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. 2014 Apr;85(4):855-70.
doi: 10.1038/ki.2013.489. Epub 2013 Dec 4.

Klotho has dual protective effects on cisplatin-induced acute kidney injury (V体育官网入口)

Monica C Panesso  1 , Mingjun Shi  2 , Han J Cho  2 , Jean Paek  2 , Jianfeng Ye  2 , Orson W Moe  3 , Ming Chang Hu  4
Affiliations

Klotho has dual protective effects on cisplatin-induced acute kidney injury

Monica C Panesso et al. Kidney Int. 2014 Apr.

"V体育2025版" Abstract

Klotho protects the kidney from ischemia-reperfusion injury, but its effect on nephrotoxins is unknown. Here we determined whether Klotho protects the kidney from cisplatin toxicity VSports手机版. Cisplatin increased plasma creatinine and induced tubular injury, which were exaggerated in Klotho haplosufficient (Kl/+) and ameliorated in transgenic Klotho overexpressing (Tg-Kl) mice. Neutrophil gelatinase-associated lipocalin and active caspase-3 protein and the number of apoptotic cells in the kidney were higher in Kl/+ and lower in Tg-Kl compared with wild-type mice. Klotho suppressed basolateral uptake of cisplatin by the normal rat kidney cell line (NRK), an effect similar to cimetidine, a known inhibitor of organic cation transport (OCT). A decrease in cell surface and total OCT2 protein and OCT activity by Klotho was mimicked by β-glucuronidase. The Klotho effect was attenuated by β-glucuronidase inhibition. On the other hand, OCT2 mRNA was reduced by Klotho but not by β-glucuronidase. Moreover, cimetidine inhibited OCT activity but not OCT2 expression. Unlike cimetidine, Klotho reduced cisplatin-induced apoptosis from either the basolateral or apical side and even when added after NRK cells were already loaded with cisplatin. Thus, Klotho protects the kidney against cisplatin nephrotoxicity by reduction of basolateral uptake of cisplatin by OCT2 and a direct anti-apoptotic effect independent of cisplatin uptake. Klotho may be a useful agent to prevent and treat cisplatin-induced nephrotoxicity. .

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"VSports在线直播" Figures

Figure 1
Figure 1. Cisplatin induces acute kidney injury
Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (10 mg/kg body weight) or vehicle (same volume of 0.9% NaCl) once into mice with three different Klotho genetic backgrounds. (A) Plasma creatinine (PCr) and (B) BUN were measured at days 0, 2, 4, 7, and 14 post injection. The results are expressed as means ± SD of 8 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **:P<0.01 vs WT cisplatin injection; #P<0.05; ##P<0.01 vs Tg-Kl cisplatin injection. At Day 4, 7, and 14 post cisplatin or vehicle injections, mice were euthanized and the kidneys were harvested and sectioned for histology from 4 animals in each group (C) Representative H&E staining in paraffin-embedded kidney sections. Renal tubular casts (asterisk); dilated renal tubules (arrow head); and infiltrated inflammatory (arrow). (D) Kidney histological scores were obtained from kidneys H&E staining by a nephropathologist blinded to the experimental conditions. Results of pathologic scores are expressed as means ± SD of 8 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 1
Figure 1. Cisplatin induces acute kidney injury
Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (10 mg/kg body weight) or vehicle (same volume of 0.9% NaCl) once into mice with three different Klotho genetic backgrounds. (A) Plasma creatinine (PCr) and (B) BUN were measured at days 0, 2, 4, 7, and 14 post injection. The results are expressed as means ± SD of 8 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **:P<0.01 vs WT cisplatin injection; #P<0.05; ##P<0.01 vs Tg-Kl cisplatin injection. At Day 4, 7, and 14 post cisplatin or vehicle injections, mice were euthanized and the kidneys were harvested and sectioned for histology from 4 animals in each group (C) Representative H&E staining in paraffin-embedded kidney sections. Renal tubular casts (asterisk); dilated renal tubules (arrow head); and infiltrated inflammatory (arrow). (D) Kidney histological scores were obtained from kidneys H&E staining by a nephropathologist blinded to the experimental conditions. Results of pathologic scores are expressed as means ± SD of 8 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 1
Figure 1. Cisplatin induces acute kidney injury
Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (10 mg/kg body weight) or vehicle (same volume of 0.9% NaCl) once into mice with three different Klotho genetic backgrounds. (A) Plasma creatinine (PCr) and (B) BUN were measured at days 0, 2, 4, 7, and 14 post injection. The results are expressed as means ± SD of 8 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **:P<0.01 vs WT cisplatin injection; #P<0.05; ##P<0.01 vs Tg-Kl cisplatin injection. At Day 4, 7, and 14 post cisplatin or vehicle injections, mice were euthanized and the kidneys were harvested and sectioned for histology from 4 animals in each group (C) Representative H&E staining in paraffin-embedded kidney sections. Renal tubular casts (asterisk); dilated renal tubules (arrow head); and infiltrated inflammatory (arrow). (D) Kidney histological scores were obtained from kidneys H&E staining by a nephropathologist blinded to the experimental conditions. Results of pathologic scores are expressed as means ± SD of 8 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 2
Figure 2. Cisplatin induces Klotho deficiency and increases NGAL expression
