Triptolide protects podocytes
Triptolide protects podocytes from puromycin
aminonucleoside induced injury in vivo and in vitro Chun-Xia Zheng 1,2,Zhao-Hong Chen 1,2,Cai-Hong Zeng 1,Wei-Song Qin 1,Lei-Shi Li 1and Zhi-Hong Liu 11Research Institute of Nephrology,Jinling Hospital,Nanjing University School of Medicine,Nanjing,China
Extracts of Tripterygium wilfordii Hook F have been used to
treat glomerulonephritis for more than 30years in China with
dramatic antiproteinuric effects.Triptolide,a diterpene
triepoxide,is one of the major active components of these
extracts.To clarify its antiproteinuric effects we induced
podocyte injury by puromycin aminonucleoside.Triptolide
effectively reduced the proteinuria induced by puromycin in
nephrotic rats without reducing the glomerular filtration rate.
The antiproteinuric effect was associated with improvement
in the foot process effacement,a decrease in the podocyte
injury marker desmin as well as the restoration of nephrin
and podocin expression and distribution.In cultured
mouse podocytes triptolide pretreatment prevented the
puromycin-induced disruption of the actin cytoskeleton
and microfilament-associated synaptopodin while protecting
nephrin and podocin expression.Triptolide suppressed
reactive oxygen species generation and p38mitogen-
activated protein kinase activation while restoring RhoA
signaling activity.These results show that triptolide
ameliorates puromycin aminonucleoside-mediated podocyte
injury in vivo and in vitro .
Kidney International (2008)74,596–612;doi:10.1038/ki.2008.203;
published online 28May 2008
KEYWORDS:triptolide;puromycin aminonucleoside;proteinuria;podocyte
Proteinuria is the main clinical manifestation of podo-cyte diseases including minimal change disease,focal segmental glomerular sclerosis,and membranous nephro-pathy.1–3It is believed that podocyte injury is a major contributor to severe proteinuria.There are expanding literatures elucidating the molecular events of podocyte injury,although,the treatment for podocyte diseases are far from satisfactory.Therefore,more effective drugs are desirable to improve the treatment for patients with podocyte diseases.Extracts of Tripterygium wilfordii Hook F (TWHF)have been used in the treatment of glomerulonephritis for more than 30years in China.Tablets made from the extracts of TWHF showed very dramatic effects on decreasing protein-uria in patients with minimal change disease,focal segmental glomerular sclerosis and membranous nephropathy.4Addi-tionally,extracts of TWHF could alleviate glomerular albumin permeability induced by protamine,tumor necrosis factor (TNF-a )and the serum from patients with focal segmental glomerular sclerosis in vitro .5It is an intriguing possibility that further characterization of the effective component of TWHF on podocyte lesion will provide a new mechanism based medicine for the treatment of podocyte diseases.Triptolide,a diterpene triepoxide,was identi?ed as one of the major active components of TWHF.Recent reports showed that triptolide has strong immuno-suppressive and anti-in?ammatory activities.6–10In previous work,we found that triptolide could effectively reduce proteinuria,alleviate glomerular immune injuries,and remarkably improve podocyte lesion in rat model with passive Heymann nephritis.11All these ?ndings implicated that bene?cial therapeutic effects of triptolide on proteinuria might be mediated,at least in part,by a protective effect on podocytes.Injection of puromycin aminonucleoside (PAN)to rats produces severe proteinuria and mimics the lesions of minimal change or focal segmental glomerular sclerosis.12It was found that PAN speci?cally injured podocytes,leading to foot process effacement,actin cytoskeleton disorganization,decreased expression and abnormal distribution of slit diaphragm proteins,including nephrin and podocin,13–15resulting in a well-described model of podocyte injury.To address the question of whether triptolide has a direct action on podocytes,an animal model of PAN was used to evaluate o r i g i n a l a r t i c l e https://www.wendangku.net/doc/4515594891.html,
&2008International Society of Nephrology
Received 23November 2007;revised 22February 2008;accepted 4
March 2008;published online 28May 2008
Correspondence:Zhi-Hong Liu,Research Institute of Nephrology,Jinling
Hospital,Nanjing University School of Medicine,Nanjing 210002,China.
E-mail:zhihong@https://www.wendangku.net/doc/4515594891.html,
2Chun-Xia Zheng and Zhao-Hong Chen have contributed equally to the
work and are to be considered first authors.
the effect of triptolide in vivo .In addition,an in vitro
study was performed using immortalized podocytes to
con?rm the direct protective effects of triptolide on podocyte
injuries and to explore the underlying mechanism of
triptolide action.
RESULTS
Triptolide reduced proteinuria in PAN nephrosis rats
Proteinuria emerged at 5days after single PAN injection,
reached the peak at 10days,was persistent at 14days,and
decreased at 21days.The antiproteinuria effect of triptolide
was evaluated by its preventing and treatment effect,
respectively.It was found that proteinuria was signi?cantly
reduced at 5days in the triptolide prevention group
compared with PAN rats (P o 0.01).The reducing effect of
triptolide on proteinuria was persistent obviously at 10days
(P o 0.01),14days (P o 0.01),and maintained at 21days
(P o 0.01).At 21days,proteinuria was restored to normal
(P 40.05,prevention group versus normal control).The
similar effect on proteinuria was observed in the triptolide
treatment group.There was no signi?cant difference bet-
ween the triptolide prevention group and treatment group
(Table 1).
Triptolide improved the abnormalities of serum albumin,
cholesterol and triglyceride in PAN nephrosis rats The restoration of serum albumin level after treatment
paralleled to that of decrease of proteinuria.The serum
albumin level was markedly elevated at 5days in triptolide
prevention group compared with PAN rats (P o 0.05).The
signi?cant effect was observed at 10days (P o 0.01).In the
triptolide prevention group,serum albumin level was
restored to normal at 21days.Meanwhile,restoration of
serum albumin in the triptolide treatment group was similar
to that in the prevention group,and there was no signi?cant
difference between the two groups (Table 2).In addition,the level of triglyceride in both the triptolide prevention and treatment groups was signi?cantly decreased compared with those in PAN rats at 10and 14days (P o 0.01),and returned to normal range at 21days (Table 3).The levels of cholesterol decreased at 14days and was back to normal at 21days both in the triptolide prevention and treatment groups (Table 4).No statistically signi?cant difference in serum creatinine levels was found among the three groups at the time when rats were killed (data not shown).The levels of aspartate aminotransferase and alanine aminotransferase remained normal in triptolide-treated rats (data not shown).Table 1|The effect of triptolide on proteinuria in PAN nephrosis rats (mg/24h)
5days
10days 14days 21days Normal control
3.59±1.90
4.06±1.17 3.99±1.46 3.64±2.02PAN model
29.22±4.46**88.74±14.16**40.19±8.18**12.71±4.77**Prevention
12.03±3.72**mm 20.24±4.06**mm 12.32±2.73**mm 5.35±1.70mm Treatment 14.97±4.54**mm
22.91±5.90**mm 14.38±3.81**mm 4.13±2.43mm PAN,puromycin aminonucleoside.*P o 0.05versus normal control;**P o 0.01versus normal control;mm P o 0.01versus the PAN model (n =5animals per group).
