Received Date : 17-Jan-2016

Revised Date : 05-Dec-2016

Accepted Date : 13-Dec-2016
Accepted Article
Article type : Original Article

Pitavastatin Suppresses Hyperglycemia-induced Podocyte Injury via Bone

Morphogenetic Protein-7 Preservation

Makoto Ohigashi1, Miyuki Kobara1, Tamotsu Takahashi1, Hiroe Toba1,

Takehiko Wada2, Tetsuo Nakata1

1
Department of Clinical Pharmacology, Division of Pathological Science, Kyoto

Pharmaceutical University, Kyoto, Japan

2
Division of Nephrology, Endocrinology and Metabolism, Tokai University

School of Medicine, Isezaki, Japan

Short title: Pitavastatin attenuates podocyte injury via BMP-7

Address correspondence to Miyuki Kobara

Department of Clinical Pharmacology, Division of Pathological Science, Kyoto

Pharmaceutical University

5 Misasagi Nakauchi-cho, Yamashina-ku, Kyoto, 607-8414, Japan

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/1440-1681.12716
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 Telephone: +81755954724 Fax: +81755954788

e-mail: kobara@mb.kyoto-phu.ac.jp
Accepted Article
Conflict of Interest Disclosures: None

Abstract

[Background] Podocytes form the essential components of the glomerular

filtration barrier and play a critical role in diabetic nephropathy. Recent

evidence suggests that HMG-CoA reductase inhibitors (statins) exert

renoprotective effects. We investigated whether pitavastatin directly

suppresses hyperglycemia-induced podocyte injury using cultured podocytes

and, if so, the mechanism of the beneficial effects. [Methods and Results]

Cultured podocytes were exposed to media containing normal (NG; 5 mmol/L)

or high (HG; 25 mmol/L) glucose for one week. HG increased the lethal

injury of podocytes and disruption of F-actin fibers, and reduced the mRNA

expression of novel podocyte markers, synaptopodin and Wilms tumor 1 (WT1),

in association with decreased bone morphogenetic protein-7 (BMP-7)

expression. Pitavastatin (100 nmol/L) reduced podocyte injury and restored

the mRNA expression of synaptopodin and WT1; however, these protective

effects were abolished by BMP-7 siRNA. Additionally, pitavastatin suppressed

HG-induced Rho kinase activation, as assessed by the phosphorylation level of

myosin phosphatase targeting subunit 1(MYTP1), and C3 exotoxin, a Rho

inhibitor, mimicked the effect of pitavastatin on BMP-7 preservation.

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 [Conclusion] Pitavastatin attenuates hyperglycemia-induced podocyte injury via

Rho-Rho kinase-dependent BMP-7 preservation.

Accepted Article
Keywords BMP-7, diabetic nephropathy, podocyte, statin

Introduction

Diabetic nephropathy is one of the serious complications of diabetes

mellitus and the most common cause of renal failure. In the early

microalbuminuria phase, diabetic nephropathy has been reported to be primarily

a glomerular disease, and growing evidence suggests that podocyte injury plays

a critical role in its progression [1, 2, 3]. Podocytes are highly specialized

kidney epithelial cells that consist of a cell body and foot processes, and they

act as a filtration barrier to albumin. The characteristic morphological changes

under hyperglycemia in podocytes are initially foot process effacement and

decreased slit diaphragms [1, 4]. Furthermore, consistent with the exacerbation

of diabetic nephropathy, apoptotic podocyte loss occurs, and this is critical

because of their low-level mitotic capacity [5].

In the progression of diabetic nephropathy, the

hyperglycemia-activated renal renin–angiotensin system (RAS) is an

exacerbating factor. Hence, in addition to the maintenance of normoglycemia,

treatments with angiotensin-converting enzyme (ACE) inhibitors or angiotensin

receptor blockers (ARBs) are the current standard therapies against diabetic

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 nephropathy. Even when these therapies effectively prevent the progression of

diabetic nephropathy, in a large number of diabetic patients, renal impairment

Accepted Article
still progresses to end-stage renal disease. Therefore, there is important to

identify additional therapeutic strategies against diabetic nephropathy.

Recently, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase

inhibitors (statins), well-known therapeutic drugs for dyslipidemia, have

exhibited renoprotection and attenuated podocyte injury in rodent models of

diabetic nephropathy and diabetic patients as pleiotropic effects [6, 7, 8, 9].

