Experimental Cell Research
Hsp90 inhibition aggravates adriamycin-induced podocyte injury through intrinsic apoptosis pathway
Junyu Dong, Zhihong Jiang∗, Guorui Ma
Department of Paediatrics, The First Affiliated Hospital of Henan University of Science & Technology, Luoyang, Henan, 471000, China
A R T I C L E I N F O
Keywords:
Hsp90 PU-H71
Adriamycin Podocyte injury
Filtration barrier function Intrinsic apoptosis pathway
A B S T R A C T
Podocyte injury leads to impaired filtration barrier function of the kidney that underlies the pathophysiology of idiopathic nephrotic syndrome (INS), the most common NS occurring in children. The heat shock protein 90 (Hsp90) is involved in the regulation of apoptosis in a variety of cell types, however, little is known about its role in podocytes and whether it associated with NS. Here, we show that Hsp90 is upregulated in glomeruli podocytes from mice with adriamycin (ADR)-induced nephropathy, and that it is also upregulated in an immortalized podocyte cell line treated with ADR in vitro, together suggesting an association of Hsp90 upregulation in po- docytes with NS pathogenesis. Functionally, Hsp90 inhibition with PU-H71 aggravates ADR-induced podocyte apoptosis and worsens the impairment of filtration barrier function. Mechanistically, Hsp90 inhibition with PU- H71 enhances the activation of intrinsic apoptotic pathway, and moreover, blockade of podocyte apoptosis with zVAD-fmk (aVAD), a pan-caspase inhibitor, abrogates effects of Hsp90 inhibition on filtration barrier function of ADR-treated podocytes, thus demonstrating that Hsp90 inhibition aggravates ADR-induced podocyte injury through intrinsic apoptosis pathway. In sum, this study reveals a detrimental role of Hsp90 inhibition in po- docyte injury, which may offer it as a potential therapeutic target in NS therapy.
1. Introduction
The idiopathic nephrotic syndrome (INS) is a leading cause of NS in children and characterized by clinical signs including massive protei- nuria, hypoalbuminemia and edema. Currently, corticosteroids, the immunosuppressive agents, remain the basic therapy for INS via tar- geting lymphocytes, however, disease frequently relapses after treat- ment [1,2], highlighting the need for alternative agents. Although the precise pathogenesis of INS remains elusive, the structural and func- tional abnormalities of podocytes have been thought to play an im- portant role [3], which paves the way for discovering novel therapeutic targets for INS treatment.
Podocytes are terminally differentiated epithelial cells required for maintaining the glomerular structural integrity and filtration barrier function of the kidney [4]. Consequently, podocyte injury increases the permeability of the glomerular basement membrane and leads to pro- teinuria. Typically, podocyte injury through apoptosis induces protei- nuria in animal models [5–7]. However, the molecular mechanisms regulating podocyte apoptosis in the context of DN are not fully un- derstood.
The heat shock protein 90 (Hsp90) is an evolutionary conserved
chaperon essential for the proteostasis of hundred protein substrates involved in a variety of cellular processes, such as stress regulation, DNA repair, proliferation and apoptosis [8]. It has been shown that Hsp90 inhibition via its inhibitor PU-H71 induces apoptosis in various types of cancer cells [9], indicating that Hsp90 may be a negative regulator of apoptosis. Interestingly, the increased level of Hsp90 transcript was found in peripheral blood mononuclear cells (PBMCs) from patients with minimal-change nephrotic syndrome (MCNS), a common histologic diagnose of INS [10]. Nevertheless, whether Hsp90 regulates apoptosis in podocytes and has a mechanistic connection to NS are largely unclear.
In this study, by advantage of adriamycin (ADR)-induced nephro- pathy model in vivo and in vitro, we show that Hsp90 is induced in podocytes and that its inhibition aggravates ADR-induced podocyte apoptosis, through which the impairment of filtration barrier function of podocytes is enhanced, therefore relating Hsp90-regulated podocyte injury to NS pathogenesis.
Corresponding author. Department of Paediatrics, The First Affiliated Hospital of Henan University of Science & Technology, No. 24 Jinghua Road, Jianxi District, Luoyang, Henan, 471000, China.
E-mail address: [email protected] (Z. Jiang).