Table 2|The effect of triptolide on serum albumin level in PAN nephrosis rats (g/l)
5days
10days 14days 21days Normal control
34.32±1.2634.16±1.6233.73±1.9435.46±0.76PAN model
24.14±3.93**17.3±3.23**23.55±4.34**30.96±2.17**Prevention
28.36±4.18**m 25.23±3.07**mm 28.35±1.99**m 35.33±0.88m Treatment 27.32±4.30**m 23.06±3.79**mm
27.74±3.28**m 34.52±2.10m PAN,puromycin aminonucleoside.*P o 0.05versus normal control;**P o 0.01versus normal control;m P o 0.05versus the PAN model;
mm P o 0.01versus the PAN model (n =5animals per group).Table 3|The effect of triptolide on triglyceride level in PAN nephrosis rats (mmol/l)5days 10days 14days 21days Normal control 1.66±0.22 1.55±0.15 1.69±0.19 1.62±0.23PAN model 3.21±1.11**7.31±2.96** 4.81±1.05** 2.00±0.78Prevention 2.14±0.44* 4.61±1.54**mm 1.87±0.36mm 1.65±0.14Treatment 2.11±0.46* 4.91±1.63**mm 2.01±0.74mm 1.64±0.40PAN,puromycin aminonucleoside.*P o 0.05versus normal control;**P o 0.01versus normal control;mm P o 0.01versus
the PAN model (n =5animals per group).Table 4|The effect of triptolide on cholesterol level in PAN nephrosis rats (mmol/l)5days 10days 14days 21days Normal control 2.37±0.34 2.23±0.43 2.28±0.29 2.30±0.29PAN model 3.54±1.03* 4.83±1.33** 3.79±1.28* 2.44±0.44Prevention 2.74±0.92 3.77±1.16* 3.09±0.99 2.34±0.35Treatment 2.77±0.91 3.89±1.27* 3.02±1.09 2.30±0.42PAN,puromycin aminonucleoside.*P o 0.05versus normal control;**P o 0.01versus normal control (n =5animals
per group).
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury o r i g i n a l a r t i c l e
The influence of triptolide on GFR
To exclude the possibility that triptolide reduced proteinuria
by decreasing glomerular ?ltration rate (GFR),we measured
GFR in normal control,PAN model,triptolide prevention,
and treatment groups (?ve rats in each group)at 5,10,
and 14days,respectively.We found no signi?cant changes of
GFR in the PAN model,triptolide prevention,and treatment
groups at 5and 14days of PAN injection,compared with
normal control.And at 10days,GFR was signi?cantly lower
in the PAN model than in normal control (P o 0.05);how-
ever,no signi?cant differences were found between PAN
model and the triptolide prevention and treatment groups
(P 40.05).GFR of both triptolide prevention and treatment
groups did not differ from that of normal level (P 40.05;
Figure 1).Therefore,the results con?rmed that triptolide
reduced proteinuria by the protective and reversing effect on
podocyte injuries in PAN nephrosis rats,rather than by
decrease of GFR.
Triptolide ameliorated podocyte foot process effacement
in PAN nephrosis rats
Light-microscopy examination showed no histological
changes of global or focal segmental glomerulosclerosis,
interstitial ?brosis,or tubular atrophy in all the three groups
(Figure 2).
Foot process effacement could be seen at 5days after
PAN injection in PAN rats (P o 0.01versus normal control).
The most extensive foot process effacement that developed
at 10days paralleled with massive proteinuria.The effaced
foot processes were just like sheet covering the glomerular
basement membrane,and the slit diaphragm gap disap-
peared.The change persisted at 14days and got recovery at
21days (Figure 3a–m).
As showed in Table 5,at each time point (5,10,14,and 21
days),foot process widths were signi?cantly decreased in the
triptolide prevention group compared with that in PAN
model rats (P o 0.01),but were still wider than normal
controls (P o 0.01).The foot process size was restored to
normal at 21days in triptolide prevention group.The similar
improvement of foot process effacement was observed in
triptolide treatment group (Table 5;Figure 3a–m).
Triptolide restored the expression and distribution of nephrin and podocin in PAN nephrosis rats By ?uorescence microscopy,the expression intensity and distribution pattern of nephrin and podocin in glomeruli were observed.The staining of nephrin and podocin was revealed as a linear pattern along glomerular capillary wall in the normal control rats.At 5days,the expression of nephrin and podocin was signi?cantly reduced and the distribution became dot-like.At 10days,the peak point of proteinuria,the changes of expression intensity and distribution both of nephrin and podocin were distinctly obvious.The linear staining pattern disappeared with remarkably decreased expression intensity of nephrin and podocin.At 14days,the expression intensity was increased,but distribution still remained dot-like both in nephrin and podocin.At 21days,the distribution of nephrin and podocin got better.Triptolide could signi?cantly improve the expression of nephrin and podocin,and reverse the redistribution of nephrin and https://www.wendangku.net/doc/4515594891.html,pared with PAN model rats,the expressions of nephrin and podocin were signi?cantly increased in both of the triptolide prevention and treatment groups at 10days,and distribution appeared linear at 14days.The expression and distribution of nephrin and podocin were signi?cantly recovered at 14days and completely restored to normal at 21days (Figure 4a–m,n–z).Triptolide decreased the expression of desmin in PAN nephrosis rats In normal control rats,trace amount of desmin was found in glomeruli.In PAN model rats,the expression of desmin increased signi?cantly at 5days,and reached its peak at 10days.At 14days,desmin expression began to decrease,and at 21days further decreased but remained still higher than that in normal https://www.wendangku.net/doc/4515594891.html,pared with PAN rats,expression of desmin was signi?cantly decreased in both the triptolide prevention and treatment groups at 5,10,14,and 21days.At 21days,expression of desmin returned to normal levels (Figure 5a–m).Triptolide protected podocytes against PAN induced cytoskeleton disruption F-actin ?laments in cultured podocytes were distributed as stress ?ber-like bundles along the axis or into the process of cells.PAN caused podocytes cytoskeleton reorganization in a dose-dependent manner.Treatment of podocytes with 25and 50m g/ml PAN for 24h only caused F-actin disorderliness and rarefaction of ?laments.Treatment with 100m g/ml PAN resul-ted in cell retraction and dramatic loss of actin stress-?ber organization (Figure 6a–d),and hence,cytoskeletal changes were accompanied by loss of synaptopodin staining (Figure 6q–s).However,when cells were preincubated with triptolide for 30min before exposure to PAN,podocytes avoided the above changes.The protective effect of triptolide on cytoskeleton was also dose dependent (Figure 6i–l).At the dose of 3ng/ml,triptolide almost completely restored normal cytoskeleton in podocytes without affecting cell survival (Figure 6m–p).0.9
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5 days 10 days 14 days
Time points (days)
Normal control
PAN model
Prevention
Treatment
Figure 1|GFR measurement in normal control,PAN nephrosis
and triptolide-treated rats.*P o 0.05versus normal control.BW,
body weight.