(A) Representative immunoblot for NGAL and Klotho in total kidney lysate in each group (total 4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. (B) Summarized densitometric analyses of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (C) Representative fluorescent immunohistochemistry for Klotho (blue) and NGAL (green) in paraffin kidney sections at day 4, 7 and 14 post injection (4 mice in each group). Arrows show NGAL signals. (D) Levels of Klotho and NGAL transcripts in the kidneys from vehicle or cisplatin injected mice at the 4th, 7th, and 14th day were analyzed by qPCR. The relative quantity of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin vehicle-injected WT mice as reference in each time point. Data are expressed as means ± SD of 6 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 2
Figure 2. Cisplatin induces Klotho deficiency and increases NGAL expression
(A) Representative immunoblot for NGAL and Klotho in total kidney lysate in each group (total 4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. (B) Summarized densitometric analyses of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (C) Representative fluorescent immunohistochemistry for Klotho (blue) and NGAL (green) in paraffin kidney sections at day 4, 7 and 14 post injection (4 mice in each group). Arrows show NGAL signals. (D) Levels of Klotho and NGAL transcripts in the kidneys from vehicle or cisplatin injected mice at the 4th, 7th, and 14th day were analyzed by qPCR. The relative quantity of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin vehicle-injected WT mice as reference in each time point. Data are expressed as means ± SD of 6 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 2
Figure 2. Cisplatin induces Klotho deficiency and increases NGAL expression
(A) Representative immunoblot for NGAL and Klotho in total kidney lysate in each group (total 4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. (B) Summarized densitometric analyses of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (C) Representative fluorescent immunohistochemistry for Klotho (blue) and NGAL (green) in paraffin kidney sections at day 4, 7 and 14 post injection (4 mice in each group). Arrows show NGAL signals. (D) Levels of Klotho and NGAL transcripts in the kidneys from vehicle or cisplatin injected mice at the 4th, 7th, and 14th day were analyzed by qPCR. The relative quantity of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin vehicle-injected WT mice as reference in each time point. Data are expressed as means ± SD of 6 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 2
Figure 2. Cisplatin induces Klotho deficiency and increases NGAL expression
(A) Representative immunoblot for NGAL and Klotho in total kidney lysate in each group (total 4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. (B) Summarized densitometric analyses of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (C) Representative fluorescent immunohistochemistry for Klotho (blue) and NGAL (green) in paraffin kidney sections at day 4, 7 and 14 post injection (4 mice in each group). Arrows show NGAL signals. (D) Levels of Klotho and NGAL transcripts in the kidneys from vehicle or cisplatin injected mice at the 4th, 7th, and 14th day were analyzed by qPCR. The relative quantity of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin vehicle-injected WT mice as reference in each time point. Data are expressed as means ± SD of 6 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 3
Figure 3. Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis
(A) Representative fluorescent immunohistochemistry TUNEL (a cell apoptosis marker, green signal), and for DAPI (a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (B) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. (C) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from 4 mice kidneys in each group at day 7 post cisplatin injection. (D) Summarized densitometric analysis of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (E) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin injected mice at the 7th day were analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to vehicle injected WT mice in each study time point. Data are expressed as means ± SD of 6 animals from each group. (F) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (4 mice per group at days 0, 4, 7 and 14 post cisplatin or vehicle injection. Arrows show positive signal of Ki67. (G) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 3
Figure 3. Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis
(A) Representative fluorescent immunohistochemistry TUNEL (a cell apoptosis marker, green signal), and for DAPI (a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (B) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. (C) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from 4 mice kidneys in each group at day 7 post cisplatin injection. (D) Summarized densitometric analysis of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (E) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin injected mice at the 7th day were analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to vehicle injected WT mice in each study time point. Data are expressed as means ± SD of 6 animals from each group. (F) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (4 mice per group at days 0, 4, 7 and 14 post cisplatin or vehicle injection. Arrows show positive signal of Ki67. (G) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 3
Figure 3. Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis
