TAK1 deficiency attenuates cisplatin-induced acute kidney injury

Abstract

Cisplatin can cause acute kidney injury (AKI), but the molecular mechanisms are not well understood. The objective of the present study was to examine the role of transforming growth factor-β-activated kinase-1 (TAK1) in the pathogenesis of cisplatin-induced AKI. Wild-type mice and proximal tubule TAK1-deficient mice were treated with vehicle or cisplatin. Compared with wild-type control mice, proximal tubule TAK1-deficient mice had less severe kidney dysfunction, tubular damage, and apoptosis after cisplatin–induced AKI. Furthermore, conditional disruption of TAK1 in proximal tubular epithelial cells reduced caspase-3 activation, proinflammatory molecule expression, and JNK phosphorylation in the kidney in cisplatin-induced AKI. Taken together, cisplatin activates TAK1-JNK signaling pathway to promote tubular epithelial cell apoptosis and inflammation in cisplatin-induced AKI. Targeting TAK1 could be a novel therapeutic strategy against cisplatin-induced AKI.

INTRODUCTION

Cisplatin (cis-diamminedichloroplatinum II) has been widely and effectively used for chemotherapy against many types of tumors (14, 25). However, it can cause a series of adverse effects in various organs, especially the kidney (8). Cisplatin-induced acute kidney injury (AKI) is a common clinical complication and is associated with high morbidity and mortality (18). The pathogenesis of cisplatin-induced AKI is incompletely understood. Recent studies have shown that cisplatin triggers inflammation and necrosis/apoptosis of renal tubular epithelial cells, which is followed by renal dysfunction and kidney damage (2, 22, 23). To improve the outcomes of patients with cancer that receive cisplatin chemotherapy, an improved understanding of the pathogenic mechanisms underlying cisplatin-induced AKI is crucial for ultimately developing and effective and safer therapy.

Transforming growth factor-β-activated kinase-1 (TAK1) is a serine-threonine kinase that has a key role in regulating immune and proinflammatory intracellular signaling pathways (29, 30). Recently, TAK1 has been reported to participate in the pathogenesis of acute injury in several organs such as the brain and liver (10, 34). Moreover, TAK1 has been implicated in the regulation of inflammation, oxidative stress, and apoptosis (1, 21). Nevertheless, its role in cisplatin-induced AKI has not been investigated. JNK, a TAK1 downstream kinase (3, 6), is involved in regulating inflammation and apoptosis (11, 24). It has been recently reported that activation of JNK contributes to cisplatin-induced renal tubular epithelial cell apoptosis in vitro (4). However, whether the TAK1-JNK signaling pathway is involved in cisplatin-induced AKI is unknown.

In the present study, we investigated the role of TAK1 in the development of cisplatin-induced AKI using mice with conditional disruption of TAK1 in proximal tubular epithelial cells. Our results show that cisplatin activates the TAK1-JNK signaling pathway, resulting in tubular epithelial cell apoptosis and inflammation in the kidney during cisplatin-induced AKI.

MATERIALS AND METHODS

Animals.

Animal experiments were performed in accordance with the guidelines of laboratory animal care and were approved by the Baylor Institutional Animal Care and Use Committee. Homozygous TAK1-floxed (TAK1^fl/fl) mice (32) were mated with mice that express Cre recombinase under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter (28) to generate mice with proximal tubule-specific disruption of TAK1. PEPCK-Cre⁺TAK1^fl/fl mice are referred to as PT-TAK1-KO mice, and littermate PEPCK-Cre⁻TAK1^fl/fl mice were used as controls (CTRL). Genotyping was performed by PCR. Male CTRL mice and TAK1-PEPCK-Cre mice, 8–12 wk old age, weighing 20–30 g, were administrated cisplatin (20 mg/kg) or vehicle (normal saline) by intraperitoneal injection. Animals were euthanized at 72 h after cisplatin injection. Kidneys were perfused and harvested.

Measurement of kidney function.

Serum creatinine was measured using a commercially available kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s instructions. Blood urea nitrogen was determined fluorometrically as previously described (13, 35).

Evaluation of kidney morphology.

Kidney tissues were fixed in 10% buffered formalin, embedded in paraffin, and cut at 4 μm thickness. Sections were deparaffinized, rehydrated, and then stained with hematoxylin and eosin. Tissue damage was examined in a blinded manner and scored (from 0−4) according to the percentage of damaged tubules: 0, no damage; 1, <25% damage; 2, 25–50% damage; 3, 50–75% damage; and 4, >75% damage, as previously reported (13, 16, 35).