J. Dong, et al. ExperimentalCellResearchxxx(xxxx)xxxx
2. Materials and methods
2.1. Animals and adriamycin-induced nephropathy
Eight-week-old male BALB/c mice were applied in this study to develop experimental nephropathy model. A total amount of 10 mg/kg body weight of adriamycin hydrochloride (ADR) (Sigma) or the equal volume of saline were injected into each mouse via the tail vein. Nine mice were included in each group. At 14 and 21 days after ADR in- jection, mice were perfused with cold PBS and the kidneys were har- vested for further analysis. The urine albumin was determined by a mouse ELISA kit (Bethyl Laboratory Inc), and meanwhile, the urine creatinine was quantified using the Creatinine Assay Kit (BioAssay Systems) according to the manufacturers’ instructions. All animal pro- cedures were conducted in accordance with the protocols approved by the Ethics Committee of The First Affiliated Hospital of Henan University of Science & Technology.
2.2. Cell culture and treatment
The immortalized murine JR07 podocytes were cultured as de- scribed previously [11]. In brief, cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.292 mg/ml glutamine (Life Technologies). Podocytes were normally expanded in medium containing 10 U/mL mouse interferon-γ (INF-γ) (Sigma-Aldrich, 11276905001) at 33 °C with 5% CO2 atmosphere, and also were al- lowed to differentiate by removing INF-γ from culture medium. The differentiation process was maintained for 10 days and culture medium was refreshed every 3 days. Differentiated podocytes were treated with
0.5 μg/ml ADR, 1 μM PU-H71 (TOCRIS, 3104) or 50 μM zVAD-fmk
(TOCRIS, 2163) alone or in combination for different time periods ac- cording to experimental purposes.
2.3. qRT-PCR analysis
The total RNA was extracted from the renal cortex or cultured po- docytes with the TRIzol reagent (ThermoFisher Scientific). The ex- tracted total RNA was reversely synthesized into cDNA using the cDNA Synthesis Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. cDNA was used to perform qRT-PCR analysis with SYBR green Mix (Bio-Rad) using a 7500 PCR Detection System (Applied Biosystems) for quantifying levels of target genes. The results were normalized to that of house-keeping gene β-Actin. The primer se- quences are available upon request.
2.4. Fractionation of mitochondria/cytosol and Western blot analysis
The total protein was extracted from the renal cortex or cultured podocytes using RIPA lysis buffer supplemented with protease cocktail inhibitors (Roche). The fractionation of mitochondria and cytosol was performed using the Mitochondria/Cytosol Fractionation Kit (Biovision, K256) according to the manufacturer’s instructions. Equal amount of total protein was separated by SDS-PAGE, and transferred to ni- trocellulose (NC) membranes. NC membranes were blocked for 1 h in 5% BSA and then incubated overnight with the primary antibodies at 4 °C. After the wash with TBST, NC membranes were incubated with HRP-conjugated secondary antibody for 1 h at RT. Protein blots were detected with the ECL Chemiluminescence Substrate Kit (Pierce), and quantified by Image J software. The antibodies were purchased from the following sources: anti-Hsp90 (Cell Signaling technology, 4874), anti-Bcl-2 (Sigma-Aldrich, B3170), Bax (Proteintech, 50599-2-Ig), cleaved caspase 9 (Cell Signaling technology, 7237) cleaved caspase 3 (Cell Signaling technology, 9661), cytochrome c (Santa Cruz, sc- 13156), β-Actin (Proteintech, 60008-1-Ig).
2.5. TUNEL assay
The TdT-mediated dUTP nick end-labeling (TUNEL) assay was carried out to detect the apoptosis of podocytes. Briefly, murine JR07 podocytes were seeded on the coverslips imbedded in 24-well plates. After treatment, apoptotic cells were stained by using the TUNEL Apoptosis Detection Kit (Roche Diagnostics) following the manufac- turer’s instructions. After the counterstaining with DAPI to visualize nuclei, images were captured using a fluorescent microscope. The TUNEL-positive cells are defined as apoptotic cells, and the number was counted in 18 randomly selected fields for each treatment group. Experiments were independently performed for three times.
2.6. Albumin influx assay
Five thousand murine JR07 podocytes were seeded on transwell filters with 3 μm size pore (Corning) and differentiated for 10 days. After differentiation, cells were treated with 1 μM PU-H71 (TOCRIS, 3104) or 50 μM zVAD-fmk or in combination in the presence or absence of 0.5 μg/ml ADR for 48 h. Cells were then washed twice with PBS supplemented with MgCl2 (1 mmol/L) and CaCl2 (1 mmol/L) to pre- serve cadherin based junctions. The transwell top chamber was filled with 0.15 ml RPMI 1640 and the bottom chamber with 1 ml RPMI 1640 containing 40 mg/ml BSA. Cells were further incubated at 37 °C for 4 h, and 100 μl media in the top chamber were collected and the albumin concentration was measured by the bicinchoninic acid assay kit (Sigma- Aldrich, B9643) according to manufacturer’s instructions.