o r i g i n a l a r t i c l e
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
To ensure that cytoskeleton disruption effect of PAN were not due to the apoptosis of podocytes,we performed Hoechst staining.Our results showed no signi?cant difference in apoptosis between PAN-treated(o0.2%of total cells), triptolide pretreatment(o0.3%of total cells),and untreated (0%)podocytes(n4300cells for each treatment)(Figure 6e–h and m–p).For subsequent experiments,triptolide was used at concentration of3ng/ml and PAN100m g/ml.
To assess whether triptolide could recover podocytes from prior injury,podocytes were pretreated with100m g/ml PAN
a
b c d
e f g
h i j
k l m
Figure2|Renal histology changes of PAN nephrosis and triptolide-treated rats.(a)Normal control.(b–k)PAN model groups of
5,10,14,and21days.(c–l)Triptolide prevention groups of5,10,14,and21days.(d–m)Triptolide treatment groups of5,10,14,and
21days.Original magnification?400.
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury o r i g i n a l a r t i c l e
for 24h.Then cells were transferred to triptolide medium
(3ng/ml)for an additional 24or 72h.Podocytes incubated
with triptolide for only 24h following PAN treatment
had sparse stress ?bers,and staining of F-actin in them
was stronger than in those without triptolide incubation.
After 72h of incubation with triptolide medium,podo-cytes reformed robust network of actin stress ?bers (Figure 7).Synchronously,disrupted distribution of synap-topodin by PAN was restored after treatment with triptolide (Figure
8).Figure 3|The effect of triptolide on foot process in PAN nephrosis rats.(a )Normal control.(b –k )PAN model groups of 5,10,14,and 21days.(c –l )Triptolide prevention groups of 5,10,14,and 21days.(d –m )Triptolide treatment groups of 5,10,14,and 21days.o r i g i n a l a r t i c l e C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
Triptolide protected podocytes against PAN-induced nephrin and podocin alteration
The effects of triptolide on the expression and distribution of nephrin and podocin were further con?rmed in cultured podocytes.PAN treatment(100m g/ml,24h)decreased the expression of nephrin and podocin in cultured podocytes. However,triptolide pretreatment could protect podocytes against PAN-induced injuries(Figure9a–c,d–f).These observa-tions were consistent with the results of nephrin and podocin expression analyzed by?ow cytometry(Figure10a and b).
Additionally,triptolide could recover PAN-induced injuries on nephrin and podocin expression in podocytes (Figure11a–f).Podocytes were treated with PAN(100m g/ml) for24h followed by triptolide medium(3ng/ml)for further 72h;the expressions of nephrin and podocin were signi?-cantly improved after triptolide treatment.This result was also con?rmed by?ow cytometry(Figure12a and b). Triptolide inhibited PAN-induced cellular ROS generation
T o unravel the mechanisms underlying the protective effect of triptolide on podocytes,PAN-induced intracellular produc-tion of reactive oxygen species(ROS)was examined.PAN (100m g/ml)signi?cantly increased ROS generation in podo-cytes.This effect was observed at30min and maintained for6h(Figure13a).ROS level at30min was twofold higher than that of basal level after PAN treatment.Pretreatment of podocytes with triptolide(3ng/ml)or antioxidant N-acetyl-cysteine(NAC,10mmol/l)before PAN exposure led to a signi?cant reduction in the cellular ROS level(Figure13b). Triptolide inhibited PAN-induced p38MAPK activation
To characterize the intracellular signaling pathway associated with the protective effects of triptolide in podocytes, phosphorylation of p38mitogen-activated protein kinase (MAPK)was analyzed.Treatment of podocytes with PAN induced a strong increase in phosphorylation of p38MAPK. It increased at3h and was maintained for12h after PAN treatment(Figure13c).Pretreatment of triptolide(3ng/ml) effectively suppressed PAN-induced phosphorylation of p38 MAPK(Figure13d).Triptolide did not affect basal phosphorylation of p38MAPK.
Cellular ROS and activations of p38MAPK are required for PAN-induced actin reorganization in podocytes
We next examined the role of ROS and p38MAPK phos-phorylation in modulating PAN-induced actin reorganization described in Figure 6.Western blots showed that NAC (10mmol/l)signi?cantly suppressed PAN-induced phosphory-lation of p38MAPK.Both NAC(10mmol/l)and p38inhibitor SB-203580(25m mol/l)effectively inhibited PAN-induced cytoskeleton disarrangement(Figure13e–h).
Triptolide restored the activity of RhoA in podocytes RhoA has been suggested to play an important role in cytoskeleton reorganization.To investigate whether RhoA-signaling pathways are involved in the effect of triptolide on podocytes,we performed RhoA activation assay.As shown in Figure14a,PAN treatment induced strong decline in RhoA activity at30min and the low level of activity was maintained till12h.When cells were pretreated with triptolide prior to PAN exposure,RhoA activity was not decreased and normal level was maintained.Total protein level of RhoA was not affected by PAN and triptolide treatment during the time of the test.However,the increase of RhoA activation was markedly inhibited by the speci?c RhoA inhibitor,C3 exoenzyme(1m g/ml).Immuno?uorescence staining was consistent with the result from western blotting,showing that inhibition of RhoA activity by C3exoenzyme abolished the protective effect of triptolide on PAN-induced F-actin dissociation(Figure14b–c).These results strongly suggested that restoration of RhoA activity mediated the protective effect of triptolide.
