cyclosporine a has improved allograft survival and the quality of life for solid - organ transplant recipients .
its effectiveness in transplantation by suppression of the immune system has led to its use in treating autoimmune diseases .
csa inhibits the immune system by binding to cyclophilin , this complex then inhibits calcineurin , which in turn inhibits the translocation of the nuclear factor of activated t cells ( nfat ) and subsequent gene transcription .
calcineurin inhibitors ( cni ) can cause nephrotoxicity involving acute renal vasoconstriction progressing on to glomerulosclerosis , tubulointerstitial fibrosis , and renal failure .   due to cni - induced nephrotoxicity ,
the use of the immunosuppressive agent sirolimus ( srl ) in transplantations is becoming more widespread .
srl has a different mechanism of immunosuppression compared to cnis , and a lesser degree of nephrotoxicity is observed with srl compared to that observed with the calcineurin inhibitors [ 4 , 5 ] . when administered with a cni
, srl has been suggested to have a protective role when administered in conjunction with csa [ 6 , 7 ] .
the rapamune us study group conducted a large multicentre clinical trial in which the efficacy of srl compared to azathioprine for reducing acute renal allograft rejection was investigated .
the use of srl reduced occurrence and the severity of biopsy - proven acute rejection episodes .
however , there have also been studies indicating enhanced nephrotoxicity when srl and csa are used in combination [ 9 , 10 ] . in another study ,
the authors showed that a combination of srl and csa significantly potentiated the nephrotoxic actions of csa by augmenting transforming growth factor  ( tgf- ) .
tgf- has been implicated as a major factor in the development of chronic cni toxicity .
increased tgf- levels have been observed in renal cells exposed to csa , in animal models of csa toxicity and in patients with cni nephropathy [ 1215 ] ; however , the nephrotoxicity caused by csa remains to be fully elucidated . in this study ,
our hypothesis was to determine whether csa or srl had direct detrimental effects on the glomerulus and identify the possible mechanisms involved , using glomerular mesangial cells .
human mesangial cells ( hmcs ) are key cells of the glomerulus and have an important role in regulating glomerular structure and function and have the potential to contribute to glomerulosclerosis by secreting profibrotic mediators , which can alter extracellular matrix ( ecm ) balance and disrupt renal function
. this may be an important mechanism in renal disease progression and the upstream signalling pathways involved in csa - induced renal dysfunction are not well characterised and warrants further investigation . one intracellular pathway that may potentially be involved in immunosuppressive - induced renal damage
is that of the mitogen - activated protein kinase ( mapk ) family , which has been implicated in tgf--induced cytotoxicity .
the analysis of the differential activation of this pathway may provide a novel insight into the mechanisms of nephrotoxicity caused by csa .
the hmcs were grown in rpmi 1640 containing 5% fcs , penicillin , streptomycin , and l - glutamine and maintained at 37c in a humidified atmosphere containing 95% air , 5% co2 .
cells used in the inhibitor studies were pretreated for 1 hour with uo126 , prior to incubation with each drug treatment .
mesangial cell viability was assessed using the resazurin conversion ( sigma - aldrich , 7017 ) cell viability assay .
the viability of the cells was expressed as a percentage of the absorbance recorded for control cells .
total rna was isolated using the trizol method from hmcs , according to the manufacturer 's protocol ( sigma - aldrich , t9424 ) . 1  g of total rna was used to synthesis cdna .
a real - time pcr taqman assay was used to quantify the relative expression levels of genes of interest and has been described previously .
briefly , cdna was amplified on the abi 7900ht sequence detection system at default thermal cycling conditions : 2  min at 50c , 10  min at 95c for enzyme activation , and then 40 cycles of 15  sec at 95c for denaturation and 1  min at 60c for annealing and extension .
were used for all genes of interest , tgf- ( hs99999918_m1 ) , ctgf ( hs00170014_m1 ) , and mmp-9 ( hs00234579_m1 ) .