(A) Representative fluorescent immunohistochemistry TUNEL (a cell apoptosis marker, green signal), and for DAPI (a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (B) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. (C) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from 4 mice kidneys in each group at day 7 post cisplatin injection. (D) Summarized densitometric analysis of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (E) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin injected mice at the 7th day were analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to vehicle injected WT mice in each study time point. Data are expressed as means ± SD of 6 animals from each group. (F) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (4 mice per group at days 0, 4, 7 and 14 post cisplatin or vehicle injection. Arrows show positive signal of Ki67. (G) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 3
Figure 3. Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis
(A) Representative fluorescent immunohistochemistry TUNEL (a cell apoptosis marker, green signal), and for DAPI (a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (B) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. (C) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from 4 mice kidneys in each group at day 7 post cisplatin injection. (D) Summarized densitometric analysis of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (E) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin injected mice at the 7th day were analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to vehicle injected WT mice in each study time point. Data are expressed as means ± SD of 6 animals from each group. (F) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (4 mice per group at days 0, 4, 7 and 14 post cisplatin or vehicle injection. Arrows show positive signal of Ki67. (G) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 3
Figure 3. Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis
(A) Representative fluorescent immunohistochemistry TUNEL (a cell apoptosis marker, green signal), and for DAPI (a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (B) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. (C) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from 4 mice kidneys in each group at day 7 post cisplatin injection. (D) Summarized densitometric analysis of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (E) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin injected mice at the 7th day were analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to vehicle injected WT mice in each study time point. Data are expressed as means ± SD of 6 animals from each group. (F) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (4 mice per group at days 0, 4, 7 and 14 post cisplatin or vehicle injection. Arrows show positive signal of Ki67. (G) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 3
Figure 3. Klotho overexpression increases cell proliferation and suppresses cisplatin-induced apoptosis
(A) Representative fluorescent immunohistochemistry TUNEL (a cell apoptosis marker, green signal), and for DAPI (a marker of cell nuclei, blue signal) in paraffin-embedded kidney sections from each group (4 mice per group) at day 4, 7 and 14 post cisplatin (CP) or vehicle injection. Arrows show TUNEL-positive nuclei. (B) Summary of TUNEL-positive cells divided by total number of DAPI-positive cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. (C) Representative immunoblot for Bax, Bcl-2, cleaved and active caspase-3, and β-actin in total kidney lysate from 4 mice kidneys in each group at day 7 post cisplatin injection. (D) Summarized densitometric analysis of all samples from vehicle or cisplatin injected mice. Data are expressed as means ± SD of 4 animals from each group. (E) Levels of Bcl-2 and Bax mRNA in the kidneys from vehicle or cisplatin injected mice at the 7th day were analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to vehicle injected WT mice in each study time point. Data are expressed as means ± SD of 6 animals from each group. (F) Representative fluorescent immunohistochemistry for Ki67 (cell proliferation marker, red) and for DAPI (marker of cell nuclei, blue) in paraffin-embedded kidney sections from each group (4 mice per group at days 0, 4, 7 and 14 post cisplatin or vehicle injection. Arrows show positive signal of Ki67. (G) Summary of positive Ki67 cells over total number of DAPI cells from 10 microscopic fields at 40X magnification. Data are expressed as means ± SD of 4 animals from each group. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 4
Figure 4. Klotho protects NRK cells from cisplatin cytotoxicity
NRK cells were plated on Transwell plate to allow separate access to apical and basal compartments. Cisplatin (CP), Klotho (Kl), or Cimetidine (Cim) were added to either apical or basolateral compartments simultaneously. Alternatively, Cisplatin was added basally for 20 minutes followed by wash-out and addition of cimetidine or Klotho to basal side. (A) Cell damage was measured by LDH release. (B) Representative fluorescent immunocytochemistry for TUNEL (apoptosis marker, green), and for DAPI (marker of cell nuclei, blue) in NRK cells from a total of 4 independent experiments. Arrows show positive TUNEL signal. (C) Summary of positive TUNEL cells divided by total number of DAPI cells from 5 microscopic fields at 40X magnification. In both (A) and (C), Data are expressed as means ± SD of 4 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups. Cim: cimetidine; Kl: Klotho; inhib.: inhibitor.