2.7. Statistical analysis
All data are presented as the mean ± SD. Statistical analysis was performed using Student’s t-test for comparing two groups, or one-way ANOVA followed by Tukey’s multiple comparisons test for comparing more than two groups. A P value less than 0.05 was considered with a significant difference.
3. Results
3.1. Hsp90 expression is induced in podocytes from ADR-induced nephropathy mice
The upregulated transcript level of Hsp90 was observed in periph- eral blood mononuclear cells (PBMCs) from patients with minimal- change nephrotic syndrome (MCNS) [12]. However, to our best knowledge, this finding is still not validated, and particularly, the renal expression pattern of Hsp90 during the development of NS is unclear either. To address it, we injected adriamycin (ADR) into mice through tail vein to induce nephrotic syndrome (NS), a rodent model of chronic kidney disease [13], and then checked the expression pattern of Hsp90 in the renal cortex. qRT-PCR analysis revealed that the transcript level of Hsp90 was significantly induced in NS mice compared with normal mice injected with vehicle (Fig. 1A). Similarly, the induced expression of Hsp90 in NS mice was also reproduced at the protein level, as de- termined by Western blot analysis (Fig. 1B). The renal cortex is en- riched with glomeruli podocytes [14]. Immunohistochemistry analysis of renal biopsies showed that in contrast to normal mice, Hsp90 ex- pression was induced in podocytes in NS mice (Fig. 1C), hence histo- logically confirming the presence of Hsp90 upregulation in podocytes from mice with ADR-induced NS. Notably, ADR indeed induced glo- merular injury and albuminuria in these mice, as indicated by the glomerular pathological changes (Fig. 1C) and increased urine al- bumin/creatinine (Fig. 1D), suggesting an in vivo correlation of Hsp90 upregulation with glomerular injury and albuminuria.
Fig. 1. Hsp90 expression is upregulated in glomeruli podocytes from ADR-in- duced nephropathy mice. (A-D) Eight-week-old male BALB/c mice were in- travenously injected with 10 mg/kg adriamycin hydrochloride (ADR) or equal volume of saline (n = 9). At 14 and 21 after ADR injections, the renal biopsies were prepared for further analysis. (A) The mRNA level of Hsp90 was measured by qRT-pCR analysis. β-Actin was used as an internal control. Data are ex- pressed as mean ± SD. One-way ANOVA followed by Tukey’s multiple com- parisons test. **, P < 0.01. (B) The protein level of Hsp90 was determined by Western blot analysis. β-Actin was used as a loading control. Results are re- presentative of 3 independent experiments, and quantification of blots is shown right. Data are expressed as mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test. **, P < 0.01. (C) Hsp90 expression was detected by immunohistochemistry analysis (brown), and representative images from each group are depicted. Arrows indicate podocytes within glomeruli fields. Scale bar, 20 μM. (D) Albuminuria indicated by the urine albumin/creatinine was measured. Data are expressed as mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test. **, P < 0.01.
3.2. Hsp90 expression is induced in podocytes by ADR treatment in vitro
ADR is a podocyte toxin applied to induce experimental murine NS [15]. We suspected that Hsp90 upregulation in podocytes from NS mice may be caused by ADR-induced podocyte injury. To test whether ADR- induced podocyte injury elevates Hsp90 expression, we treated an im- mortalized murine JR07 podocyte cell line with ADR in vitro. We found that under the treatment of ADR, the transcript level of Hsp90 was increased in JR07 podocytes in a time-dependent manner (Fig. 2A). Moreover, consistent with this result, the protein level of Hsp90 was also upregulated in JR07 podocytes treated by ADR (Fig. 2B). There- fore, these results suggest that the observed Hsp90 induction in podo- cytes from NS mice may be caused by ADR-induced injury, and in turn,also hint that Hsp90 is involved in podocyte injury in the context of ADR-induced NS pathology.