We further examined whether p38MAPK pathway cross-talked with RhoA-signaling pathways in regulating PAN-induced cytoskeleton disruption.Western blot results showed that C3exoenzyme(1m g/ml)did not alter p38MAPK phosphorylation in response to PAN,and neither NAC (10mmol/l)nor SB-203580(25m mol/l)altered RhoA activity (Figure15).Our data suggested that p38MAPK and RhoA are two independent signaling pathways involved in regula-tion of PAN-induced cytoskeleton disorganization. DISCUSSION
The concept that podocyte has the major role in the develop-ment of proteinuria and progression of glomerulosclerosis, leads us to?nd new approaches to target podocyte lesions. These efforts have generated numerous?ndings showing that certain reagents,such as retinoids,?uvastatin and darbe-poetin,decreased proteinuria by ameliorating podocyte injury.16–18Here,we reported another reagent,triptolide, showing potent ef?cacy in reducing proteinuria and restoring podocyte injury in PAN-induced nephrosis.
Table5|The effect of triptolide on foot process width in PAN nephrosis rats(nm)
5days10days14days21days Normal control271±64268±60267±57277±66 PAN model1808±316**4929±1002**2231±664**876±264** Prevention863±209**mm1307±279**mm881±211**mm270±54mm Treatment880±218**mm1444±291**mm906±245**mm274±61mm PAN,puromycin aminonucleoside.
**P o0.01versus normal control;mm P o0.01versus the PAN model(n=5animals per group).
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury o r i g i n a l a r t i c l e
Results of this study demonstrated that triptolide effectively reduced proteinuria and ameliorated podocyte foot process effacement in PAN nephrosis rats.Recovery from podocyte injury was further proved by reduction of desmin expression in PAN rats after treatment with triptolide.In addition,the alterations of slit diaphragm proteins,nephrin and podocin,induced by PAN were remarkably restored by triptolide.In vitro studies further con?rmed that triptolide protected and reversed PAN-induced cytoskeleton disruption,as well as distribution of nephrin and podocin expression,in podocytes.It was found that the above action of triptolide may be mediated through ROS–p38MAPK and RhoA pathway.PAN-induced nephrosis is characterized by heavy protein-uria and podocyte foot process effacement.Studies have correlated podocyte slit diaphragm proteins and cytoskeletal abnormalities with the onset of proteinuria in PAN-induced nephrosis.13,19Recently,it has been revealed that the slit diaphragm proteins complex (nephrin–podocin–CD2AP)is an important component in maintaining glomerular ?ltra-tion barrier.In PAN-induced nephrosis models,the expres-sion and distribution of nephrin and podocin were found abnormal before the onset of proteinuria.20,21In addition to the morphological changes of foot processes and expressional alterations of slit diaphragm proteins,upregulation of various proteins,such as desmin,B7-1,and heat-shock protein 27,has been noted in podocytes.22–24Among them,desmin staining has been used frequently as a marker of podocyte injury because of its notable changes.In this study,we applied a minimal change disease model induced by PAN for our observations,which was demon-strated by light microscopy and electron microscopy.After PAN injection,proteinuria peaked at 10days in nephrotic rats.Simutaneously,podocyte foot process effacement was the most obvious and decreased expression and redistribu-tion of nephrin and podocin were also remarkable at this point.Coincidently,the podocyte injury marker,desmin,was increased conspicuously.The results provided evidence that massive proteinuria had correlations with podocyte injuries.In comparison,at 10days after triptolide treatment of PAN nephrosis rats,we found that proteinuria reduced signi?cantly,accompanied by improvement of podocyte foot process effacement and restoration of both nephrin and podocin expression as well as desmin expression.All these ?ndings indicated that the antiproteinuric effect of triptolide was strongly associated with recovery of podocyte injury.Oxidative stress initiation and subsequent apoptosis have been thought to be associated with podocyte impairment induced by PAN.25,26In our study,we have not found any apoptotic cells in the kidney tissues of nephrotic rats at any time points at the dose of 8mg/100g body weight,or not in triptolide prevention and treatment rats (data not shown).It was reported that calcineurin inhibitors reduced proteinuria at the cost of signi?cantly decreased GFR.27,28By compa-rison,we observed that triptolide had no effect on GFR levels at the administered dose,which further proved
its Figure 4|The effect of triptolide on nephrin and podocin
expression and distribution in PAN nephrosis rats.(a –m )Nephrin
and (n –z )podocin.(a ,n )Normal control.(b –k )and (o –x )PAN model
groups of 5,10,14,and 21days.(c –l ,p –y )Triptolide prevention
groups of 5,10,14,and 21days.(d –m ,q –z )Triptolide treatment
groups of 5,10,14,and 21days.Original magnification ?400.
o r i g i n a l a r t i c l e
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
independent action of reducing https://www.wendangku.net/doc/4515594891.html,bined with histological evidence,the result also suggested that triptolide had no nephrotoxicity at the dosage used.
To verify that triptolide has directly protective effect on podocyte injuries,and to explore the underlying mechanism of triptolide action,we further performed in vitro studies. Actin cytoskeleton plays a central role in regulating podocyte structure and acts as the critical cytoskeletal protein anchoring focal adhesion and slit diaphragm proteins for maintaining the barrier function.These cytoskeletal proteins are responsible for attachment between adjacent podo-cytes.29–31Any alterations in actin might lead to changes in the structure and function of podocytes.19In this study, it was found that the structure of actin cytoskeleton and synaptopodin was disrupted by PAN.Pretreatment with triptolide could prevent disruption of actin?laments and
a
b c d
e f g
h i j
k l m
Figure5|The effect of triptolide on the expression of desmin in PAN nephrosis rats.(a)Normal control.(b–k)PAN model groups of 5,10,14,and21days.(c–l)Triptolide prevention groups of5,10,14,and21days.(d–m)Triptolide treatment groups of5,10,14,and
21days.Original magnification?400.
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury o r i g i n a l a r t i c l e
synaptopodin induced by PAN.Furthermore,we observed
the restoration effect of triptolide on cytoskeleton disrup-
tion induced by PAN.Triptolide enhanced recovery of
PAN-induced actin ?laments and synaptopodin disruption.