Immunohistochemistry.

Immunohistochemical staining was performed on paraffin sections. Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was quenched with 3% H₂O₂. After being blocked with 5% normal serum, sections were incubated with primary antibodies in a humidified chamber overnight. After being washed, sections were incubated with appropriate secondary antibodies and ABC solution sequentially using the ABC kit (Vector Laboratories). Immunoreactivities were then visualized by incubation in DAB solution for an appropriate period of time. Nuclear staining was performed with hematoxylin. Slides were dehydrated, cleared, and mounted. Images from these slides were acquired and analyzed by NIS Element software with a Nikon microscope image system in a blinded manner (13).

Apoptosis assay.

Apoptosis was evaluated using a TUNEL assay kit (Millipore, Billerica, MA). The number of TUNEL-positive cells per high-power field (HPF) were quantified in a blinded fashion (13, 16, 35).

Quantitative real-time RT-PCR.

Total RNA was extracted from kidney tissues by using TRIzol Reagent (Invitrogen, Carlsbad, CA). Aliquots (1 μg) of total RNA were reverse transcribed using SuperScript II reverse transcriptase (Bio-Rad, Hercules, CA). Real-time PCR was performed using IQ SYBR Green Supermix reagent (Bio-Rad) with a Bio-Rad real-time PCR machine according to the manufacturer’s instructions. The comparative threshold cycle (C_T) method (ΔΔC_T) was used to quantify gene expression, and relative quantification was calculated as $2^{-\Delta \Delta {\text{C}}_{\text{T}}}$. Expression levels of target genes were normalized to the GAPDH level in each sample. The primer sequences were as follows: IL-6, forward 5′-AGGATACCACTCCCAACAGACCTG-3′ and reverse 5′-CTGCAAGTGCATCATCGTTGTTCA-3′; TNF-α, forward 5′-CATGAGCACAGAAAGCATGATCCG-3′ and reverse 5′-AAGCAGGAATGAGAAGAGGCTGAG-3′; monocyte chemoattractant protein (MCP)-1, forward 5′-TCACCTGCTGCTACTCATTCACCA-3′ and reverse 5′ TACAGCTTCTTTGGGACACCTGCT-3′; macrophage inflammatory protein (MIP)-2, forward 5′-AAAGTTTGCCTTGACCCTGAAGCC-3′ and reverse 5′-TCCAGGTCAGTTAGCCTTGCCTTT-3′; and GAPDH, forward 5′-CCAATGTGTCCGTCGCGTGGATCT-3′ and reverse 5′-GTTGAAGTCGCAGGAGACAACC-3′.

Western blot analysis.

Total proteins were extracted using RIPA buffer containing cocktail proteinase and phosphatase inhibitors and quantified with a Bio-Rad protein assay. An equal amount of protein (50 μg) was separated on SDS-polyacrylamide gels in a Tris-SDS buffer system and then transferred onto nitrocellulose membranes. Blotting was performed according to standard procedures with primary antibodies overnight followed by incubation with appropriate fluorescence-conjugated secondary antibodies. Proteins of interest were analyzed using an Odyssey IR scanner, and signal intensities were quantified using ImageJ software (National Institutes of Health).

Statistical analysis.

Data are presented as means ± SE. Multiple-group comparisons were performed by ANOVA followed by the Bonferroni procedure for comparison of means. P values of <0.05 were considered statistically significant.

RESULTS

TAK1 deficiency in proximal tubule reduces cisplatin-induced AKI.

To investigate the role of TAK1 in kidney tubular epithelial cells in the pathogenesis of cisplatin-induced AKI, we generated conditional TAK1 knockout (KO) mice in which the TAK1 gene is specifically disrupted in kidney proximal tubules using the Cre-LoxP system. Homozygous TAK1-floxed (TAK1^fl/fl) mice were mated with mice that express Cre recombinase under the control of the PEPCK promoter. PEPCK-Cre⁺TAK1^fl/fl mice were referred to as PT-TAK1-KO mice and their littermate PEPCK-Cre⁻TAK1^fl/fl mice were used as CTRL mice. PT-TAK1-KO mice and CTRL mice were treated with vehicle or cisplatin. CTRL mice displayed severe renal dysfunction, as reflected by marked elevations of serum creatinine and blood urea nitrogen at 72 h after cisplatin treatment. In contrast, PT-TAK1-KO mice exhibited much less severe renal dysfunction with lower serum creatinine and blood urea nitrogen (Fig. 1, A and B). Furthermore, PT-TAK1-KO mice developed substantially less severe histological kidney injury, as manifested by less severe tubular epithelial cell injury, tubular dilation, and intratubular cast formation (Fig. 1, C and D).