Figure 4
Figure 4. Klotho protects NRK cells from cisplatin cytotoxicity
NRK cells were plated on Transwell plate to allow separate access to apical and basal compartments. Cisplatin (CP), Klotho (Kl), or Cimetidine (Cim) were added to either apical or basolateral compartments simultaneously. Alternatively, Cisplatin was added basally for 20 minutes followed by wash-out and addition of cimetidine or Klotho to basal side. (A) Cell damage was measured by LDH release. (B) Representative fluorescent immunocytochemistry for TUNEL (apoptosis marker, green), and for DAPI (marker of cell nuclei, blue) in NRK cells from a total of 4 independent experiments. Arrows show positive TUNEL signal. (C) Summary of positive TUNEL cells divided by total number of DAPI cells from 5 microscopic fields at 40X magnification. In both (A) and (C), Data are expressed as means ± SD of 4 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups. Cim: cimetidine; Kl: Klotho; inhib.: inhibitor.
Figure 5
Figure 5. Klotho suppresses OCT-mediated cisplatin update by NRK cells
(A) Representative immunoblots for OCT1 and OCT2 protein expression in total cell lysate of NRK cells from 3 independent experiments. (B) Representative RT-PCR for oct1, oct2, and β-actin transcripts in NRK cells and kidneys of normal Spraque-Dalwey rat. Same findings were seen in 3 independent experiments. (C) Representative confocal images showing fluorescence-labeled cisplatin uptake by NRK cells. Cisplatin was labeled with Oregon Green 488 dye (Invitrogen) and added to apical or basolateral medium with Klotho or cimetidine or vehicle. Arrows show the presence of cisplatin in the cytoplasmic compartment of NRK cells. Same results were seen in 3 independent experiments. (D) 14C-TEA was basally or apically incubated with cimetidine or Klotho simultaneously or 14C-TEA was basally incubated for 20 minutes followed by wash-out and addition of cimetidine or Klotho to basal medium. Data are expressed as means ± SD of 4 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups. TEA: tetraethylammonium.
Figure 5
Figure 5. Klotho suppresses OCT-mediated cisplatin update by NRK cells
(A) Representative immunoblots for OCT1 and OCT2 protein expression in total cell lysate of NRK cells from 3 independent experiments. (B) Representative RT-PCR for oct1, oct2, and β-actin transcripts in NRK cells and kidneys of normal Spraque-Dalwey rat. Same findings were seen in 3 independent experiments. (C) Representative confocal images showing fluorescence-labeled cisplatin uptake by NRK cells. Cisplatin was labeled with Oregon Green 488 dye (Invitrogen) and added to apical or basolateral medium with Klotho or cimetidine or vehicle. Arrows show the presence of cisplatin in the cytoplasmic compartment of NRK cells. Same results were seen in 3 independent experiments. (D) 14C-TEA was basally or apically incubated with cimetidine or Klotho simultaneously or 14C-TEA was basally incubated for 20 minutes followed by wash-out and addition of cimetidine or Klotho to basal medium. Data are expressed as means ± SD of 4 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups. TEA: tetraethylammonium.