3.3. Hsp90 inhibition aggravates ADR-induced podocyte apoptosis through intrinsic pathway
The persistence of ADR-induced podocyte injury leads to cell loss from apoptosis that contributes to the progressive kidney damage in NS murine model [16,17]. Besides, Hsp90 is a molecular chaperon protein involved in cellular adaptive responses to lethal conditions and pro- tection against apoptosis [18]. To establish a functional connection between Hsp90 and ADR-induced podocyte injury, we next focused on investigating whether the treatment of PU-H71, a widely-used inhibitor of Hsp90 [19,20], affects ADR-induced podocyte apoptosis. As ex- pected, TUNEL assay showed that ADR treatment alone triggered pro- minent podocyte apoptosis (Fig. 3A). Moreover, strikingly, PU-H71 increased ADR-induced podocyte apoptosis, whereas, had no obvious effect in the absence of ADR treatment (Fig. 3A), suggesting that Hsp90 inhibition aggravates ADR-induced podocyte apoptosis. It has been previously reported that Hsp90 suppresses the intrinsic apoptotic pathway [21]. In concert with this, we found that Hsp90 inhibition with PU-H71 enhanced ADR-activated intrinsic apoptotic pathway in podo- cytes, as evidenced by the decreased expression of Bcl-2 and con- comitant increased expression of Bax, cleaved caspase 9, and cleaved caspase 3 (Fig. 3B, lane 3 vs. lane 4), which was further confirmed by the increased mitochondrial release of cytochrome c (Fig. 3C, lane 3 vs. lane 4). Additionally, in accordance with results obtained from TUNEL assay (Fig. 3A), Hsp90 inhibition alone did not obviously affect the expression of these protein markers of intrinsic apoptotic pathway (Fig. 3B and C, lane 1 vs. lane 2). Altogether, these data demonstrate that Hsp90 inhibition via PU-H71 aggravates ADR-induced podocyte apoptosis, in which the activation of intrinsic apoptotic pathway is promoted.
3.4. Hsp90 inhibition worsens ADR-impaired filtration barrier function of podocytes
ADR-induced podocyte injury impairs the filtration barrier function that leads to albuminuria in NS mice [13,22]. Since Hsp90 inhibition aggravates ADR-induced podocyte apoptosis, we wondered whether the filtration barrier function of podocytes is also deteriorated under this circumstance. To test this, we took advantage of an in vitro assay evaluating the paracellular albumin permeability, where albumin is allowed to permeate across the podocyte monolayer, and the extent of which is reversely depended on the filtration barrier function of po- docyte monolayer (Fig. 4A). As shown, ADR treatment increased the paracellular albumin permeability across podocyte monolayer, sug- gesting an impaired filtration barrier function (Fig. 4B). Furthermore, the ADR-increased paracellular albumin permeability was significantly
Fig. 2. Hsp90 expression is upregulated in podocytes treated with ADR in vitro. (A-B) The immortalized murine JR07 podocytes were allowed to differentiate for 10 days and then treated with 0.5 μg/ml ADR for different time periods as indicated (n = 3).
(A) The mRNA level of Hsp90 was measured by qRT-pCR analysis. β-Actin was used as an internal control. Data are expressed as mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test. **, P < 0.01; *, P < 0.05. (B) The protein level of Hsp90 was determined by Western blot analysis. β-Actin was used as a loading control. Results are representative of 3 in-
dependent experiments, and quantification of blots is shown right. Data are expressed as mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test. **, P < 0.01.
Fig. 3. Hsp90 inhibition aggravates ADR-induced podocyte apoptosis through intrinsic pathway. (A-C) The immortalized murine JR07 podocytes were allowed to differentiate for 10 days and then treated with 0.5 μg/ml ADR for 48 h in the presence or absence of 1 μM PU-H71 (n = 5). (A) The apoptosis was assessed by TUNEL assay. The representative images (upper) and statistical analysis of the percentage of TUNEL positive cells (green) within the total cells (blue) (lower) are presented. Student's t-test. **, P < 0.01; *, P < 0.05. (B) The protein expression of Bax, Bcl-2, cleaved caspase 9, and cleaved caspase 3 was determined by Western blot analysis. β-Actin was used as a loading control. Results are representative of 3 independent experiments. (C) The protein expression of cytochrome c in the cytosol and mitochondrial fractions was determined by Western blot analysis. β-Actin and VDAC 1 was used as a loading control, respectively. Results are representative of 3 independent experiments.
augmented by Hsp90 inhibition with PU-H71 in a dose-dependent manner (Fig. 4B), therefore these observations illustrate that in addition to aggravating the ADR-induced apoptosis, Hsp90 inhibition also wor- sens the ADR-impaired filtration barrier function of podocytes.