Fig. 1.

Genetic deficiency of transforming growth factor-β-activated kinase-1 (TAK1) in proximal tubules (PTs) protects the kidney against cisplatin-induced acute kidney injury. A: effect of conditional TAK1 deficiency on serum creatinine in control and phosphoenolpyruvate carboxykinase-Cre⁺TAK1^fl/fl knockout (PT-TAK1-KO) mice at 72 h after cisplatin or saline administration. n = 6 in each group. **P < 0.01 vs. the vehicle-treated control group; +P < 0.05 vs. the cisplatin-treated KO group; #P < 0.05 vs. the cisplatin-treated control group. B: effect of conditional TAK1 deficiency on blood urea nitrogen (BUN) in control and PT-TAK1-KO mice at 72 h after cisplatin or vehicle administration. n = 6 in each group. **P < 0.01 vs. the vehicle-treated control group; ++P < 0.01 vs. the cisplatin-treated KO group; #P < 0.05 vs. the cisplatin-treated control group. C: representative photomicrographs of hematoxylin and eosin staining for kidney sections of control and PT-TAK1-KO mice at 72 h after cisplatin or vehicle treatment. Scale bar = 50 μm. D: quantitative assessment of tubular damage in wild-type (WT) and KO mice at 72 h after cisplatin treatment. n = 6 in each group. *P < 0.05 vs. the cisplatin-treated control group.

TAK1 deficiency in proximal tubules inhibits tubular cell apoptosis in cisplatin-induced AKI.

We next performed TUNEL staining to evaluate whether TAK1 deficiency in proximal tubules influences tubular cell apoptosis in the kidney. Cisplatin treatment resulted in a significant increase in the number of tubular apoptotic cells in kidneys of CTRL mice. Remarkably, the number of tubular apoptotic cells was reduced in the kidneys of cisplatin-treated PT-TAK1-KO mice (Fig. 2, A and B).

Fig. 2.

Transforming growth factor-β-activated kinase-1 (TAK1) deficiency in proximal tubules (PTs) inhibits apoptosis in cisplatin-induced acute kidney injury. A: representative photomicrographs of kidney sections stained for apoptotic cells in control and phosphoenolpyruvate carboxykinase-Cre⁺TAK1^fl/fl knockout (PT-TAK1-KO) mice at 72 h after cisplatin or vehicle treatment. Scale bar = 50 μm. B: quantitative analysis of apoptotic cells in kidneys of control and PT-TAK1-KO mice after cisplatin or vehicle treatment. n = 6 in each group. **P < 0.01 vs. the vehicle-treated control group; ++P < 0.01 vs. the cisplatin-treated KO group; #P < 0.05 vs. the cisplatin-treated control group.

TAK1 deficiency in proximal tubules suppresses caspase-3 activation in cisplatin-induced AKI.

We assessed the effect of TAK1 deficiency in proximal tubules on caspase-3 activation in the kidney. Immunohistochemical staining was performed using an antibody against cleaved caspase-3. The results revealed that caspase-3 was activated in kidney tubular epithelial cells of CTRL mice treated with cisplatin. In contrast, disruption of TAK1 in proximal tubules significantly reduced caspase-3 activation (Fig. 3, A and B). We next performed Western blot analysis using antibody against cleaved caspase-3. Consistent with immunohistochemical staining results, the results of Western blot analysis showed that caspase-3 was significantly activated in kidneys of CTRL mice after cisplatin treatment compared with vehicle-treated control mice. Conditional TAK1 deficiency in proximal tubules significantly inhibited caspase-3 activation in kidneys of cisplatin-treated mice (Fig. 3, C and D). These results suggest that TAK1 promotes caspase-3 activation in AKI induced by cisplatin.

Fig. 3.