In addition,the decreased expression of nephrin and podocin
induced by PAN could be protected by triptolide.This effect
not only was observed in triptolide pretreated cells,but also
could be seen even podocyte injury occurred.The ?nding that triptolide protected and reversed podocyte injury added more clinical application of using triptolide for patients with podocyte diseases.We further asked what is the underlying mechanism by which triptolide exerted the protective effect on podocytes.Overproduction of ROS has been proved to
mediate Figure 6|Triptolide pretreatment protected podocytes against PAN-induced cytoskeleton disruption.(a –h )PAN caused podocytes cytoskeleton reorganization in a dose-dependent manner without causing cell apoptosis.Normal control (a ,e ),25m g/ml PAN (b ,f ),50m g/ml PAN (c ,g ),and 100m g/ml PAN (d ,h ).(i –p )Triptolide pretreatment protected podocytes actin cytoskeleton from PAN-induced injury in a dose-dependent manner.100m g/ml PAN treated podocytes (i ,m ),0.5ng/ml triptolide preincubation (j ,n ),1ng/ml triptolide preincubation (k ,o ),3ng/ml triptolide preincubation (l ,p ).Red fluorochrome corresponds to F-actin and blue to the nucleus.Original magnification ?400.(q –s )Triptolide pretreatment protected podocytes synaptopodin from PAN-induced injury.Untreated podocytes (q ),PAN (100m g/ml,24h)treated podocytes (r ),podocytes were preincubated for 30min with triptolide before PAN exposure (s ).Green fluorochrome corresponds to synaptopodin and red to the nucleus.Original magnification ?400.
o r i g i n a l a r t i c l e C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
succedent podocyte injury induced by PAN toxicity.32–34
Besides podocytes,triptolide has previously been shown to
inhibit ROS generation in PC12neuronal cell,which shares
many cell biological characteristics with podocytes.35–37We
observed that ROS generation induced by PAN in podocytes
was signi?cantly inhibited by triptolide treatment.Further-
more,our study found that triptolide inhibited p38MAPK
activation induced by PAN.p38was the major MAP kinase
activated by oxidative stress in several cells under various
stimuli.38–40It was believed that activation of p38MAPK was
related to podocyte injury and development of proteinuria,as
well as actin cytoskeleton disruption.Pretreatment of PAN
nephrosis rats with p38MAPK inhibitor completely inhibited
p38MAPK activation and proteinuria.41A study by Susztak
et al .39also has shown that glucose-induced ROS caused
podocyte apoptosis and depletion via p38MAPK pathway.In
our study,we showed that both antioxidant NAC and p38
MAPK inhibitor SB-203580successfully reduced PAN-
induced podocyte damage.In addition,NAC could effectively
suppress PAN-induced phosphorylation of p38MAPK.These results indicated that ROS generation and subsequent p38MAPK phosphorylation mediated PAN-induced podocyte injury.Therefore,our observations demonstrated that triptolide prevented podocyte from damage by inhibiting ROS generation and p38MAPK phosphorylation.Small GTPase-Rho-mediated signal transduction is a ubiquitous pathway in various kinds of cells.It is a direct upstream signaling that controls actin ?lament reorganiza-tion,directing cellular behaviors and phenotypic alterations.RhoA also plays an import role in podocyte cytoskeleton organization.19Proper regulation of Rho GTPase is required for maintaining differentiation of podocytes.42We hypothe-sized that RhoA might also contribute to the protective effects of triptolide against PAN-induced lesions.We found that triptolide pretreatment restored RhoA activity,which was suppressed in PAN-treated podocytes.In addition,C3exoenzyme,a highly selective inhibitor of RhoA,abolished the effect of triptolide,as shown by western blotting and immuno?uorescence staining.These ?ndings strongly suggested that restoration of RhoA activity mediated the protective effect of triptolide.Although these results suggested that the protective effect of triptolide on PAN-induced podocyte injury targeted at the RhoA-and p38MAPK-signaling pathways,it needed further elucidation of the relationship between RhoA-and p38MAPK-signaling pathways.Moreover,several studies have suggested that the two pathways of RhoA and p38MAPK could crosstalk and/or cooperate with each other.43,44Therefore,we performed a set of experiments to examine these possibilities.First,we found that C3exoenzyme did not affect phosphorylation of p38MAPK.And then,we observed that NAC and p38MAPK inhibitor,SB-203580,did not affect PAN-induced RhoA activity.Taken together,these data supported the fact that p38MAPK and RhoA are two independent signaling pathways in regulation of PAN-induced cytoskeleton disorganization.In conclusion,triptolide showed a prominent antiprotein-uric effect on PAN-induced nephrosis.This effect was characterized by improvement of foot process effacement,recovery of podocyte injury marker,desmin,and restoration of nephrin and podocin redistribution.In vitro study further proved that triptolide could protect podocyte from PAN-induced cytoskeleton disruption,abnormality of nephrin and podocin expression.The above action of triptolide might contribute to inhibition of ROS generation and subsequent p38MAPK pathway,as well as restoration of RhoA activity.MATERIALS AND METHODS Reagents Triptolide (C 20H 24O 6,molecular weight 360)was obtained from Chinese National Institute for Control of Pharmaceutical and Biological Products (Beijing,China).The purity of triptolide was detected by high-performance liquid chromatography to be 99.99%.Triptolide was reconstituted in 0.01%dimethyl sulfoxide and freshly diluted with culture medium before use.Dimethyl
sulfoxide’s Figure 7|Triptolide reversed PAN-induced podocytes actin
cytoskeleton disruption.Untreated podocytes (a ).PAN
(100m g/ml,24h)-treated podocytes (b ).Podocytes were treated
with PAN (100m g/ml,24h)followed by the culture medium with
triptolide for further 24and 72h (c ,e ).Podocytes were treated
with PAN (100m g/ml,24h)followed by culture medium without
triptolide for further 24and 72h (d ,f ).Red fluorochrome
corresponds to F-actin.Original magnification ?400.
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
o r i g i n a l a r t i c l e
concentration in in vitro studies was less than 0.002%(v/v).PAN
was purchased from Sigma (St Louis,MO,USA).
Animal experimental design
Male Sprague–Dawley rats (Laboratory Animals Center of Nanjing
University School of Medicine,Nanjing,China)weighing 150–180g
were fed standard diet.All experiments conducted on rats were in
accordance with the guidelines for the care and use of laboratory
animals approved by Nanjing University School of Medicine.PAN
nephrosis was induced by a single intravenous injection of PAN
(8mg/100g body weight).Rats were divided into normal control,
PAN model,triptolide prevention group,and triptolide treatment
group.For the prevention group,triptolide treatment (200m g/kg per
day orally)was started 2days before PAN injection and continued
throughout the experiment.While for the treatment group,
triptolide was started 1day after PAN injection.On each time
point of 5,10,14,and 21days,24h urine collection was performed
using a metabolic cage and blood samples were collected for
biochemical analysis.Simultaneously,rats were killed under
ketamine anesthesia.Renal tissues were processed for light
microscopy,immuno?uorescence staining,and electron microscopy.
Urinary protein concentration was measured with Coomassie
brilliant blue G-250.Serum albumin,cholesterol,triglyceride,
alanine aminotransferase,aspartate aminotransferase,and serum
creatinine levels were measured by HITACHI7080(Tokyo,Japan).