Previous studies in our, and other laboratories have indicated that statins

inhibited renal ACE in rodent models of diabetic nephropathy and Heymann

nephritis [10, 11], indicating that the pleiotropic protective effects of statins are,

in part, attributed to the inhibition of RAS. However, it was recently reported

that adding a statin to a combination of ACE inhibitor and ARB leads to a

further reduction of proteinuria and podocyte injury in uninephrectomized

streptozotocin-induced diabetic rats and diabetic patients [12, 13], suggesting

that, in addition to RAS inhibition, other renoprotective mechanisms may be

promoted by statins.

Bone morphogenetic protein-7 (BMP-7) is a member of the BMP

family within the TGF-β superfamily. BMP-7 is crucial in kidney

development and localizes to podocytes and tubular epithelial cells in the

mature kidney [14, 15]. Several studies have shown that transgenically

expressed BMP-7 and exogenously administered rhBMP-7 reduce the severity

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 of renal diseases in rodent models, such as ureteral obstruction-induced renal

injury [16], lupus nephritis [17], and diabetic nephropathy [18, 19]. Moreover,
Accepted Article
in diabetic nephropathy, the transgenic expression of BMP-7 in glomerular

podocytes and proximal tubules prevents podocyte dropout in association with

reduced albuminuria [19]. Recently, statins were reported to preserve renal

BMP-7 expression in association with the maintenance of podocyte proteins,

Wilm’s tumor-1 (WT-1), in unilateral ureteric obstruction models [20].

However, whether statins directly preserve BMP-7 protein in podocytes remains

to be elucidated. Therefore, we tested the hypothesis that pitavastatin attenuates

hyperglycemia-induced podocyte injury via the restoration of BMP-7 levels

using cultured mouse podocytes.

Results

Effect of high glucose on podocyte BMP-7 expression

Differentiated mouse podocytes expressed BMP-7 mRNA and protein,

and the incubation of podocytes with high glucose clearly reduced these

expression levels (Fig. 1). Treatment with pitavastatin significantly attenuated

the impaired BMP-7 expression in the high glucose condition (Fig. 1).

However, pitavastatin did not affect the mRNA level of BMP-7 in the normal

glucose condition (Appended Fig. 1).

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 Effect of pitavastatin on high-glucose- induced podocyte apoptosis

Nuclear staining with 4', 6’-diamidino-2 phenylindole (DAPI) showed

Accepted Article
that stimulation with high glucose significantly increased the ratio of apoptotic

nuclei by 2.5-fold compared with incubation with normal glucose (Fig. 2).

Treatment of podocytes with pitavastatin suppressed the high-glucose-induced

podocyte apoptosis (Fig. 2). We further examined whether the preservation of

BMP-7 was involved in the apoptosis reduction by pitavastatin using

BMP-7-deleted podocytes. As shown in Fig. 2, BMP-7 siRNA abolished the

protective effects of pitavastatin, as negative control RNA had no effect.

Effect of pitavastatin on high-glucose-induced caspase-3 activation

Caspase-3 activity in the podocytes incubated with high glucose was

significantly increased by 65% compared with that in the

normal-glucose-treated control (Fig. 3A). Treatment with pitavastatin

significantly inhibited high-glucose-stimulated caspase-3 activation. Moreover,

in agreement with the results for apoptotic nuclei, BMP-7 siRNA abolished the

protective effects of pitavastatin (Fig. 3A).

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 Effect of pitavastatin on high-glucose-induced lactate dehydrogenase (LDH)

release
Accepted Article
Lactate dehydrogenase activity in the culture media after high-glucose

stimulation was significantly increased by 28% compared with that in the

normal-glucose-treated control (Fig. 3B). Treatment with pitavastatin

significantly suppressed high-glucose-stimulated LDH release, and BMP-7

siRNA also reduced the suppresive effects of pitavastatin (Fig. 3B).

Effect of pitavastatin on the morphological changes in phase-contrast images

and F-actin structure

In phase-contrast images, podocytes under the normal-glucose

condition displayed a characteristic flattened morphology, whereas those under

the high-glucose condition shrank and detached podocytes often appeared.

Pitavastatin markedly attenuated high-glucose-induced morphological changes.