Transforming growth factor-β-activated kinase-1 (TAK1) deficiency in proximal tubules (PTs) suppresses caspase-3 activation in cisplatin-induced acute kidney injury. A: representative photomicrographs of kidney sections stained for cleaved caspase-3 (brown) and counterstained with hematoxylin (blue) in control and phosphoenolpyruvate carboxykinase-Cre⁺TAK1^fl/fl knockout (PT-TAK1-KO) mice. Scale bar = 50 μm. B: quantitative analysis of cleaved caspase-3 in kidneys of control and PT-TAK1-KO mice. n = 6 in each group. **P < 0.01 vs. the vehicle-treated control group; ++P < 0.01 vs. the cisplatin-treated PT-TAK1-KO group; ##P < 0.01 vs. the cisplatin-treated control group. C: representative Western blots showing cleaved caspase-3 protein in kidneys of wild-type (WT) and PT-TAK1-KO mice. D: quantitative analysis of cleaved caspase-3 protein in kidneys of WT and PT-TAK1-KO mice after vehicle or cisplatin treatment. n = 6 in each group. **P < 0.01 vs. the vehicle-treated control group; ++P < 0.01 vs. the cisplatin-treated KO group; #P < 0.05 vs. the cisplatin-treated control group.

TAK1 deficiency in proximal tubules suppresses proinflammatory molecule expression in cisplatin-induced AKI.

Inflammation characterized by expression of chemokines and cytokines in the kidney is involved in the pathogenesis of cisplatin-induced AKI (27, 33, 34, 35). We performed quantitative real-time RT-PCR to examine whether conditional disruption of TAK1 in proximal tubules affects expression levels of known proinflammatory molecules that play a critical role in cisplatin-induced AKI. The results showed that mRNA levels of IL-6, TNF-α, MCP-1, and MIP-2 were increased significantly in the kidneys of CTRL mice at 72 h after cisplatin treatment compared with vehicle-treated control mice (Fig. 4). In contrast, mRNA levels of IL-6, TNF-α, MCP-1, and MIP-2 were significantly diminished in the kidneys of PT-TAK1-KO mice at 72 h after cisplatin treatment. These results indicate that proximal tubular TAK1 stimulates inflammatory cytokine and chemokine expression in the kidney during cisplatin-induced AKI.

Fig. 4.

Transforming growth factor-β-activated kinase-1 (TAK1) deficiency in proximal tubules (PTs) suppresses gene expression of proinflammatory molecules in the kidney. A: quantitative analysis of IL-6 mRNA expression in the kidney. B: quantitative analysis of TNF-α mRNA expression in the kidney. C: quantitative analysis of monocyte chemoattractant protein (MCP)-1 mRNA expression in the kidney. D: quantitative analysis of macrophage-inflammatory protein (MIP)-2 mRNA expression in the kidney. n = 6 in each group. **P < 0.01 vs. the vehicle control group; ++P < 0.01 vs. the cisplatin-treated phosphoenolpyruvate carboxykinase-Cre⁺TAK1^fl/fl knockout (PT-TAK1-KO) group; ##P < 0.01 vs. the cisplatin-treated control group.

TAK1 deficiency in proximal tubules reduces JNK activation in cisplatin-induced AKI.

JNK has been implicated in many cellular responses including apoptosis and inflammation (30, 31). TAK1 is an upstream signal for JNK activation (32). To explore the mechanisms by which TAK1 contributes to cisplatin-induced AKI, we examined whether TAK1 affects JNK activation. Western blot analysis showed that phosphorylation of JNK was markedly increased in the kidneys of CTRL mice after cisplatin treatment compared with vehicle-treated CTRL mice. In comparison, levels of phosphorylation of JNK were significantly attenuated in kidneys of PT-TAK1-KO mice treated with cisplatin compared with cisplatin-treated CTRL mice (Fig. 5, A and B). These data suggest that TAK1 activates JNK to promote cisplatin-induced kidney injury.

Fig. 5.

Transforming growth factor-β-activated kinase-1 (TAK1) deficiency in proximal tubules (PTs) inhibits JNK1 phosphorylation in cisplatin-induced acute kidney injury. A: representative Western blots showing the phosphorylation level of JNK1 in kidneys of control and phosphoenolpyruvate carboxykinase-Cre⁺TAK1^fl/fl knockout (PT-TAK1-KO) mice. P-JNK, phosphorylated JNK. B: quantitative analysis of the phosphorylation level of JNK1 in kidneys of control and PT-TAK1-KO mice. n = 6 in each group. **P < 0.01 vs. the vehicle-treated control group; ++P < 0.01 vs. the cisplatin-treated KO group; #P < 0.05 vs. the cisplatin-treated control group.