Light microscopy Kidney tissues were ?xed in 10%formalin,dehydrated in graded alcohol,and embedded in paraf?n.Sections measuring 2m m were cut and stained with hematoxylin and eosin,periodic acid–Schiff regent,periodic acid–sliver methenamine,and Masson’s trichrome.All slides were evaluated by the same pathologist who was unaware of the nature of the experimental groups,using a Nikon E800microscope.Transmission electron microscopy Blocks of renal cortical tissue (l mm 3)were ?xed in cold 3.75%glutaraldehyde for 4h and post?xed in phosphate-buffered 1%osmium tetroxide for 2h.And then specimens were embedded in Epon 812ultrathin sections (70nm).The specimens were examined and photographed with Hitachi 7500transmission electron micro-scopy (Tokyo,Japan).GFR measurement GFR was measured according to method in the literature.45.After urine collection,rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (30mg/kg)and were placed on a heated table to maintain core temperature between 371C and 381C.Trachea,both jugular veins,and right carotid artery were catheterized with polyethylene tubing PE-240and PE-50.Then the rats were treated with an infusion of 4%inulin (Farco
Chemical Figure 8|Triptolide reversed podocyte synaptopodin distribution in PAN-induced injury.Untreated podocytes (a ).Podocytes were treated with PAN (100m g/ml,24h)followed by culture medium for further 72h (b ).Podocytes were treated with PAN (100m g/ml,24h)followed by triptolide medium for further 72h (c ).Green fluorochrome corresponds to synaptopodin and red to the nucleus.Original magnification ?400.
o r i g i n a l a r t i c l e C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
Supplies,Hong Kong,China)diluted in 0.9%saline solution at
2.0ml/h by left jugular vein.Urine was drained from the bladder
with a PE-50tube.After surgery,rats were maintained under
euvolemic conditions by infusing 10ml/kg of body weight of
isotonic rat plasma by left jugular vein.After an equilibrium period
of 120min,urine was collected for 60min and blood samples were taken by right carotid artery at the beginning and end of the urine collection period.Mean inulin concentrations in urine and plasma were determined with anthrone reagent.Appropriate plasma and urine blanks,collected before inulin infusion,were run for
each Control PAN Triptolide pretreatment +PAN
Figure 9|Triptolide protected podocytes against PAN-induced nephrin and podocin alteration.(a –c )Green fluorochrome corresponds to nephrin and red to the nucleus.(d –f )Green fluorochrome corresponds to podocin and red to the nucleus.Untreated podocytes (a ,d ).PAN (100m g/ml)treated podocytes (b ,e ).Podocytes were preincubated for 30min with triptolide before PAN exposure (c ,f ).Original magnification ?400.
C o u n t 125
1007550
25
C o u n t 125
10075
50
25
C o u n t 125100
7550250C o u n t
1251007550250C o u n t 1251007550250C o u n t
150
100500102103104105FITC-A
102103104105FITC-A 102103104105FITC-A 102103104105
FITC-A
102103104105FITC-A 102103104105
FITC-A
P2P2P2
P2P2P2Control
PAN Triptolide pretreatment +PAN Figure 10|Effects of triptolide pretreatment on pococytes nephrin and podocin expression analyzed by flow cytometry.MFI of nephrin (a ).MFI of podocin (b ).Panels on the left represent untreated podocytes.Middle panels represent PAN (100m g/ml)treated podocytes.Panels on the right represent podocytes preincubated for 30min with triptolide before PAN exposure.
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury o r i g i n a l a r t i c l e
determination.Inulin was calculated by the standard formula GFR
(ml/min *100g body weight)?U *V/P ,where U is the concentration
of inulin in urine,V is urine ?ow rate,and P is the concentration of
inulin in plasma.
Foot processes width measure Each photograph was analyzed with SimplePCI software (Compix Co.,Irvine,CA,USA).The curved length of the peripheral capillary basement membrane was measured,the number of slit
diaphragms Control PAN PAN + triptolide treatment
Figure 11|Triptolide restored the expression of podocytes nephrin and podocin in PAN-induced injury.Green fluorochrome corresponds to nephrin and red to the nucleus (a –c ).Green fluorochrome corresponds to podocin and red to the nucleus (d –f ).Untreated podocytes (a ,d ).Podocytes were treated with PAN (100m g/ml,24h)followed by culture medium for further 72h (b ,e ).Podocytes were treated with PAN (100m g/ml,24h)followed by culture medium with triptolide for further 72h (c ,f ).Original magnification ?400.
C o u n t 150
100500C o u n t 150100
50
C o u n t 150
100500C o u n t 150100500C o u n t 150
100
50
C o u n t 150100500102103104105FITC-A 102103104105FITC-A 102103104105FITC-A 102103104105
FITC-A
102103104105FITC-A
102103104105
FITC-A
P2P2P2P2P2P2
Control
PAN PAN + triptolide treatment Figure 12|Triptolide restored podocytes nephrin and podocin expression analyzed by flow cytometry.MFI of nephrin (a ).MFI of podocin (b ).Panels on the left represent untreated podocytes.Middle panels represent podocytes were treated with PAN (100m g/ml,24h)followed by culture medium without triptolide for further 72h.Panels on the right represent podocytes were treated with PAN (100m g/ml,24h)followed by culture medium with triptolide for further 72h.
o r i g i n a l a r t i c l e C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
overlying the capillary basement membrane was counted,and the
average foot process width (W p,nm)was calculated using the
formula in the literature.46
Immunofluorescence staining
Cryosections measuring 4m m were stained with mouse anti-rat
nephrin antibody,monoclonal antibody 5-1-6(1:400;a gift from
professor Hiroshi Kawachi in Niigata University,Japan),and rabbit
anti-rat podocin antibody (1:200;Sigma),followed by treatment
with ?uorescein isothiocyanate-conjugated rabbit anti-mouse IgG
(1:50;Dako,Carpinteria,CA,USA)and swine anti-rabbit IgG (1:50;
Dako).Sections were examined by immuno?uorescence microscopy
(Nikon Eclipse E800,Tokyo,Japan).All exposure parameters were
kept constant for each section.