In addition, stimulation by high glucose markedly disrupted the F-actin

cytoskeleton compared with the incubation with normal glucose, and treatment

with pitavastatin preserved F-actin fibers. These morphological protections

from pitavastatin were abolished by BMP-7 siRNA (Fig. 4).

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 Effect of pitavastatin on the expression of podocyte markers
Accepted Article
As a consequence of podocyte damage, the reduction of podocyte

markers has been well documented. We next examined synaptopodin and

WT1 mRNA expression, as podocyte markers, after normal- or high-glucose

incubation. Incubation with high glucose significantly decreased synaptopodin

and WT1 mRNA expression, and the treatment with pitavastatin significantly

reversed these high-glucose-induced reductions. In addition, BMP-7 siRNA

also reduced the preservational effects of pitavastatin (Fig. 5B, C).

Preservation of BMP-7 by pitavastatin via suppression of Rho-Rho kinase

pathway

To examine the mechanisms by which pitavastatin preserved BMP-7

expression, we examined Rho kinase activity using the phosphorylation level of

myosin phosphatase targeting subunit 1 (MYPT-1), as some pleiotropic actions

by statins have been reported to be due to inhibition of the Rho-Rho kinase

pathway [9, 21, 22]. Incubation with high glucose significantly increased

phosphorylated MYPT-1, indicating Rho kinase activation, and treatment with

pitavastatin significantly attenuated high-glucose-induced activation (Fig. 6A).

Moreover, C3 exotoxin, a Rho inhibitor, also preserved BMP-7 mRNA

expression after the high-glucose condition in association with the restoration of

synaptopodin and WT1 mRNA, and the reduction of podocyte apoptosis (Fig.

6B and Appended Fig. 2). Therefore, these results indicate that pitavastatin

preserved BMP-7 due to suppression of the Rho-Rho kinase pathway.

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 Discussion

The present study demonstrated that high glucose increased podocyte

Accepted Article
death, disrupted cytoskeletal fibers, and reduced podocyte marker mRNA

expression, synaptopodin and WT-1, in association with BMP-7 reduction.

Pitavastatin (100 nmol/L) reduced podocyte injury and restored podocyte

marker expression accompanied by BMP-7 preservation. However, the

protective effects were abolished by the silencing of BMP-7. Moreover,

pitavastatin suppressed high glucose-induced Rho kinase activation, and C3

exotoxin, a Rho inhibitor, mimicked the effect of pitavastatin on BMP-7

preservation. In conclusion, pitavastatin attenuates hyperglycemia-induced

podocyte injury due to Rho-Rho kinase-dependent BMP-7 preservation.

In diabetic nephropathy, podocyte injury is critical in the progression of

glomerulosclerosis. In histological examinations of diabetes animal models

and diabetic patients, foot process effacement of podocytes and their subsequent

detachment from the glomerular basement membrane have been reported [23,

24, 25, 26]. In differentiated podocyte markers, we assessed synaptopodin and

WT1 in the present study. Synaptopodin is reduced in association with foot

process effacement in early diabetic injury [18], and WT1 plays an essential

role in podocyte function and integrity [27]. We found that pitavastatin

reversed high-glucose-induced synaptopodin and WT1 reduction, preserved

F-actin fibers, and reduced lethal injury of podocytes, suggesting that statins

preserve the podocyte number and function in diabetic nephropathy.

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 In the present study, hyperglycemia-induced podocyte death and

podocyte marker reduction were attenuated by pitavastatin, and these beneficial

Accepted Article
effects were reduced by the silencing of BMP-7. Statins protect against renal

injury by a mechanism that is independent of cholesterol-lowering actions, the

so-called pleiotropic actions [6, 9, 28, 29, 30]. The representative pleiotropic

actions of statins are anti-inflammatory and anti-oxidative actions. Pravastatin

suppresses inflammatory transcription factor NF-κB and the associated

enhancement of MCP-1 in podocytes [28]. In the present study, pitavastatin

may have exhibited some anti-inflammatory effects. However, except for

autocrine effects, inflammatory cells were not included in the present in vitro

study, and so these effects would have been limited. In addition, as

anti-oxidative properties, statins have been shown to ameliorate albuminuria,

mesangial expansion, and podocyte injury by the suppression of NADPH

oxidase activity in a mouse model of diabetic nephropathy [29], and to preserve

copper/zinc superoxide dismutase activity in an insulin-resistant rat model [30].