the ribosomal 18s gene was used as an endogenous control for normalisation of the target genes .
a negative control containing all reaction components except for superscript for each set of samples was used .
a tgf-1 elisa was used to determine the effect csa had on secreted tgf-1 protein levels in the media of hmcs .
the specificity and sensitivity of the assay was assessed using 5  ng of tgf-1 as a positive control and sterile water as a negative control .
transfection reagent - dna complex was prepared by adding serum - free medium to a sterile eppendorf tube .
fugene ( roche ) reagent was added directly to the tube and then 1  g of tgf- smad responsive ( caga ) luciferase reporter dna plasmid ( a gift from dr .
roel goldschmeding ) and 1  g of control plasmid ( renilla ) ( promega ) . for multiple - well
following transfection , cells were treated with each immunosuppressive agent for the required time period .
cells were lysed using passive lysis buffer ( promega ) and put into fresh tubes .
the luminometer was programmed to perform a 2-second premeasurement delay followed by a 10-second measurement .
equal volumes of cell lysate and luciferase reagent were added together and the luminescence reading recorded . stop and glo reagent was added to halt the reaction and another reading performed .
control lysates were made from untransfected cells to determine background luminescence levels and a positive control of cells treated with 5  ng tgf- was used .
total protein was isolated from hmcs using the ripa buffer method ( sigma - alridch , r0278 ) according to the manufacturer 's protocol .
expression levels of renal proteins following csa treatment was determined by western blot and has been described previously [ 15 , 20 , 21 ] .
proteins of interest were detected using the following antibodies according to the manufacturer 's protocol ( rabbit anti - erk 1/2 , cell signalling technology , 9211s and 9211 ) .
data was analysed by one - way analysis of variance ( anova ) , and multiple comparisons between control and treatment groups were made using the bonferroni posttest .
the following scheme was used throughout the work ; * p < 0.05 , * * p < 0.01 , * * * p < 0.001 .
microscopic examination highlighted that treatment with csa or srl alone for 24 hours had no significant effect on hmc morphology ( figure 1(a ) ) .
however , 24-hour treatment with csa / srl combination did cause alterations in hmc morphology ( figure 1(a ) ) .
an elongation of normal hmc shape accompanied with gaps in the cell monolayer and cell death ( indicated by round floating cells ) was observed .
treatment for 24 hour with csa or srl alone had no significant effect on hmc viability .
treatment with csa / srl drastically reduced hmc viability ( 100 versus 54  3.9 , p < 0.05 )
. the concentrations of csa and srl used in this study were chosen following dose response studies , and it approximates to concentrations used in vivo and reflect concentrations in the kidney .
treatment with csa for 24 hours caused a significant increase in tgf-1 secretion ( 843.1  7.1 versus 1089.5  26.1 
pg / ml ; p < 0.05 ) as demonstrated by elisa ( figure 1(b ) ) .
however , cotreatment of csa / srl caused the most significant rise in tgf-1 secretion ( 843.1  7.1 versus 1247.4  10.4  pg / ml ; p
the increase in tgf-1 secretion observed with csa / srl was significantly greater than the increase observed with csa alone ( 1089.5  26.1 versus 1247.4  10.4  pg / ml ; p < 0.05 ) . to further examine the role of tgf- , we decided to investigate the effects of csa , srl , and csa / srl cotreatment on a smad - responsive luciferase construct .
csa treatment caused a significant increase in smad activation ( 100 versus 156.9  1.7 luciferase units ; p < 0.05 ) .
srl administration did not significantly alter smad activation ( 100 versus 123.5  4.3 luciferase units ) .
treatment with csa / srl cotreatment caused the greatest increase in smad activation ( 100 versus 210.1  15.9 luciferase units ; p < 0.01 ) .
the increase in smad activation in the presence of csa / srl cotreatment was significantly greater than with csa alone ( 156.9  1.7 versus 210.1  15.9 luciferase units ; p < 0.01 ) ( figure 1(c ) ) .