3.5. Blockade of intrinsic apoptotic pathway abrogates effect of Hsp90 inhibition on ADR-impaired filtration barrier function of podocytes
In the end, to examine a potential causal link between Hsp90 in- hibition-aggravated apoptosis and -worsened impairment of filtration
barrier function of podocytes, we treated podocytes with zVAD-fmk, a pan-caspase inhibitor [23], in order to eliminate the promoted activa- tion of podocyte apoptosis. As anticipated, zVAD-fmk abolished PU- H71-increased expression of cleaved caspase 3 (Fig. 5A) and the number of apoptotic cells (Fig. 5B) when podocytes were treated with ADR, indicating that Hsp90 inhibition-aggravated apoptosis induced by ADR was indeed blocked by zVAD-fmk. More importantly, as shown by albumin influx assay, keeping pace with the blocked apoptosis, PU- H71-augmented impairment of filtration barrier function of podocytes by ADR was accordingly abrogated in the presence of zVAD-fmk
Fig. 4. Hsp90 inhibition deteriorates filtra- tion barrier function of ADR-treated podo- cytes. (A) The schematic diagram of para- cellular albumin permeability assay, where albumin in the bottom chamber is allowed to permeate across the podocyte monolayer seeded on the upper chamber without the addition of albumin. (B) The immortalized murine JR07 podocytes were allowed to differentiate for 10 days and then treated with increasing concentrations of PU-H71 for 48 h in the presence or absence of
0.5 μg/ml ADR. Podocytes were further in-
cubated at 37 °C for 4 h in transwell chamber, in which the transwell upper chamber was filled with 0.15 ml RPMI 1640 and the bottom chamber was filled with 1 ml RPMI 1640 containing 40 mg/ml BSA. The results of albumin infiltrating the podocyte monolayer (mg/ml) are shown. Each column represents the mean value from 5 replicates. Data are expressed as mean ± SD from 3 independent experiments. Student t-test. **, P < 0.01; NS, not significant.
Fig. 5. Blockade of podocyte apoptosis abrogates effect of Hsp90 inhibition on ADR-impaired filtration barrier function. (A-C) The immortalized murine JR07 podocytes were treated with or without 1 μM PU-H71 or 50 μM zVAD-fmk or in combination in the presence of 0.5 μg/ml ADR for 48 h. (A) The protein expression of cleaved caspase 3 was determined by Western blot analysis. β-Actin was used as a loading control. Results are representative of 3 independent experiments. (B) The apoptosis was assessed by TUNEL assay. The statistical analysis of the percentage of TUNEL positive cells within the total cells is shown. Student's t-test. **, P < 0.01; NS, not significant. (C) Podocytes were further incubated at 37 °C for 4 h in transwell chamber, in which the transwell upper chamber was filled with
0.15 ml RPMI 1640 and the bottom chamber was filled with 1 ml RPMI 1640 containing 40 mg/ml BSA. The results of albumin infiltrating the podocyte monolayer (mg/ml) are shown. Each column represents the mean value from 5 replicates. Data are expressed as mean ± SD from 3 independent experiments. Student t-test. **, P < 0.01; NS, not significant.
(Fig. 5C). Hence, this collection of evidence proves that the impairment of filtration barrier function of podocytes promoted by PU-H71-medi- ated Hsp90 inhibition relies on the enhanced podocyte apoptosis, which implicates a protective role of Hsp90 against ADR-induced podocyte injury and the following increased permeability.
4. Disscussion
It is increasingly recognized that in addition to the disorder of T-cell function, the dysfunction of podocytes within glomerulus is another pathogenic factor that plays a pivotal role in the pathogenesis of childhood INS [3]. As known, podocyte injury or loss inevitably dis- rupts the normal filtration barrier function and causes proteinuria and progressive glomerular damage, and eventually, renal failure [24,25]. In this sense, it's reasonable that developing therapeutics to prevent podocyte injury holds potential promise for the treatment of kidney glomerular diseases. However, at present, this prospect is largely ob- scured by our limited understanding of the mechanisms leading to podocyte injury.
Podocytes are primary injured targets in ADR-induced nephropathy in mice [13,15]. In our current study, through applying an animal model, we found that the expression of Hsp90 was induced in glomeruli podocytes from nephropathy mice. In addition, Hsp90 expression was also induced in immortalized murine JR07 podocytes treated with ADR in vitro. These two lines of experimental observations sharing the si- milarity of podocyte injury suggest that Hsp90 expression in podocytes responses to ADR treatment by upregulation, and that Hsp90 may play a functional role associated with this pathological process. We propose that Hsp90 plays a protective role against ADR-induced podocyte injury based on our subsequent findings: 1) Hsp90 inhibition with PU-H71 promotes the activation of intrinsic apoptotic pathway and aggravates ADR-induced podocyte apoptosis; 2) Hsp90 inhibition with PU-H71 worsens ADR-induced impairment of filtration barrier function of po- docytes; 3) Blockade of podocyte apoptosis with the pan-caspase in- hibitor zVAD-fmk abrogates the detrimental effect of Hsp90 inhibition on ADR-impaired filtration barrier function of podocytes. Taken to- gether, these findings suggest that Hsp90 could protect podocytes from ADR insult by reducing apoptosis via suppressing the intrinsic apoptotic pathway. Furthermore, our study may also offer Hsp90 as a possible therapeutic target for minimizing podocyte injury when coping with glomerular diseases, including childhood INS.