However, in our previous experiment using streptozotocin-induced diabetic rats,

pitavastatin was superior to tempol, a novel radical scavenger, in BMP-7

preservation, urinary protein suppression, and podocyte marker expression [31].

Moreover, in the presence of diabetes, statins may suppress receptors for

advanced glycation end product (RAGE) expression, resulting in a decrease of

advanced glycation end product (AGE)-induced renal implications [32]. In the

present study, we did not measure AGE-RAGE pathway-related parameters, so

we cannot exclude the possibility that pitavastatin reduced the exacerbation of

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 the AGEs effects in the present model and led to beneficial effects. On the

other hand, Thallas-Bonke et al. reported that AGEs are upstream factors of
Accepted Article
oxidative stress activation, and antioxidants prevent AGE-mediated damage in

diabetic nephropathy [33], whereas in our previous report [31], the BMP-7

preservation effect of pitavastatin was not mimicked by antioxidants.

Considered together, the BMP-7 preservation effects we noted on statin

treatment were an independent pleiotropic effect, in part, based on previously

reported actions.

The crucial mechanisms of BMP-7 for podocyte protection remain

unresolved. The present results indicate that BMP-7 preservation is critical in

treatment with pitavastatin. Previously, rhBMP-7 was reported to attenuate a

high-glucose-induced reduction of podocyte markers, such as synaptopodin and

podocin, using cultured podocytes [18]. Hence, our results are in agreement

with these findings. However, we could not clearly elucidate the role of

BMP-7 in the restoration of the podocyte structure and markers, synaptopodin

and WT1. Mitu et al. reported that BMP-7 increased smad 5 phosphorylation

and activated smad 5 regulating gene expression as a transcription factor,

leading to the preservation of actin filaments under a high-glucose condition

[34]. Hence, in the present study, similar to this previous report, smad 5 may

be a downstream signal of BMP-7.

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 The present results show that the preservation of BMP-7 by pitavastatin

attenuates high-glucose-induced podocyte injury, and a possible mechanism of

Accepted Article
BMP-7 preservation is the suppression of the high-glucose-activated Rho-Rho

kinase pathway. Statins suppress the mevalonate pathway, leading to a

decrease in isoprenoid production, such as falnecil pyrolic acid and gelanil

gelanil pyrolic acid, as well as cholesterol biosynthesis, and the reduced

synthesis of these isoprenoids inactivates small GTPase (Rho, Rac, and Ras)

[35]. In these isoprenoids, Rho was activated in the kidneys of rodent diabetes

models, including STZ-induced diabetic rats and db/db mice, as well as

high-glucose-stimulated cultured mesangial cells [21, 36, 37, 38, 39, 40]. In

addition, statins may ameliorate renal injury due to the inhibition of Rho

activity [9, 21, 22]. Our present results are consistent with those of prior

studies in which high glucose promoted the Rho-Rho kinase pathway, as

assessed based on the phosphorylated form of MYPT-1, and this effect was

attenuated by treatment with pitavastatin, as shown in Fig. 6. To determine the

association between Rho and BMP-7 expression, we next examined the effect of

C3 exoenzyme, a Rho inhibitor, on BMP-7 expression in the

hyperglycemia-stimulated podocytes, as Rho has been reported to participate in,

and contribute to, BMP-7 signaling during nerve growth cone attraction [41].

C3 exoenzyme mimicked the preservation of BMP-7 by pitavastatin against

high-glucose-induced BMP-7 regression. On the basis of these findings,

pitavastatin preserves BMP-7 expression due to the suppression of

high-glucose-induced Rho-Rho kinase activation, leading to the attenuation of

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 podocyte injury. In addition, Hamasaki et al. recently reported that simvastatin

suppressed uterine sensitization-associated gene-1 (USTG-1), a dominant

Accepted Article
antagonist of BMP-7 produced by renal distal tubular epithelial cells, leading to

BMP-7 preservation using a mouse renal fibrosis model [42]. Hence, in

patients with diabetic nephropathy, statins may enhance renal BMP-7 due to

USTG-1 suppression in renal distal tubular epithelial cells as well as Rho-Rho

kinase inhibition in podocytes.