the observation of increased smad signalling prompted an investigation into a downstream mediator of tgf- , connective tissue growth factor ( ctgf ) .
treatment for 24 hours with csa showed a trend towards an increase in ctgf gene expression , which was not statistically significant .
however , treatment with csa / srl cotreatment caused a significant increase in ctgf gene expression ( 1 versus 2.0  0.02 ; p < 0.01 ) ( figure 2(a ) ) .
csa treatment for 24 hours caused a significant decrease in matrix metalloproteinase 9 ( mmp-9 ) gene expression ( 1.0 versus 0.18  0.15 ; p < 0.05 ) .
csa / srl cotreatment also caused a significant decrease in mmp-9 gene expression ( 1 versus 0.37  0.2 ; p < 0.05 , figure 2(b ) ) . in order to determine the mechanism of csa / srl co - treatment - induced alterations , we investigated the effects of csa , srl , and csa / srl on erk 1/2 activation .
csa significantly increased erk 1/2 phosphorylation after 15 , 30 , and 60 minutes administration ( figure 3(a ) ) .
srl also significantly increased erk 1/2 phosphorylation after 15 and 30 and 60 minutes treatment ( figure 3(b ) ) .
csa / srl co - treatment caused a synergistic increase in erk 1/2 phosphorylation at 15 , 30 , and 60 minutes .
however , the combination of csa / srl co - treatment also significantly increased erk 1/2 phosphorylation at 24 and 48 hours , unlike the csa and srl individual treatments ( figure 3(c ) ) .
the addition of uo126 prevented the csa , srl , and csa / srl co - treatment - induced stimulation of erk 1/2 ( figure 4 ) .
total levels of erk 1/2 protein were unaltered following csa , srl , or csa / srl combination in the presence or absence of uo126 treatment .
it was upon the notable activation of erk 1/2 phosphorylation that the effect of 24-hour csa / srl treatment in the presence of the erk 1/2 inhibitor uo126 was examined ( figure 5 ) .
erk 1/2 inhibition appeared to partially ameliorate the csa / srl co - treatment - induced morphological alterations in hmcs .
the csa / srl - induced reduction in hmc viability was prevented to some extent by erk inhibition ( 54  3.9 versus 88  3.5 , p < 0.001 ) .
the addition of the erk inhibitor alone caused no effect on hmc viability or morphology .
inhibition of erk 1/2 phosphorylation prevented the csa alone and csa / srl co - treatment - induced increases in tgf- secretion ( figure 4 ) .
( csa + uo126 ,  518  23.5  versus 1089  26.1 , csa , p < 0.001 ) .
( csa / srl + uo126 ,  424  54.2  versus  1247  10.4 , csa / srl , p < 0.001 ) .
erk inhibition also dramatically reduced the csa / srl - induced increase in ctgf gene expression to below control levels ( csa / srl , 1.7  0.2 versus 0.6  0.14 , csa / srl + uo126 ) .
erk inhibition also prevented the previously observed csa / srl co - treatment - induced decrease in mmp-9 gene expression ( 0.37  0.25 versus 1.6  0.2 ; p < 0.001 ) ( figure 4 ) .
erk inhibition also restored the csa- and srl - induced decreases in mmp-9 and restored mmp-9 values to control levels .
the results presented in this body of work demonstrated that the combination treatment of csa and srl did not result in an attenuation of the cni - associated changes in hmcs but in fact resulted in synergistic nephrotoxic - like effects .
it appeared that the primary causative factor for the csa / srl - induced mesangial cell dysfunction was a significant dual increase in tgf- and ctgf expression .
we also observed enhanced activation of the erk 1/2 mapk pathway , the tgf- signalling pathway , and alteration of ecm regulators .  
altered hmc morphology and viability gave the first indication that csa and srl combination treatment was exerting a detrimental effect when administered to hmcs in the present study .