Figure 6
Figure 6. Mechanism of action of Klotho on OCT2 in CHO cells
CHO cells were transiently transfected with empty pcDNA3.1 vector, human OCT2-V5 plasmid, or human Klotho plasmid. Two days post-transfection, cells were subjected to 14C-TEA uptake, immunocytochemistry, immunoblot, and RT-qPCR. β-glucuronidase (100μg/ml), sialidase (0.1 IU/ml), DSAL (10 μM), or cimetidine (0.1 mM) were added into culture medium 16 hours prior to assays. (A) 14C-TEA uptake by CHO cells. Data are expressed as means ± SD of 4 independent experiments. (B) Representative image (X, Y and Z scanning) of fluorescent immunocytochemistry for V5 (OCT2, green), for Klotho (blue) and for phalloidin (marker of actin, red) in CHO cells from 4 independent experiments. CHO cell without Klotho expression had strong OCT2 expression (showed by arrow); whereas CHO cells expressing high Klotho had weak OCT2 expression (showed by arrow). (C) Summary of arbitrary units of OCT2 signal vs. Klotho signal in OCT2/V5 and/or Klotho transiently transfected CHO cells. Each point was an average of arbitrary units from 5 different randomized fields where each field has at least 4 positive transfected cells at 100X magnification. OCT2-positive cells (green symbols); Klotho-positive cells (blue symbols) and double positive cells (orange symbols). Y axis is arbitrary unit of OCT2 signal calculated by green density (OCT2) over red density (actin) using Image J program; X axis is arbitrary unit of Klotho signal which was calculated by green density (OCT2) over red density (actin) using Image J program. Data are expressed as means ± SD of 4 independent experiments. (D) Representative immunoblots for OCT2/V5 by V5 antibody, Klotho by KM2076 antibody, and β-actin in total membrane protein extracted from CHO cells. Identical results were seen in 3 independent experiments. Bottom panel is a summary of densitometric analysis of bands of V5 and β-actin from all 3 independent experiments. (E) Levels of human OCT2, rat oct1 and oct2 mRNA in NRK cells transiently transfected with human OCT2/V5 plasmid and Klotho plasmid were quantitatively analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to OCT2/V5 transfected CHO cells. Data are expressed as means ± SD of 3 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *: P<0.05; or **: P<0.01 between two groups. β-glu: β-glucuronidase; DSAL: D-Saccharic acid 1,4-lactone.
Figure 6
Figure 6. Mechanism of action of Klotho on OCT2 in CHO cells
CHO cells were transiently transfected with empty pcDNA3.1 vector, human OCT2-V5 plasmid, or human Klotho plasmid. Two days post-transfection, cells were subjected to 14C-TEA uptake, immunocytochemistry, immunoblot, and RT-qPCR. β-glucuronidase (100μg/ml), sialidase (0.1 IU/ml), DSAL (10 μM), or cimetidine (0.1 mM) were added into culture medium 16 hours prior to assays. (A) 14C-TEA uptake by CHO cells. Data are expressed as means ± SD of 4 independent experiments. (B) Representative image (X, Y and Z scanning) of fluorescent immunocytochemistry for V5 (OCT2, green), for Klotho (blue) and for phalloidin (marker of actin, red) in CHO cells from 4 independent experiments. CHO cell without Klotho expression had strong OCT2 expression (showed by arrow); whereas CHO cells expressing high Klotho had weak OCT2 expression (showed by arrow). (C) Summary of arbitrary units of OCT2 signal vs. Klotho signal in OCT2/V5 and/or Klotho transiently transfected CHO cells. Each point was an average of arbitrary units from 5 different randomized fields where each field has at least 4 positive transfected cells at 100X magnification. OCT2-positive cells (green symbols); Klotho-positive cells (blue symbols) and double positive cells (orange symbols). Y axis is arbitrary unit of OCT2 signal calculated by green density (OCT2) over red density (actin) using Image J program; X axis is arbitrary unit of Klotho signal which was calculated by green density (OCT2) over red density (actin) using Image J program. Data are expressed as means ± SD of 4 independent experiments. (D) Representative immunoblots for OCT2/V5 by V5 antibody, Klotho by KM2076 antibody, and β-actin in total membrane protein extracted from CHO cells. Identical results were seen in 3 independent experiments. Bottom panel is a summary of densitometric analysis of bands of V5 and β-actin from all 3 independent experiments. (E) Levels of human OCT2, rat oct1 and oct2 mRNA in NRK cells transiently transfected with human OCT2/V5 plasmid and Klotho plasmid were quantitatively analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to OCT2/V5 transfected CHO cells. Data are expressed as means ± SD of 3 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *: P<0.05; or **: P<0.01 between two groups. β-glu: β-glucuronidase; DSAL: D-Saccharic acid 1,4-lactone.