In conclusion, pitavastatin ameliorated high-glucose-induced podocyte

injury. Pitavastatin preserved BMP-7 expression, and the silencing of BMP-7

abolished its protective effects. The high-glucose-induced Rho-Rho kinase

activation was attenuated by pitavastatin, and a Rho inhibitor also preserved

BMP-7 expression under the high glucose condition. Thus, the beneficial

effects of pitavastatin on podocytes can be attributed to the preservation of

BMP-7 protein due to the suppression of Rho- Rho kinase activation. Statins

are widely used agents for the treatment of dyslipidemia, and our results provide

support for the clinical use of effectively applied statins late in the progression

of early-stage diabetic nephropathy.

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 Methods

Cell culture
Accepted Article
The present experiments were performed using murine podocyte cell

lines kindly provided by Dr. Stuart J. Shankland and Dr. T. Wada [43]. The

cells were cultured at 33°C with RPMI1640 containing 10% FBS and

recombinant mouse interferon-γ (50 U/mL, Toyobo Co.). After reaching

confluence, the cells were thermo-shifted to 37°C and incubated with

interferon-free medium on type I collagen–coated dishes for 8 – 10 days to

induce differentiation into the podocyte lineage. Then, the podocytes were

incubated with a medium containing a normal concentration of glucose (NG; 5

mmol/L) or a high concentration of glucose (HG; 25 mmol/L) in the presence

or absence of pitavastatin (100 nmol/L) for one week. To exclude an

osmotic effect from the high glucose, D-mannitol (20 mmol/L) was added to

the medium containing the normal glucose concentration. The culture

medium was changed on the third day.

Gene expression of BMP-7 and podocyte markers

The determinations of BMP-7, synaptopodin, and WT-1 mRNAs were

performed by reverse transcription (RT)-PCR. Total RNA was extracted from

the cultured podocytes using Isogen regent (NIPPON GENE, Tokyo, Japan)

according to the manufacturer’s instructions and was subsequently treated with

RNase-free DNase. Total RNA (0.5 µg) was reversed-transcribed with oligo

(dT) primers using a High Capacity cDNA Reverse Transcript Kit (Applied

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 Biosystems, Foster City, CA, USA). The cDNA fragments were amplified by

PCR using Taq DNA polymerase (TaKaRa Ex Taq; TaKaRa, Kyoto, Japan).
Accepted Article
The gene-specific primers were: for BMP-7, forward primer

5’-TGTGGCAGAAAACAGCAGCA-3’ and reverse primer

5’-TCAGGTGCAATGATCCAGTCC-3’; synaptopodin,

5’-ACCAGCCAGATAGAGCAAAG-3’ and

5’-GTCTGCACTAGGTCCAGCAA-3’; WT-1,

5’-TACCCAGGCTGCAATAAGAG-3’ and

5’-AAGCAGTTTGCACACTTTCC-3’. Parallel amplification of mouse

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as an

internal control (forward primer 5’-CCTTCATTGACCTCAACTACATGG-3’,

reverse primer 5’-CCTGCTTCACCACCTTCTTGAT-3’). After

electrophoresis and visualization by ethidium bromide (0.5 µg/mL) staining, the

intensity of each band was quantified by scanning densitometry. The

presented results were normalized to the expression levels of GAPDH.

Protein expression of BMP-7 and (phospho)-MYPT-1

The protein expressions of BMP-7, phospho-MYPT-1, and MYPT-1

were examined by Western blotting. Podocytes were lysed in an RIPA buffer

(Nacalai Tesque, Inc.) on ice for 10 min. Cell extracts were clarified by

centrifugation at 10,000 g for 10 min at 4°C, and the supernatant was used as

the total podocyte lysate. Protein concentrations of cell extracts were

examined by the Lowry methods. Samples containing equal amounts of

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 protein were subjected to SDS-polyacrylamide gel electrophoresis followed by

electroblotting onto PVDF membranes (ATTO, Tokyo, Japan). The

Accepted Article
membranes were blocked with 5% BSA (BMP-7), or 3% non-fat dry milk