these initial findings of enhanced toxicity were surprising as it had been previously reported that srl had a protective role and could reduce the toxicity observed with cni administration [ 22 , 23 ] .
however , more recently , experimental evidence has emerged suggesting that a combination of csa and srl might not be protective . in a rat model ,
kidney function and morphology were assessed following short - term combination therapy with csa , and srl .
striped fibrosis and glomerular filtration rate ( gfr ) were significantly worse in rats receiving csa and srl combination according to the study by nielsen et al . .  
we observed that the secretion of tgf- was significantly increased following immunosuppressive drug treatment in the present model .
tgf- is a major factor involved in csa - induced renal fibrosis and renal disease .
tgf- has been shown to be upregulated in a number of in vitro and in vivo experimental models following csa treatment and in patients undergoing csa therapy [ 14 , 15 , 25 ] . increased tgf- may have contributed to the adverse effects observed in the present study .
the lack of an increase in tgf- as observed with srl alone in the current study may help to explain the reduced nephrotoxic side effects associated with srl .   it is accepted now that altered pharmacokinetics have a role in contributing to the observed toxicity with a combination of srl and csa . an in vitro study in human renal epithelial cells demonstrated that srl prolonged the intracellular accumulation of csa when given in combination .
the authors showed that srl inhibited p - glycoprotein - mediated efflux contributing to csa nephrotoxicity .
however , the reasons for the augmented nephrotoxicity resulting from a combination of srl and csa still remain to be fully defined , with some studies attributing the effect to pharmacokinetics and others to hyperglycaemia [ 10 , 26 , 27 ] .
another known contributor to csa nephrotoxicity is tgf-.   in the present hmc model , srl had a synergistic effect on tgf- secretion when coadministered with csa .
however , similar increases in tgf- were also reported in a study on csa and srl combination therapy by shihab et al . .
in that study , rats were given doses of csa alone , srl alone , or a combination of csa and srl .
the authors observed a worsening in renal function in the group that were given csa and srl in combination compared to csa and srl alone .
the csa / srl group also exhibited tubular injury , interstitial fibrosis , and arteriolopathy as indicated by histological analysis .
elevated tgf- protein levels were also observed in the csa and srl co - treatment groups , and the levels were significantly higher than with csa or srl alone . to our knowledge , however , this is the first time that this effect has been observed in hmcs .  
ctgf was upregulated in the present hmc model following csa / srl co - treatment .
ctgf has been found to be upregulated in many inflammatory glomerular diseases and in patient biopsies with numerous different conditions including glomerulonephritis and focal segmental glomerulosclerosis .
it appeared that a novel dual elevation of tgf- and ctgf in the hmcs following csa / srl co - treatment contributed to the enhanced csa / srl - induced mesangial cell alterations .
this dual elevation of tgf- and ctgf was not observed following treatment with csa or srl alone .   in the current study , csa
the observed reduction in mmp-9 in the present mesangial cell model may have facilitated ecm accumulation by preventing ecm degradation .
it is known that ecm regulators are extremely context specific and chronologically variable . in models of chronic kidney disease ,
decreased mmp-9 has been shown to aid disease progression . in a rat ischemia - reperfusion model , caron et al . observed that mmp-9 was significantly upregulated and may have contributed to glomerular injury .
in another model , transgenic mice overexpressing renin developed hypertension - induced renal dysfunction . isolated glomeruli from these mice exhibited elevated tgf- accompanied with decreased mmp-9 gene expression .  
these alterations in hmcs following csa / srl treatment are at least in part due to enhanced signalling through the erk 1/2 pathway .
previous studies in our laboratory have indicated that the mapk pathways are also activated in response to csa in madin darby canine kidney ( mdck ) epithelial cells [ 32 , 33 ] .
a biphasic activation of erk 1/2 following csa / srl co - treatment was observed in the hmcs and may be the mechanism by which the synergistic nephrotoxic effect is exerted by the csa / srl co - treatment .