DISCUSSION

Cisplatin is a broadly used agent for the treatment of solid tumors (31). A common complication of cisplatin administration is AKI, which limits its clinical use (8). TAK1 is a key component of signal cascades that regulate apoptosis and inflammation (1, 21). However, the role of TAK1 in cisplatin-induced AKI is not known. In the present study, we have demonstrated that 1) conditional disruption of TAK1 in proximal tubules protects the kidney from cisplatin-induced AKI, 2) conditional disruption of TAK1 in proximal tubules reduces apoptosis and inhibits caspase-3 activation in cisplatin-induced AKI, 3) conditional disruption of TAK1 in proximal tubules inhibits proinflammatory molecule production in the kidney after cisplatin administration, and 4) conditional disruption of TAK1 in proximal tubules suppresses JNK activation. These results suggest that TAK1 promotes the pathogenesis of cisplatin-induced AKI through activation of the JNK signal pathway and regulation of apoptosis and inflammation.

Recent studies have reported that a diverse set of intra- and extracellular stimuli can activate TAK1, which has a central role in regulating cell death and survival (17, 21). Accumulating evidence indicates that TAK1 regulates expression of proinflammatory cytokines and chemokines in various tissue injuries (15, 19), but its role of TAK1 in cisplatin-induced AKI remains to be determined. In the present study, we demonstrated, for the first time, that conditional disruption of TAK1 in proximal tubular epithelial cells preserves renal function and reduces tubular damage after cisplatin-induced AKI. These findings uncover a crucial role of TAK1 signaling in the pathogenesis of cisplatin-induced AKI.

It is well recognized that tubular epithelial cell apoptosis contributes to kidney injury in various animal models of AKI (9). Cisplatin can induce apoptosis of kidney tubular epithelial cells (22, 23). In the present study, we demonstrated that conditional disruption of TAK1 in renal tubular epithelial cells reduces cisplatin-induced apoptotic cell death in the kidney. Caspase-3 is a zymogen that is cleaved to form active caspase-3 through death ligand and mitochondrial pathways. Caspase-3 activation is a key mechanism resulting in cisplatin-induced apoptotic cell death in tubular epithelial cells (5, 22, 23). In the present study, we showed that conditional disruption of TAK1 in proximal tubules inhibits cisplatin-induced caspase-3 activation in the kidney. These results indicate that TAK1 signaling activates caspase-3 to stimulate apoptotic cell death in cisplatin-induced AKI.

Recent studies have demonstrated that proinflammatory cytokines and chemokines play a crucial role in the pathogenesis of cisplatin-induced AKI (12, 16, 26). In the present study, we assessed mRNA expression levels of IL-6, TNF-α, MCP-1, and MIP-2 in the kidney using real-time RT-PCR. Our results revealed that conditional disruption of TAK1 in proximal tubules reduces gene expression levels of IL-6, TNF-α, MCP-1, and MIP-2 in the kidney after the administration of cisplatin. These results indicate that renal proximal tubules are a major source of proinflammatory cytokines and chemokines in cisplatin-induced AKI.

The JNK pathway has been implicated in the development of apoptosis and inflammation in response to various cellular stresses (7, 11). Activation of TAK1 triggers several downstream signaling cascades, including the JNK pathway (20). However, it is unclear whether TAK1 regulates JNK activation in the kidney in cisplatin-induced AKI. In the present study, we demonstrated that conditional disruption of TAK1 in proximal tubules inhibits JNK phosphorylation in the kidney in response to cisplatin administration. Furthermore, inhibition of TAK1 with 5Z-7-oxozeaenol abolishes cisplatin-induced JNK activation and proinflammatory molecule expression in cultured tubular epithelial cells (data not shown). These results suggest that TAK1 may activate JNK to promote tubular injury and inflammation.

In summary, our study uncovers an important role of TAK1 in the pathogenesis of cisplatin-induced AKI. In response to cisplatin administration, the TAK1-JNK signaling pathway is activated, which causes tubular epithelial cell apoptosis and inflammation. These results indicate that the TAK1 signaling pathway could be a novel therapeutic target for cisplatin-induced AKI.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-95835 and United States Department of Veterans Affairs Grant I01BX02650 (to Y. Wang).

DISCLAIMERS

The contents of the article do not represent the views of the United States Department of Veterans Affairs or the United States government.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

J.Z. and Y.W. conceived and designed research; J.Z. and C.A. performed experiments; J.Z. and C.A. analyzed data; J.Z., C.A., X.J., Z.H., and R.L.S. interpreted results of experiments; J.Z. and C.A. prepared figures; J.Z. drafted manuscript; C.A. and Y.W. edited and revised manuscript; J.Z., C.A., X.J., R.L.S., and Y.W. approved final version of manuscript.