The upregulation of Hsp90 expression was observed at both tran-
script level and protein level. This suggests that its transcription is very likely to be activated in podocytes upon ADR treatment. Previous lit- eratures have documented that as a master regulator of Hsp90 level in eukaryotic cells, the transcription of Hsp90 is principally induced by
the stress-related transcription factor heat shock factor 1 (HSF1) through binding to the heat shock elements (HSEs) located within Hsp90 promoters [26,27]. HSF1 is a stress-responsive transcription factor which induces hundreds of genes in response to environmental stresses [28]. It has been reported that ADR treatment induces IER5 upregulation, which in turn mediates HSF1 activation for the protection
of cancer cells from ADR-elicited DNA damage stress [29]. Although other transcription factors, such as the nuclear factor-κB subunit p65 (NF-κB), C/EBPδ, STAT3 and STAT1, are also involved in the regulation
of Hsp90 transcription under the stimulation of interferons [27], we suspect that the upregulated expression of Hsp90 in ADR-treated po- docytes is very possibly attributed to the induced HSF1 activation. However, more studies would be required to test whether this is the case in the future. Furthermore, whether Hsp90 is induced in the po- docytes from other nephropathy animal models or human clinical samples is uncertain. Addressing these questions could provide clues to show whether Hsp90 has more clinical relevance and significance in the pathogenesis of nephropathy.
Generally, Hsp90 is an anti-apoptotic protein that helps cells to endure lethal conditions, and its chaperone role for mediating the sta- bility of several transcriptional factors and signal transducing kinases involved in apoptotic process is considered to play a central role, such as Akt, Blc-2, p53 and HIF1α [18]. In cancer cells, overexpression of Hsp90 prevents apoptosis triggered by various stimuli [30,31], and its downregulation or inhibition sensitizes cells to apoptosis [32]. There- fore, it might be unsurprising that Hsp90 inhibition with PU-H71 pro- motes ADR-induced podocyte apoptosis through an intrinsic apoptotic pathway. However, on the other hand, Hsp90 inhibition alone can not induce obvious podocyte apoptosis in the absence of ADR, suggesting that under the unstressed condition, the endogenous level of Hsp90 may be indispensable for controlling cell apoptosis.
Through in vitro albumin influx assay, we reveal that Hsp90 in-
hibition-promoted podocyte apoptosis further aggravates the impair- ment of filtration barrier function of ADR-treated podocytes. As far as we know, this is the first time associating Hsp90 function with filtration barrier function of podocytes. The maintenance of podocyte function and integrity is essential for preventing the development of proteinuria [33,34]. Currently, the development of HSP90 inhibitors as anticancer agents is in progress [35]. According to our findings, we guess that the administration of Hsp90 inhibitors may deteriorate the condition of proteinuria. Hence, we suppose that Hsp90 inhibitors should be used with cautions in the future when cancer patients are diagnosed with proteinuria.
In summary, we associate Hsp90 function with ADR-induced ne-
phropathy, and uncover detrimental roles of Hsp90 inhibition in ag- gravating ADR-induced podocyte injury, including podocyte apoptosis
and filtration barrier dysfunction. Therefore, boosting Hsp90 expres- sion or activity might be beneficial in reducing podocyte injury and alleviating proteinuria.
Authors contributions
DJY, JZH and MGR designed and conducted experiments, and in- terpreted results. DJY wrote the manuscript.
Funding
No funds.
Declaration of competing interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
References
[1] N. Larkins, S. Kim, J. Craig, E. Hodson, Steroid-sensitive nephrotic syndrome: an evidence-based update of immunosuppressive treatment in children, Arch. Dis. Child. 101 (2016) 404–408.
[2] E.M. Hodson, J.F. Knight, N.S. Willis, J.C. Craig, Corticosteroid Therapy for Nephrotic Syndrome in Children, The Cochrane database of systematic reviews, 2000, p. CD001533.
[3] K. Kaneko, S. Tsuji, T. Kimata, T. Kitao, S. Yamanouchi, S. Kato, Pathogenesis of childhood idiopathic nephrotic syndrome: a paradigm shift from T-cells to podo- cytes, World journal of pediatrics : WJP 11 (2015) 21–28.