this is further supported by the fact that erk 1/2 inhibition resulted in significant attenuation of this csa / srl - induced hmc dysfunction . considering the well - characterised roles of erk 1/2 signalling in cell survival and proliferation ,
our finding that enhanced erk 1/2 signalling is central to csa / srl cytotoxicity is interesting .
this apoptosis was accompanied by activation of the erk 1/2 mapk pathway . pharmacological inhibition of erk 1/2
similarly , in cisplatin - induced cytotoxicity , erk 1/2 has been shown to play a central role .
several mechanisms of erk 1/2-mediated cytotoxicity have been proposed including erk - dependent activation of caspases   and induction of autophagy .
importantly from the perspective of this study , in examples of erk - mediated cytotoxicity , erk activation is unusually prolonged ( up to 72 hours ) .
therefore , the prolonged erk activation observed in this study , in the presence of csa / srl co - treatment , may be a key mechanistic difference compared to the two immunosuppressants in isolation .
this suggests that the erk 1/2 pathway may play a major role in tgf- signalling .
erk 1/2 also appeared to play a role in ctgf expression in this mesangial cell model as ctgf gene expression was attenuated by erk inhibition .
these observations have been observed in other models of csa nephrotoxicity [ 39 , 40 ] .  
our proposed mechanism for the enhanced csa / srl nephrotoxicity is that csa / srl strongly activates erk 1/2 , which promotes tgf- cellular secretion .
tgf- may also promote decreased ecm degradation via decreased mmp9 , which contributes to the observed mesangial cell dysfunction .
these findings are important in determining future immunosuppression strategies for srl therapy in clinical organ transplantation .
currently , there are two main strategies : de novo use of srl in combination with reduced amounts of csa or complete conversion from a cni - based protocol to srl in cases where well - recognized adverse effects of cnis ( such as impaired renal function ) are prevalent .
however , there is much debate on this issue and longer - term studies in larger cohorts of patients are required to determine whether replacement of csa with srl provides any significant improvement in patient and graft survival .
the results shown in this study would suggest that long - term use of srl in patients will have serious nephrotoxic effects .
this work was supported by the health research board , science foundation ireland , enterprise ireland , and the programme for research in third level institutions administered by the higher education authority and by the eu 7th framework grant  syskid  , health - f2 - 2009 - 241544 . c. slattery is funded by a government of ireland research fellowship from the irish research council for science , engineering and technology .
the results presented in this paper have not been published previously in whole or part , except in abstract form .
 end stage renal disease ( esrd ) is an ever increasing problem worldwide . 
 however the mechanisms underlying disease progression are not fully elucidated . 
 this work addressed nephrotoxicity induced by the immunosuppressive agents ' cyclosporine a ( csa ) and sirolimus ( srl ) . 
 nephrotoxicity is the major limiting factor in long term use of csa . 
 srl causes less nephrotoxicity than csa . 
 therefore investigations into the differential effects of these agents may identify potential mechanisms of nephrotoxicity and means to prevent esrd induced by therapeutic drugs . using elisa , western 
 blotting , quantitative pcr and a reporter gene assay we detailed the differential effects of csa and srl in human renal mesangial cells . 
 csa treatment increased profibrotic tgf-1 secretion in human mesangial cells whereas srl did not , indicating a role for tgf- in csa toxicity . 
 however we observed a synergistic nephrotoxic effect when csa and srl were co - administered . 
 these synergistic alterations may have been due to an increase in ctgf which was not evident when the immunosuppressive drugs were used alone . 
 the csa / srl combination therapy significantly enhanced smad signalling and altered the extracellular matrix regulator matrix metalloproteinase 9 ( mmp-9 ) . 
 inhibition of the erk 1/2 pathway , attenuated these csa / srl induced alterations indicating a potentially significant role for this pathway . 
1. Introduction
2. Methods
3. Results
4. Discussion
 Funding
 Conflict of Interests