Immunohistochemistry
After de-waxing,2m m sections were stained with mouse anti-rat
desmin antibody (1:200;Dako),followed by incubation with the
Dako Envision system (ready to use;Dako).Sections were then
visualized with 3-amino-9-ethyl-carbazole and counterstained with hematoxylin.TUNEL staining Apoptosis cells were detected with the In Situ Cell Death Detection kit,Fluorescein (Roche Applied Science,Indianapolis,IN,USA)according to the manufacturer’s protocol.Brie?y,4m m cryostat kidney sections were ?xed with 4%paraformaldehyde and incubated with permeabilization solution for 2min on ice.The negative control sections were incubated only in label solution and the positive control sections were treated with 50U/ml DNaseI (Sigma)prior to labeling procedures.Then sections were incubated with enzyme solution.Finally,sections were observed by immuno-?uorescence microscopy.Mouse podocyte culture Conditionally immortalized mouse podocytes were a kind gift from Professor Stuart J Shankland (University of Washington,Seattle,WA,USA).Podocytes were cultured in RPMI-1640
medium ???????700
600500400300200
100C D C F f l u o r e s c e n c e (M F I )C D C F f l u o r e s c e n c e (M F I )PAN (100 μg/ml)PAN (100 μg/ml)
PAN (100 μg/ml)PAN (100 μg/ml)PAN(100 μg/ml)Triptolide(3 ng/ml)Triptolide
(3 μg/ml)PAN (100 μg/ml)Triptolide
(3 ng/ml)
NAC(10 mmol/l)01361224h 0.501361224h 0.501361224h 0.5 1.2100.80.60.40.2
1.2
10
0.80.60.4
0.2
P <0.01P <0.01
P <0.01P <0.01––
–
––––––––––
–––
–++++++++
++++++Phospho-p38p38GAPDH Phospho-p38
p38
GAPDH p -p 38/t -p 38 1.2100.80.60.40.2p -p 38/t -p 38Figure 13|Roles of the ROS-and p38MAPK-signaling pathways in the protective effect of triptolide.Time course of PAN-induced cellular ROS generation (a ).Triptolide inhibited ROS generation in PAN-treated podocytes (b ).Time course of PAN on p38MAPK activation (c ).Triptolide diminished p38MAPK activation in PAN-treated podocytes (d ).(e –h )Cellular ROS and p38MAPK activation are required for PAN-induced actin reorganization in podocytes.Untreated podocytes (e );PAN (100m g/ml,24h)treated podocytes (f );podocytes were preincubated for 30min with NAC before PAN exposure (g );podocytes were preincubated for 30min with SB-203580before PAN exposure (h ).All above results presented are representative of three independent experiments.Values are expressed as
mean ±s.d.;*P o 0.01versus time point 0.
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury o r i g i n a l a r t i c l e
containing 10%fetal bovine serum,100U/ml penicillin,and
100m g/ml streptomycin (Gibco-BRL,Gaithersburg,MD,USA).
Podocytes were expanded by culture in medium containing 10U/ml
mouse interferon-g (R&D Systems,Minneapolis,MN,USA)at
331C.Removal of interferon-g and switching growth temperature to
371C for 10B 14days caused podocytes to stop proliferating and
undertake a differentiated phenotype.14
Reagents treatments
Podocyte injury was induced by treatment with 25B 100m g/ml
PAN for 24h.The protective and recovering effects of triptolide
were observed.For the protective effect experiments,podocytes
were preincubated for 30min with triptolide (0.5B 3ng/ml)
before PAN exposure.There were three groups,including
normal control,PAN injury,and triptolide pretreatment groups.
While for recovering effect experiments,the culture medium
was changed to medium with or without triptolide (3ng/ml)
after 24h of PAN treatment,antioxidant (NAC,10mmol/l),
inhibitor of p38MAPK,SB-203580(25m mol/l),and RhoA inhi-
bitor,and C3exoenzyme (1m g/ml),were administered 30min
before PAN.
Apoptosis detection by Hoechst 33342
Differentiated podocytes were grown on chamber slides.After
treatment,cells were ?xed in 4%paraformaldehyde and mounted in
10m mol/l Hoechst 33342.Apoptosis was de?ned as the presence of
nuclear condensation on Hoechst staining,and the percentage of the
cells with nuclear condensation was calculated on at least 300
consecutive cells.All experiments were performed a minimum of
three times.
Immunofluorescence staining of podocytes Cells were ?xed in 4%paraformaldehyde and then permeablized with 0.5%Triton X-100.For cytoskeletal staining,cells were incubated with 0.5ng/ml rhodamine–phalloidin (Cytoskeleton Company,Denver,CO,USA)for 40min.As for synaptopodin and slit diaphragm proteins staining,cells were stained with rabbit anti-mouse synatopodin antibody (ready to use;Fitzgerald,Concord,CA,USA),rabbit anti-mouse nephrin antibody (1:50,a gift from professor David J Salant in Boston University,Boston,MA,USA),and rabbit anti-mouse podocin antibody (1:50;Sigma),followed by treatment with ?uorescein isothiocyanate-conjugated swine anti-rabbit IgG (1:50;Dako).Cells were then counterstained with 100nmol/l propidium iodide.Cells were observed and imaged by confocal microscopy (LSM 510;Carl Zeiss,Jena,Germany).Flow cytometry analysis of nephrin and podocin expression Nephrin and podocin expression were quanti?ed by ?ow cytometry (FACS Aria;Becton Dickinson,San Jose,CA,USA).Cells were harvested and ?xed in 4%paraformaldehyde and permeated with 0.1%saponin.Aliquots of 1?106cells/ml were incubated for 30min with antibodies of nephrin (1:50)and podocin (1:50),and then incubated with ?uorescein isothiocyanate-conjugated swine anti-rabbit IgG (1:50,Dako)for 30min.Quantitative changes in antigenic expression were assessed using mean ?uorescence intensity (MFI).ROS assay The intracellular production of ROS was assayed using the ?uoroprobe CM-H 2DCFDA (chloromethyl-2,7-dichlorodihydro-?uorescein diacetate;Molecular Probes,Eugene,OR,USA).
To 0 h
1 h 3 h 6 h 1
2 h 24 h 0.5 h ?????PAN PAN (100 μg/ml)Triptolide (
3 ng/ml)(100 μg/ml)PAN (100 μg/ml)RhoA GTP
R h o A G T P /R h o A RhoA
GAPDH
RhoA GTP RhoA GAPDH ––––––++++++––
––––++++++C3 exoenzyme (1 μg/ml)PAN (100 μg/ml)Triptolide (3 ng/ml)C3 exoenzyme
(1 μg/ml)
2.51.50.5
210R h o A G T P /R h o A 2.5
1.50.5
21
001h 3612240.5P <0.01P <0.01Control PAN Triptolide pretreatment
C3 exoenzyme(–)C3 exoenzyme(+)
Figure 14|Role of the RhoA-signaling pathway in the protective effect of triptolide.Time course of RhoA activity inhibited by PAN in podocytes (a ).Triptolide restored RhoA activity in PAN-treated podocytes,and RhoA inhibitor C3exoenzyme abolished the effect of triptolide (b ).C3exoenzyme blocked the protective effect of triptolide on cytoskeleton in podocytes (red fluorochrome corresponds to F-actin)(c ).All the above results are representative of three independent experiments.Values are expressed as mean ±s.d.;*P o 0.01versus time point 0.
o r i g i n a l a r t i c l e C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
examine the effect of PAN on ROS generation by podocytes,podocytes were stimulated with 100m g/ml PAN for 0.5,1,3,6,12,and 24h.To determine the effect of triptolide on PAN-stimulated ROS generation,podocytes were preincubated for 30min with triptolide (3ng/ml)before PAN exposure.After treatment,cells were loaded with 10m mol/l CM-H 2DCFDA.Cells were immediately analyzed by ?ow cytometry.For each sample,10,000events were collected and the content of ROS was assessed by MFI.