[4] J.H. Miner, Podocyte biology in 2015: new insights into the mechanisms of podo- cyte health, Nat. Rev. Nephrol. 12 (2016) 63–64.
[5] D. Kim, S. Lim, M. Park, J. Choi, J. Kim, H. Han, K. Yoon, K. Kim, J. Lim, S. Park, Ubiquitination-dependent CARM1 degradation facilitates Notch1-mediated podo- cyte apoptosis in diabetic nephropathy, Cell. Signal. 26 (2014) 1774–1782.
[6] S. Shi, L. Yu, C. Chiu, Y. Sun, J. Chen, G. Khitrov, M. Merkenschlager, L.B. Holzman,
W. Zhang, P. Mundel, E.P. Bottinger, Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis, J. Am. Soc. Nephrol. : JASN (J. Am. Soc. Nephrol.) 19 (2008) 2159–2169.
[7] T. Niranjan, B. Bielesz, A. Gruenwald, M.P. Ponda, J.B. Kopp, D.B. Thomas,
K. Susztak, The Notch pathway in podocytes plays a role in the development of glomerular disease, Nat. Med. 14 (2008) 290–298.
[8] F.H. Schopf, M.M. Biebl, J. Buchner, The HSP90 chaperone machinery, Nat. Rev. Mol. Cell Biol. 18 (2017) 345–360.
[9] C. Gallerne, A. Prola, C. Lemaire, Hsp90 inhibition by PU-H71 induces apoptosis through endoplasmic reticulum stress and mitochondrial pathway in cancer cells and overcomes the resistance conferred by Bcl-2, Biochim. Biophys. Acta 1833 (2013) 1356–1366.
[10] M. Bonilla-Felix, C. Parra, T. Dajani, M. Ferris, R.D. Swinford, R.J. Portman,
R. Verani, Changing patterns in the histopathology of idiopathic nephrotic syn- drome in children, Kidney Int. 55 (1999) 1885–1890.
[11] R.F. Ransom, N.G. Lam, M.A. Hallett, S.J. Atkinson, W.E. Smoyer, Glucocorticoids protect and enhance recovery of cultured murine podocytes via actin filament stabilization, Kidney Int. 68 (2005) 2473–2483.
[12] D. Sahali, A. Pawlak, A. Valanciute, P. Grimbert, P. Lang, P. Remy, A. Bensman,
G. Guellaen, A novel approach to investigation of the pathogenesis of active minimal-change nephrotic syndrome using subtracted cDNA library screening, J. Am. Soc. Nephrol. : JASN (J. Am. Soc. Nephrol.) 13 (2002) 1238–1247.
[13] V.W. Lee, D.C. Harris, Adriamycin nephropathy: a model of focal segmental glo- merulosclerosis, Nephrology 16 (2011) 30–38.
[14] W. Kriz, B. Kaissling, Chapter 20 - structural organization of the mammalian kidney, in: R.J. Alpern, O.W. Moe, M. Caplan (Eds.), Seldin and Giebisch's the Kidney, fifthed., Academic Press, 2013, pp. 595–691.
[15] J.W. Pippin, P.T. Brinkkoetter, F.C. Cormack-Aboud, R.V. Durvasula, P.V. Hauser,
J. Kowalewska, R.D. Krofft, C.M. Logar, C.B. Marshall, T. Ohse, S.J. Shankland, Inducible rodent models of acquired podocyte diseases, Am. J. Physiol. Renal physiology 296 (2009) F213–F229.
[16] K.N. Campbell, J.S. Wong, R. Gupta, K. Asanuma, M. Sudol, J.C. He, P. Mundel, Yes- associated protein (YAP) promotes cell survival by inhibiting proapoptotic dendrin signaling, J. Biol. Chem. 288 (2013) 17057–17062.
[17] M.S. Zou, J. Yu, G.M. Nie, W.S. He, L.M. Luo, H.T. Xu, 1, 25-dihydroxyvitamin D3 decreases adriamycin-induced podocyte apoptosis and loss, Int. J. Med. Sci. 7 (2010) 290–299.
[18] D. Lanneau, A. de Thonel, S. Maurel, C. Didelot, C. Garrido, Apoptosis versus cell differentiation: role of heat shock proteins HSP90, HSP70 and HSP27 (2007) 53–60 Prion 1.
[19] E. Caldas-Lopes, L. Cerchietti, J.H. Ahn, C.C. Clement, A.I. Robles, A. Rodina,
K. Moulick, T. Taldone, A. Gozman, Y. Guo, N. Wu, E. de Stanchina, J. White,
S.S. Gross, Y. Ma, L. Varticovski, A. Melnick, G. Chiosis, Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 8368–8373.