Figure 6
Figure 6. Mechanism of action of Klotho on OCT2 in CHO cells
CHO cells were transiently transfected with empty pcDNA3.1 vector, human OCT2-V5 plasmid, or human Klotho plasmid. Two days post-transfection, cells were subjected to 14C-TEA uptake, immunocytochemistry, immunoblot, and RT-qPCR. β-glucuronidase (100μg/ml), sialidase (0.1 IU/ml), DSAL (10 μM), or cimetidine (0.1 mM) were added into culture medium 16 hours prior to assays. (A) 14C-TEA uptake by CHO cells. Data are expressed as means ± SD of 4 independent experiments. (B) Representative image (X, Y and Z scanning) of fluorescent immunocytochemistry for V5 (OCT2, green), for Klotho (blue) and for phalloidin (marker of actin, red) in CHO cells from 4 independent experiments. CHO cell without Klotho expression had strong OCT2 expression (showed by arrow); whereas CHO cells expressing high Klotho had weak OCT2 expression (showed by arrow). (C) Summary of arbitrary units of OCT2 signal vs. Klotho signal in OCT2/V5 and/or Klotho transiently transfected CHO cells. Each point was an average of arbitrary units from 5 different randomized fields where each field has at least 4 positive transfected cells at 100X magnification. OCT2-positive cells (green symbols); Klotho-positive cells (blue symbols) and double positive cells (orange symbols). Y axis is arbitrary unit of OCT2 signal calculated by green density (OCT2) over red density (actin) using Image J program; X axis is arbitrary unit of Klotho signal which was calculated by green density (OCT2) over red density (actin) using Image J program. Data are expressed as means ± SD of 4 independent experiments. (D) Representative immunoblots for OCT2/V5 by V5 antibody, Klotho by KM2076 antibody, and β-actin in total membrane protein extracted from CHO cells. Identical results were seen in 3 independent experiments. Bottom panel is a summary of densitometric analysis of bands of V5 and β-actin from all 3 independent experiments. (E) Levels of human OCT2, rat oct1 and oct2 mRNA in NRK cells transiently transfected with human OCT2/V5 plasmid and Klotho plasmid were quantitatively analyzed by qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin and compared to OCT2/V5 transfected CHO cells. Data are expressed as means ± SD of 3 independent experiments. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *: P<0.05; or **: P<0.01 between two groups. β-glu: β-glucuronidase; DSAL: D-Saccharic acid 1,4-lactone.
Figure 7
Figure 7. Klotho decreased cationic styryl dye (ASP+, a substrate of OCT) uptake by kidney slices
Kidney slices from Kl/+, WT, and Tg-Kl (C) mice were co-incubated with ASP+ with vehicle, Klotho, or cimetidine for 2 hour. Kidney sections were photographed for ASP+ signal (green fluorescent) with laser confocal microscopy.