(phospho-MYPT-1 and MYPT-1), in Tris-buffered saline containing 0.1%

Tween 20, and then incubated with rabbit polyclonal antibody against BMP-7

(1:1,000, Abcam, Cambridge, UK), phospho-MYPT-1 (1:500, Upstate

Biochemistry, Lake Placid, NY, USA), or MYPT-1 (1:500, Santa Cruz

Biotechnology, TX, USA). Then, the membranes were incubated with

HRP-linked anti-rabbit IgG. (1:2,000, Oromega, Madison, USA). The

detection of chemiluminescence was carried out with enhanced

chemiluminescence (ECL, Amersham Biosciences Corp., Buckinghamshire,

England). The bands were quantified by scanning densitometry. The optical

density of BMP-7 protein was normalized with respect to the amount of actin

(1:5,000, mouse anti-actin monoclonal antibody, Chemicon International,

Temecula, USA; 1:2,500, HRP-linked anti-mouse IgG, Amersham Bioscienses

Corp.). The phosphorylation ratio of MYPT-1 was assessed by the optical

density of phospho-MYPT-1 normalized with respect to that of MYPT-1.

Determination of podocyte apoptosis

Cultured podocytes were fixed with 2% paraformaldehyde, and

permeabilized with 0.5% Triton X-100. Nuclei were then stained with DAPI.

Apoptotic cells were identified by the distinctive condensed or fragmented

nuclei. The numbers of nuclei were counted in ten randomized and

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 non-overlapping fields at a magnification of x400, and apoptotic cell counts are

expressed as a percentage of the total number of nuclei counted.

Accepted Article
Caspase-3 activity

Caspase-3 activity was determined using an APOCYTO Caspase-3

Colorimetric Assay Kit (MBL, Nagoya, Japan). In accordance with the

manufacturer’s protocol, podocytes lysates were incubated with 10 mmol/L

DTT and the labeled caspase-3 substrate DEVD-p-nitroanilide (DEVD-pNA)

for 24 hours at 37°C. Cleavage of the substrate was quantified by measuring

absorbance at 405 nm using a microplate reader.

LDH activity

LDH activity in culture media was measured enzymatically using the

Spotchem kit (Arkray Co., Shiga, Japan) as an index of podocyte death. The

activity of LDH was expressed as IU/L.

Podocyte morphology

Podocyte morphology was examined using an inverted phase contrast

microscope with a 20× objective lens (Olympus IX71, Tokyo, Japan).

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 F-actin staining

Cultured podocytes were fixed with 2% paraformaldehyde,

Accepted Article
permeabilized with 0.2% Triton X-100, and blocked with 1% bovine serum

albmin. F-actin was visualized using fluorescent phalloidin (100 nmol/L.

Acti-stain 555 Fluoresent Phalloidin, Xytoskelton, Inc., Denver, USA).

Confocal images were obtained with the fluorescent microscope LSM510 and a

40x objective lens.

Transient silencing of BMP-7

Negative control RNA and mouse BMP-7-specific siRNA duplexes

were purchased from Invitrogen (Carlsbad, CA, USA). SiRNA was used to

transfect the podocytes using Lipofectamine RNAiMAX (Invitrogen, CA)

according to the manufacturer’s instructions. Podocytes were provided with

fresh media 4 hours after transfection, and then the podocytes were subjected to

high glucose in the presence of pitavastatin. In the preliminary examination,

siRNA suppressed BMP-7 expression by 72% in cultured podocytes (Appended

Fig 3).

Statistical Analysis

All results are expressed as the mean ± SE. Statistical analyses were

performed by one-way ANOVA combined with Fisher’s post-hoc test. Values

of p<0.05 were considered to be significant.

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 Acknowledgements

The authors appreciate the supply of the murine podocyte cell line by
Accepted Article
Dr. Stuart J. Shankland. There are no conflicts of interest to declare.

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 Figure Legends

Fig. 1
Accepted Article
Effects of pitavastatin on BMP-7 mRNA and protein expression after 7 days of

high-glucose incubation in podocytes. The representative expressions of

BMP-7 mRNA (A, top panel) and protein (B, top panel), and densitometric

analyses of their levels (A and B, bottom panels, n=7-8). NG, normal-glucose

group; HG, high-glucose group; HG+Pit, pitavastatin-treated high-glucose

group. * p<0.05 versus NG, # p<0.05 versus HG.

Fig. 2

Effect of pitavastatin on podocyte apoptosis and the role of BMP-7 in its effect.

(A) Podocytes were incubated with normal glucose or high glucose in the

presence or absence of pitavastatin for 7 days, and then stained with DAPI.