[20] M. Trendowski, Pu-H71, An improvement on nature's solutions to oncogenic Hsp90 addiction, Pharmacol. Res. 99 (2015) 202–216.
[21] A. Rodina, M. Vilenchik, K. Moulick, J. Aguirre, J. Kim, A. Chiang, J. Litz,
C.C. Clement, Y. Kang, Y. She, N. Wu, S. Felts, P. Wipf, J. Massague, X. Jiang,
J.L. Brodsky, G.W. Krystal, G. Chiosis, Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer, Nat. Chem. Biol. 3 (2007) 498–507.
[22] E. Heikkila, J. Juhila, M. Lassila, M. Messing, N. Perala, E. Lehtonen, S. Lehtonen,
J. Sjef Verbeek, H. Holthofer, Beta-Catenin Mediates Adriamycin-Induced Albuminuria and Podocyte Injury in Adult Mouse Kidneys, Nephrology, Dialysis, Transplantation vol. 25, official publication of the European Dialysis and Transplant Association - European Renal Association, 2010, pp. 2437–2446.
[23] R.A. Lockshin, Z. Zakeri, Caspase-independent cell deaths, Curr. Opin. Cell Biol. 14 (2002) 727–733.
[24] F. Cellesi, M. Li, M.P. Rastaldi, Podocyte injury and repair mechanisms, Curr. Opin. Nephrol. Hypertens. 24 (2015) 239–244.
[25] L. Barisoni, Podocyte biology in segmental sclerosis and progressive glomerular injury, Adv. Chron. Kidney Dis. 19 (2012) 76–83.
[26] C. Prodromou, Mechanisms of Hsp90 regulation, Biochem. J. 473 (2016) 2439–2452.
[27] M. Taipale, D.F. Jarosz, S. Lindquist, HSP90 at the hub of protein homeostasis: emerging mechanistic insights, Nat. Rev. Mol. Cell Biol. 11 (2010) 515–528.
[28] L. Whitesell, S. Lindquist, Inhibiting the transcription factor HSF1 as an anticancer strategy, Expert Opin. Ther. Targets 13 (2009) 469–478.
[29] Y. Asano, T. Kawase, A. Okabe, S. Tsutsumi, H. Ichikawa, S. Tatebe, I. Kitabayashi,
F. Tashiro, H. Namiki, T. Kondo, K. Semba, H. Aburatani, Y. Taya, H. Nakagama,
R. Ohki, IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis, Sci. Rep. 6 (2016) 19174.
[30] Q. Xu, J. Tu, C. Dou, J. Zhang, L. Yang, X. Liu, K. Lei, Z. Liu, Y. Wang, L. Li, H. Bao,
J. Wang, K. Tu, HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma, Mol. Canc. 16 (2017) 178.
[31] M. Chatterjee, S. Jain, T. Stuhmer, M. Andrulis, U. Ungethum, R.J. Kuban,
H. Lorentz, K. Bommert, M. Topp, D. Kramer, H.K. Muller-Hermelink, H. Einsele,
A. Greiner, R.C. Bargou, STAT3 and MAPK signaling maintain overexpression of heat shock proteins 90alpha and beta in multiple myeloma cells, which critically contribute to tumor-cell survival, Blood 109 (2007) 720–728.
[32] A.R. Goloudina, O.N. Demidov, C. Garrido, Inhibition of HSP70: a challenging anti- cancer strategy, Canc. Lett. 325 (2012) 117–124.
[33] C. Wei, C.C. Moller, M.M. Altintas, J. Li, K. Schwarz, S. Zacchigna, L. Xie, A. Henger,
H. Schmid, M.P. Rastaldi, P. Cowan, M. Kretzler, R. Parrilla, M. Bendayan, V. Gupta,
B. Nikolic, R. Kalluri, P. Carmeliet, P. Mundel, J. Reiser, Modification of kidney barrier function by the urokinase receptor, Nat. Med. 14 (2008) 55–63.
[34] C. Faul, M. Donnelly, S. Merscher-Gomez, Y.H. Chang, S. Franz, J. Delfgaauw,
J.M. Chang, H.Y. Choi, K.N. Campbell, K. Kim, J. Reiser, P. Mundel, The actin cy- toskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A, Nat.PU-H71 Med. 14 (2008) 931–938.
[35] R. Garcia-Carbonero, A. Carnero, L. Paz-Ares, Inhibition of HSP90 molecular cha- perones: moving into the clinic, Lancet Oncol. 14 (2013) e358–369.