Figure 8
Figure 8. OCT1 and 2 are expressed in mouse kidney
(A) Representative fluorescent immunohistochemistry for OCT1 and OCT2 (green), Klotho (blue), and phalloidin (actin, red) in paraffin-embedded kidney sections from WT mice. (B) Representative immunoblots for OCT1, OCT2, and β-actin in total kidney lysate from 3 mice in each genotype. Right panel is a summarized densitometric analysis OCT1 and OCT2 protein over β-actin. Data are expressed as means ± SD of 3 animals from each genotype. (C) Representative fluorescent immunohistochemic stain for OCT2 (green) and Klotho (blue) in paraffin-embedded kidney sections from mice in each genotype. (D) Representative RT-PCR for oct1, oct2, and β-actin transcripts in the kidney from mice with each genotype. (E) The levels of oct2 mRNA in mouse kidneys were quantitatively analyzed by RT-qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin compared to WT mice. Data are expressed as means ± SD of 3 animals from each genotype. All experiments were repeated independently 3 times. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 8
Figure 8. OCT1 and 2 are expressed in mouse kidney
(A) Representative fluorescent immunohistochemistry for OCT1 and OCT2 (green), Klotho (blue), and phalloidin (actin, red) in paraffin-embedded kidney sections from WT mice. (B) Representative immunoblots for OCT1, OCT2, and β-actin in total kidney lysate from 3 mice in each genotype. Right panel is a summarized densitometric analysis OCT1 and OCT2 protein over β-actin. Data are expressed as means ± SD of 3 animals from each genotype. (C) Representative fluorescent immunohistochemic stain for OCT2 (green) and Klotho (blue) in paraffin-embedded kidney sections from mice in each genotype. (D) Representative RT-PCR for oct1, oct2, and β-actin transcripts in the kidney from mice with each genotype. (E) The levels of oct2 mRNA in mouse kidneys were quantitatively analyzed by RT-qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin compared to WT mice. Data are expressed as means ± SD of 3 animals from each genotype. All experiments were repeated independently 3 times. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 8
Figure 8. OCT1 and 2 are expressed in mouse kidney
(A) Representative fluorescent immunohistochemistry for OCT1 and OCT2 (green), Klotho (blue), and phalloidin (actin, red) in paraffin-embedded kidney sections from WT mice. (B) Representative immunoblots for OCT1, OCT2, and β-actin in total kidney lysate from 3 mice in each genotype. Right panel is a summarized densitometric analysis OCT1 and OCT2 protein over β-actin. Data are expressed as means ± SD of 3 animals from each genotype. (C) Representative fluorescent immunohistochemic stain for OCT2 (green) and Klotho (blue) in paraffin-embedded kidney sections from mice in each genotype. (D) Representative RT-PCR for oct1, oct2, and β-actin transcripts in the kidney from mice with each genotype. (E) The levels of oct2 mRNA in mouse kidneys were quantitatively analyzed by RT-qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin compared to WT mice. Data are expressed as means ± SD of 3 animals from each genotype. All experiments were repeated independently 3 times. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 8
Figure 8. OCT1 and 2 are expressed in mouse kidney
(A) Representative fluorescent immunohistochemistry for OCT1 and OCT2 (green), Klotho (blue), and phalloidin (actin, red) in paraffin-embedded kidney sections from WT mice. (B) Representative immunoblots for OCT1, OCT2, and β-actin in total kidney lysate from 3 mice in each genotype. Right panel is a summarized densitometric analysis OCT1 and OCT2 protein over β-actin. Data are expressed as means ± SD of 3 animals from each genotype. (C) Representative fluorescent immunohistochemic stain for OCT2 (green) and Klotho (blue) in paraffin-embedded kidney sections from mice in each genotype. (D) Representative RT-PCR for oct1, oct2, and β-actin transcripts in the kidney from mice with each genotype. (E) The levels of oct2 mRNA in mouse kidneys were quantitatively analyzed by RT-qPCR with specific primers. The relative quantification of transcripts was calculated as 2−(ΔΔCt) by normalization to cyclophilin compared to WT mice. Data are expressed as means ± SD of 3 animals from each genotype. All experiments were repeated independently 3 times. Statistical significance was assessed by one-way ANOVA followed by Student-Newman-Keuls test, and significant differences were accepted when *P<0.05; **P<0.01 between two groups.
Figure 9
Figure 9. Klotho suppressed cisplatin excretion in vivo
Ten μl/gram BW of 14C-TEA (0.1 μCi/μl, 3.5 mCi/mmol) was once injected intravenously into Kl/+, WT and Tg-Kl mice, blood and urine samples were collected at the indicated times. (A) Time course of blood concentration of 14C-TEA. (B) Time course of urinary excretion of 14C-TEA. (C) Cumulated urinary 14C-TEA over 60 minutes. (D) Creatinine clearance. In A–C, data are expressed as means ± SD of 3 animals from each genotype and statistical analysis were run by one-way ANOVA followed by Student-Newman-Keuls test. Significant differences were accepted when *P<0.05; **P<0.01 compared to Kl/+ mice; and when #:P<0.05; ##:P<0.01 compared to WT mice. (E) Two hours after injection, eight organs were harvested and homogenized for measurement of 14C-TEA uptake and total protein. Uptake of 14C-TEA normalized to protein was calculated and significances between mice with three different genotypes were analyzed by one-way ANOVA followed by Student-Newman-Keuls test. Significant differences were accepted when *P<0.05; **P<0.01 between two groups. (F) Tissues and organs were sectioned at 10 μm thickness and subjected to autoradiography.