Western blot analysis of total and phospho-p38MAPK Cells were lysed in cold cell lysis buffer (50m M Tris,150m M NaCl,10m M ethylenediaminetetraacetic acid,1%Triton X-100)contain-ing protease and phosphatase inhibitors.The cell lysates (50m g)were heated for 5min at 951C in sample buffer,separated on 10%SDS–polyacrylamide gel,and transferred onto nitrocellulose mem-brane.Membranes were probed with rabbit polyclonal antibody to p38MAPK,phosphospeci?c p38MAPK,and glyceraldehyde-3-phosphate dehydrogenase (1:1000;Santa Cruz Biotechnology,Waltham,MA,USA)and then were reprobed with horseradish peroxidase-conjugated anti-rabbit antibody (1:1000;Santa Cruz Biotechnology)and visualized by enhanced chemiluninecence detection.
RhoA activation assay by pull-down experiment RhoA activation was studied using the RhoA activation kit (Cytoskeleton Inc.,Denver,CO,USA)according to the manufac-turer’s recommendations.After cell lysis,about 300m g of protein was incubated with 50m g of Rhotekin-RBD protein beads (GST fusion protein containing the RhoA-binding domain of Rhotekin).The bound proteins were fractionated on 12%SDS-polyacrylamide-gel electrophoresis and immunoblotted with anti-RhoA monoclonal antibody.The level of active RhoA was determined after normal-ization with the total RhoA presented in the cell lysates.Statistical analysis Statistical analyses were performed with SPSS software (version 11.5).Results were expressed as mean ±s.d.Student’s t -test was used to compare difference between groups.P o 0.05was considered statistically signi?cant and P o 0.01was considered of high statistical signi?cance.DISCLOSURE All the authors declared no competing interests.ACKNOWLEDGMENTS This study was supported by the Foundations of Medical Research (06G040and 06Z35).We thank Professor Hiroshi Kawachi at Niigata University,Japan,for providing mouse anti-rat nephrin antibody,and thank Professor David J Salant at Boston University,USA,for providing rabbit anti-mouse nephrin antibody.Conditionally immortalized mouse podocytes were kindly provided by Professor Stuart J Shankland at University of Washington,USA.REFERENCES 1.Cattran DC.Idiopathic membranous glomerulonephritis.Kidney Int 2001;9:1983–1994.2.Ly J,Alexander M,Quaggin SE.A podocentric view of nephrology.Curr Opin Nephrol Hypertens 2004;13:299–305.3.de Zoysa JR,Topham PS.Podocyte biology in human disease.Nephrology 2005;10:362–367.4.Li LS,Zhang X,Chen GY.Clinical study of Tripterygium wilfordii Hook in the treatment of nephritis.Chin J Intern Med 1981;20:216–220.5.Sharma M,Li JZ,Sharma R et al.Inhibitory effect of Tripterygium wilfordii multiglycoside on increased glomerular albumin permeability in vitro .Nephrol Dial Transplant 1997;12:2064–2068.6.Yang Y,Liu Z,Tolosa E et al.Triptolid induces apoptotic death of T lymphocyte.Immunopharmacology 1998;40:139–149.7.Liu Q,Chen T,Chen G et al.Immunosuppressant triptolide inhibits dendritic cell-mediated chemoattraction of neutrophils and T cells through inhibiting Stat3phosphorylation and NF-k B activation.Biochem Biophys Res Commun 2006;345:1122–1130.8.Sylvester J,Liacini A,Li WQ et al.Tripterygium wilfordii Hook F extract suppresses proinflammatory cytokine induced expression of matrix metalloproteinase genes in articular chondrocytes by inhibiting activating protein-1and nuclear factor kappaB activities.Mol Pharmacol 2001;59:1196–1205.9.Kim YH,Lee SH,Lee JY et al.Triptolide inhibits murine inducible nitric oxide synthase expression by down regulating lipopolysaccharide-induced activity of nuclear factor-kappa B and c-Jun NH(2)-terminal kinase.Eur J Pharmacol 2004;494:1–9.10.Dai YQ,Jin DZ,Zhu XZ et al.Triptolide inhibits COX-2expression via NF-kappa B pathway in astrocytes.Neurosci Res 2006;55:154–160.11.Qin W,Liu Z,Zeng C et al.Therapeutic effect of triptolide on podocyte injury in passive Heymann nephritis.Chin J Nephrol Dial Transplant 2007;16:101–109.12.Hagiwara M,Yamagata K,Capaldi RA et al.Mitochondrial dysfunction in focal segmental glomerulosclerosis of puromycin aminonucleoside nephrosis.Kidney Int 2006;69:1146–1152.13.Guan N,Ding J,Deng J et al.Key molecular events in puromycin aminonucleoside nephrosis rats.Pathol Int 2004;54:703–711.14.Mundel P,Reiser J,Zu ′n ?iga Mej?′a Borja A et al.Rearrangements of the cytoskeleton and cell contacts induce process formation
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NAC PAN Control C3 exoenzyme SB203580
NAC PAN PAN
C3 exoenzyme SB203580
RhoA
RhoA GTP
R h o A
G
T
P /
R
h
o A Phospho-p38
p38
GAPDH
2.5
1.2
1.50.52
1100Control NAC PAN PAN
C3 exoenzyme SB203580
Control ??
?
?
??
0.8
0.60.4
0.2
p -p
38
/
t
-
p 38Figure 15|ROS-p38MAPK and RhoA mediated PAN-induced podocyte damage in parallel.Neither NAC nor SB-203580altered RhoA activity in response to PAN,and inhibition of RhoA activity by C3exoenzyme did not alter p38MAPK
phosphorylation.All the above results are representative of three independent experiments.Values are expressed as mean ±s.d.;*P o 0.01versus control.
C-X Zheng et al.:Triptolide protects PAN-induced podocyte injury
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