<0.05 versus controls (n=3). (E) RT-PCR shows that TGF-β1 repressed Klotho mRNA expression in tubular epithelial cells. HKC-8 cells were treated with TGF-β1 (2 ng/ml) for various periods of time as indicated. Expression of Klotho, TNF-α, and β-actin mRNA was analyzed. (F and G) Repression of Klotho by TGF-β1 depends on Smad3 signaling. HKC-8 cells were pretreated with TGF-β type I receptor inhibitor (SB431542; 10 μM) or Smad3 inhibitor (SIS3; 10 μM) for 1 hour, followed by incubation with TGF-β1. Representative Western blot (F) and quantitative data (G) are presented. *P<0.05 versus controls (n=3); †P<0.05 versus TGF-β1 alone (n=3). SB, SB431542. (H) TGF-β1 does not induce Wnt expression in tubular epithelial cells. HKC-8 cells were treated with TGF-β1 (2 ng/ml) for various periods of time as indicated. Expression of various Wnt mRNA was assessed by RT-PCR. (I–L) Western blot analyses and quantitative data show that Klotho inhibited β-catenin activation and its target gene expression induced by TGF-β1. HKC-8 cells were transfected with empty vector (pcDNA3) or Klotho expression vector (pV5-mKlotho) as indicated, followed by incubation with TGF-β1 (2 ng/ml). Western blot (I) and quantitative data for active β-catenin (J), PAI-1 (K), and Snail1 (L) are presented. (M and N) Klotho abolished TGF-β1–mediated fibrogenic actions. HKC-8 cells were transfected with empty vector (pcDNA3) or Klotho expression vector (pV5-mKlotho) as indicated, followed by incubation with TGF-β1 (2 ng/ml). Total cell lysates were immunoblotted with specific antibodies against fibronectin, α-SMA and actin, respectively. *P<0.05 versus pcDNA3 controls; †P<0.05 versus TGF-β1 alone (n=3).

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Expressing, In Vitro, Western Blot, Incubation, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Transfection, Plasmid Preparation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: Ectopic expression of secreted Klotho in vivo inhibited renal β-catenin activation and the expression of its target genes. (A) Western blot analyses show Klotho expression in mouse kidneys at 24 h after intravenous injection of naked plasmid encoding secreted Klotho (pV5-sKlotho). Secreted Klotho was readily detected in the kidneys after injection of pV5-sKlotho plasmid. (B) ELISA shows increased Klotho protein in the urine after injection of pV5-sKlotho plasmid. Urine was collected at 24 hours after plasmid injection. Urinary Klotho was expressed as ng/mg creatinine. *P<0.05 versus pcDNA3 (n=6). (C) Experimental design. Arrows indicate the injection of pV5-sKlotho plasmid. Narrow arrows indicate the timing of UUO surgery. Both preventive protocol (UUO/K-1) and therapeutic protocol (UUO/K+3) were used. (D–G) Western blot analyses and quantitative data show renal expression of β-catenin and its target genes, such as Snail1 and PAI-1, in different groups as indicated. Numbers (1 and 2) indicate each individual animal in a given group. *P<0.05 versus sham controls; †P<0.05 versus UUO alone (n=5–7).

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Expressing, In Vivo, Activation Assay, Western Blot, Injection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: Ectopic expression of secreted Klotho in vivo attenuates renal fibrosis in obstructive nephropathy. (A) Representative micrographs show that secreted Klotho inhibited α-SMA expression and reduced fibronectin and collagen I deposition in the obstructed kidneys after UUO. Myofibroblast activation was assessed by immunostaining for α-SMA on paraffin sections. Frozen kidney sections were stained with specific antibodies against fibronectin and collagen I, respectively. Scale bar, 50 μm. (B–D) Western blot analyses show the expression of α-SMA and fibronectin in different groups. Numbers (1 and 2) indicate each individual animal. Western blot (B) and quantitative data for α-SMA (C) and fibronectin (D) are shown. *P<0.05 versus sham controls; †P<0.05 versus UUO alone (n=5–7). (E) Representative micrographs show kidney fibrotic lesions in different groups. Paraffin sections were used for Masson trichrome staining. Scale bar, 50 μm. (F) Graphical presentation of kidney fibrotic lesions in different groups after quantitative determination. *P<0.05 versus sham controls; †P<0.05 versus UUO alone (n=5–7).

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Expressing, In Vivo, Activation Assay, Immunostaining, Staining, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: Secreted Klotho ameliorates kidney injury in adriamycin (ADR) nephropathy. (A) Experimental design. Green arrows indicate the injection of secreted Klotho expression vector (pV5-sKlotho). Red arrows indicate adriamycin injection. (B) Urinary albumin levels in different groups. Urinary albumin levels in mice at 3 weeks after adriamycin injection. Urinary albumin was expressed as mg/mg creatinine. *P<0.05 versus controls (n=5–7). (C) Representative micrographs show kidney injury at 3 weeks after adriamycin injection in different groups of mice as indicated. Images of PAS staining with different magnification were shown. Scale bar, 50 µm. (D) Klotho reduces serum creatinine levels in adriamycin nephropathy. Serum creatinine was assessed at 3 weeks after adriamycin injection. *P<0.05 versus controls; †P<0.05 versus adriamycin alone (n=5–7). (E) Klotho preserves glomerular integrity in adriamycin nephropathy. Representative micrographs show nephrin and WT1 staining at 3 weeks after various treatments as indicated. (F and G) Klotho preserves nephrin protein expression in adriamycin nephropathy. Representative Western blot (F) and quantitative data (G) are presented. *P<0.05 versus controls; †P<0.05 versus adriamycin alone (n=5–7). CTL, control.

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Injection, Expressing, Plasmid Preparation, Staining, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Loss of Klotho Contributes to Kidney Injury by Derepression of Wnt/ β -Catenin Signaling

doi: 10.1681/ASN.2012080865

Figure Lengend Snippet: Secreted Klotho reduces renal fibrotic lesions in adriamycin nephropathy. (A–C) Western blot analyses show the expression of Klotho, β-catenin, and Snail1 in different groups as indicated. Western blot (A) and graphical presentation of Klotho (B), β-catenin and Snail1 (C) are shown. *P<0.05 versus controls; †P<0.05 versus adriamycin alone (n=5–7). (D) Representative micrographs show renal expression and localization of β-catenin, fibronectin, and PAI-1 at 3 weeks after adriamycin injection. Frozen kidney sections were stained with specific antibodies against β-catenin, fibronectin, and PAI-1. Scale bar, 50 µm. (E) Representative micrographs show collagen deposition in different groups as indicated. Paraffin sections were used for Masson trichrome staining. Scale bar, 50 μm. (F) Graphical presentation shows kidney fibrotic lesions after quantitative determination. *P<0.05 versus controls; †P<0.05 versus adriamycin alone (n=5–7). CTL, control.

Article Snippet: HKC-8 cells were transiently transfected with various expression vectors, such as HA-tagged Wnt1 expression plasmid (pHA-Wnt1; Upstate Biotechnology, Lake Placid, NY), human membrane Klotho (pV5-mKlotho; Addgene) or secreted Klotho (pV5-sKLotho) by using Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY).

Techniques: Western Blot, Expressing, Injection, Staining