Additionally, to clarify the roles of BMP-7, podocytes were also treated with

BMP-7 siRNA or negative control RNA in addition to pitavastatin.

Arrowheads indicate a typical feature of apoptotic podocytes. Bars indicate

100 µm. (B) Percentage of apoptotic podocytes. Apoptotic podocytes were

calculated as described in Methods (n=7). BMP-7 siRNA, BMP-7 specific

siRNA; NC RNA, negative control RNA. * p<0.05 versus NG, # p<0.05

versus HG, †p<0.05 versus HG+Pit+NC RNA.

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 Fig. 3

Effect of pitavastatin and the role of BMP-7 in its effect on caspase-3 activity in
Accepted Article
podocytes and LDH activity in culture media. (A) Podocytes were incubated

with normal or high glucose in the presence or absence of pitavastatin for 7 days.

Podocytes were also treated with BMP-7 siRNA or negative control RNA in

addition to pitavastatin. Caspase-3 activities in podocytes were measured as

described in Methods (n=7). (B) LDH activity in culture media was assessed

as described in Methods (n=8). BMP-7 siRNA, BMP-7-specific siRNA; NC

RNA, negative control RNA. * p<0.05 versus NG, # p<0.05 versus HG, †

p<0.05 versus HG+Pit+NC RNA.

Fig. 4

Effect of pitavastatin and the role of BMP-7 in its effect on the morphology and

structure of F-actin filaments in podocytes. Podocytes were incubated with

normal or high glucose in the presence or absence of pitavastatin for 7 days, and

then the podocyte morphology was assessed by phase contrast image

microscopy (A) and the structure of F-actin filaments (B). Podocytes were

also treated with BMP-7 siRNA or negative control RNA in addition to

pitavastatin. Bars indicate 100 µm (A) and 50 µm (B).

Fig. 5

Effect of pitavastatin and the role of BMP-7 in its effect on synaptopodin and

WT-1 mRNA expression in podocytes. Total RNA was extracted from

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 podocytes after incubation with normal or high glucose in the presence or

absence of pitavastatin for 7 days. Podocytes were also treated with BMP-7
Accepted Article
siRNA or negative control RNA in addition to pitavastatin. Expression levels

of mRNA were examined by RT-PCR as described in Methods. Bar graphs

indicate mRNA expression quantified by densitometric analysis. (A)

synaptopodin mRNA. (B) WT-1 mRNA (n=8). * p<0.05 versus NG, # p<0.05

versus HG, † p<0.05 versus HG+Pit+NC RNA.

Fig. 6

(A) Effect of pitavastatin on the phosphorylation of myosin phosphatase

targeting subunit 1 (MYPT1) in podocytes. The bar graph indicates the ratio

of phosphorylated-MYPT1 to total MYPT1 protein expression (n=7). (B) The

effect of C3 exotoxin, a Rho inhibitor, on BMP-7 mRNA expression in

high-glucose-stimulated podocytes. The bar graph indicates mRNA

expression quantified by densitometric analysis (n=7). * p<0.05 versus NG, #

p<0.05 versus HG.

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 Appended Figure Legends
Accepted Article
Appended Fig. 1

Effects of pitavastatin on BMP-7 mRNA expression after 7 days of

normal-glucose incubation in podocytes (n=6).

Appended Fig. 2

Effect of C3 exotoxin on podocyte apoptosis (A), synaptopodin mRNA

(B), and WT-1 mRNA (C) expression after 7 days of high-glucose incubation in

podocytes (n=6). Statistical analyses were performed by one-way ANOVA

combined with Fisher’s post-hoc test. Values of p<0.05 were considered

significant. # p<0.05 versus HG.

Appended Fig. 3

Effect of BMP-7-specific siRNA on BMP-7 mRNA expression in

podocytes. Podocytes were incubated with 10 nmol/L siRNA or negative

control RNA in a reduced medium for 4 hours, and the medium was then

exchanged to the usual RPMI (n=5). Statistical analysis was performed by

unpaired students’ t test. Values of p<0.05 were considered significant.

BMP-7 siRNA, BMP-7 specific siRNA; NC RNA, negative control RNA. *

p<0.05 versus NG+NC RNA.

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Accepted Article

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Accepted Article

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Accepted Article

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