Figure 9
Figure 9. Klotho suppressed cisplatin excretion in vivo
Ten μl/gram BW of 14C-TEA (0.1 μCi/μl, 3.5 mCi/mmol) was once injected intravenously into Kl/+, WT and Tg-Kl mice, blood and urine samples were collected at the indicated times. (A) Time course of blood concentration of 14C-TEA. (B) Time course of urinary excretion of 14C-TEA. (C) Cumulated urinary 14C-TEA over 60 minutes. (D) Creatinine clearance. In A–C, data are expressed as means ± SD of 3 animals from each genotype and statistical analysis were run by one-way ANOVA followed by Student-Newman-Keuls test. Significant differences were accepted when *P<0.05; **P<0.01 compared to Kl/+ mice; and when #:P<0.05; ##:P<0.01 compared to WT mice. (E) Two hours after injection, eight organs were harvested and homogenized for measurement of 14C-TEA uptake and total protein. Uptake of 14C-TEA normalized to protein was calculated and significances between mice with three different genotypes were analyzed by one-way ANOVA followed by Student-Newman-Keuls test. Significant differences were accepted when *P<0.05; **P<0.01 between two groups. (F) Tissues and organs were sectioned at 10 μm thickness and subjected to autoradiography.
Figure 9
Figure 9. Klotho suppressed cisplatin excretion in vivo
Ten μl/gram BW of 14C-TEA (0.1 μCi/μl, 3.5 mCi/mmol) was once injected intravenously into Kl/+, WT and Tg-Kl mice, blood and urine samples were collected at the indicated times. (A) Time course of blood concentration of 14C-TEA. (B) Time course of urinary excretion of 14C-TEA. (C) Cumulated urinary 14C-TEA over 60 minutes. (D) Creatinine clearance. In A–C, data are expressed as means ± SD of 3 animals from each genotype and statistical analysis were run by one-way ANOVA followed by Student-Newman-Keuls test. Significant differences were accepted when *P<0.05; **P<0.01 compared to Kl/+ mice; and when #:P<0.05; ##:P<0.01 compared to WT mice. (E) Two hours after injection, eight organs were harvested and homogenized for measurement of 14C-TEA uptake and total protein. Uptake of 14C-TEA normalized to protein was calculated and significances between mice with three different genotypes were analyzed by one-way ANOVA followed by Student-Newman-Keuls test. Significant differences were accepted when *P<0.05; **P<0.01 between two groups. (F) Tissues and organs were sectioned at 10 μm thickness and subjected to autoradiography.
Figure 10
Figure 10. Summary of findings and proposed scheme for Klotho effect on cisplatin-induced cytotoxicity
Klotho OCT2-independent effect is related to anti-apoptosis (right panel). Klotho OCT2-depedent effects (let panel) can be glucuronidase-dependent or glucuronidase-independent. Glucuronidase-independent effect is associated with downregulation of OCT2 mRNA. Glucuronidase-dependent effects are more complex. Klotho could decrease total cell OCT2 protein by functioning as a glucuronidase independent of OCT2 mRNA. Klotho also reduces primarily glycosylated surface OCT2 protein in 2 hours effect, and both glycosylated and unglycosylated surface OCT2 in 2 days effect. Klotho can potentially directly decrease OCT2 activity without alteration of OCT2 protein (dash line). Collectively, Klotho suppresses OCT2 transport function and consequently reduces cisplatin uptake. On the other hand, Klotho functions as an anti-apoptotic agent and anti-oxidant independently of cisplatin uptake to protect cells against cisplatin-induced cytotoxicity.
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