clinically , calcineurin inhibitors ( e.g. , cyclosporine a , csa ) have been used to reduce proteinuria in focal segmental glomerulosclerosis ( fsgs ) , minimal change disease ( mcd ) , and other proteinuric kidney diseases .
t - cell dysfunction is associated with some forms of proteinuria , including a subset of mcd in children .
this concept originally stemmed from the so called  shalhoub hypothesis  that  lipoid nephrosis  is produced by a systemic abnormality of t - cell function . for decades
, fsgs was thought to be an immunologic disease resulting from the noxious effect of a lymphokine on the podocyte .
this was the primary reason for using csa as an immunosuppressive drug , to continue to endorse this mechanism of action despite studies demonstrating that calcineurin inhibition reduced proteinuria in nonimmunologic glomerulopathies .
actually , csa can also reduce proteinuria in human and experimental alport 's syndrome , a nonimmunological disease , raising doubts of this hypothesis [ 4 , 5 ] . moreover , although csa effectively lowered the level of proteinuria in human membranous nephropathy , the study of ambalavanan et al .
showed a more numerous and lager electron - dense immune deposits than before csa 's therapy , indicating that whilst csa exerts an antiproteinuric effect , the drug does not modify the histologic aggravation of the glomerular lesions .
a recent study showed that csa has a direct antiproteinuric effect on podocytes .csa blocked calcineurin - mediated dephosphorylation of the actin - organizing protein synaptopodin , a podocyte foot process cytoskeletal component , facilitating its degradation by cathespin l. mechanistically , this study thus identified a new calcineurin signaling pathway in kidney podocytes and attributed the antiproteinuric effect of csa to its inhibition of calcineurin - mediated degradation of synaptopodin .
thus , this antiproteinuric effect was shown to be independent of t cells , at least partially .
although arguing against an antiproteinuric role of csa through the suppression of t cells , this study did not rule out the involvement of nfat proteins downstream of calcineurin in kidney podocytes .
our results , along with those from wang et al .   and nijenhuis et al . , suggested that conditional nfatc1 activation in podocytes per se is sufficient to induce proteinuria in mice .
thus , both studies provided in vivo evidence that nfat activation in podocytes may be a critical pathogenic molecular event leading to proteinuria or fsgs .
altogether , these advances in podocyte research indicate that calcineurin - nfat signal or calcineurin - synaptopodin axis has a direct proteinuric effect on podocytes , and these observations raise the possibility of developing specific antiproteinuric drugs that lack the unwanted effects of calcineurin or nfat inhibition .
proteinuria , a cardinal sign and a prognostic marker of kidney disease , affects several hundred million people worldwide .
podocytes , endothelial cells , and the glomerular basement membrane ( gbm ) constitute the glomerular filtration barrier , a highly specialized structure for selective ultrafiltration .
the common denominator in a variety of kidney diseases , including mcd and fsgs , is podocyte injury involving a massive loss of protein in the urine ( proteinuria ) [ 13 , 14 ] .
several studies showed that the podocyte has a central role in the development of proteinuria and idiopathic nephrotic syndrome .
several pathogenic pathways involved in effacement of the podocyte foot processes and the development of proteinuria have been discovered .
studies in hereditaryproteinuric syndromeshaveuncovered that mutations of podocyte proteins , including -actinin-4 , cd2ap [ 16 , 17 ] , nephrin , plce1 , podocin , trpc 6 [ 21 , 22 ] , formin protein inf2 , and myo1e   lead to proteinuria , podocyte foot processes effacement and podocyte actin cytoskeleton disruption [ 14 , 25 ] .
other proteins regulate the podocyte actin cytoskeleton and are important for the glomerular filtration barrier .
these proteins include rho gdialpha [ 26 , 27 ] , podocalyxin , fat1 , nck1/2 , and synaptopodin .
trpc6 is a member of the large transient receptor potential superfamily of nonselective cation channels [ 32 , 33 ] .
mutations in the gene that encodes trpc6 have been identified in families with autosomal - dominant fsgs [ 3436 ] .
many calcium - dependent signals , including calcineurin , may be potential targets of trpc6 activation in podocytes since trpc6 are involved in the regulation of intracellular calcium concentration in response to the activation of g - protein - coupled receptors and receptor tyrosine kinases . meanwhile , some trpc6 mutations found in human beings with fsgs result in increased amplitude and duration of calcium influx into hek293 cells [ 21 , 22 ] .
trpc6-deficient mice do not show any obvious renal phenotype . however , transient induction of trpc6 overexpression by in vivo gene delivery results in proteinuria .
the actin - binding protein synaptopodin , which is highly expressed in podocytes , is a key regulator of podocyte function since bigenic heterozygosity for synaptopodin and cd2-associated protein results in proteinuria and fsgs .
synaptopodin induces stress fibers by stabilizing the gtpase rhoa   and suppresses filopodia by disrupting cell - division cycle-42-insulin receptor substrate p53-mena signaling complexes . recently , urokinase receptor ( upar ) and its soluble form ( supar ) have been shown to be involved in the pathogenesis of proteinuria and fsgs [ 39 , 40 ] .
upar is a glycosylphosphatidylinositol - anchored protein that has been shown to be a proteinase receptor for urokinase but has also been involved in nonproteolytic pathways , mainly through interactions with other plasma membrane proteins such as integrins .
upar and 3 integrin colocalize in podocytes and thus form a complex with 3 integrin , thereby causing the activation of 3 integrin . in vivo gene delivery of constitutively active 3 integrin is sufficient to induce proteinuria in mice ; conversely , inhibition of upar expression and 3 integrin activation has an antiproteinuric effect .
interestingly , supar , a soluble form of upar has been identified as a circulating fsgs factor that is elevated in the serum of approximately two - thirds of primary fsgs patients .
supar - mediated activation of 3 integrin on podocyte foot processes may be the mechanism of injury caused by high supar blood concentrations .
the calcineurin is ubiquitously expressed in all mammalian tissues and is a ca - dependent serine / threonine phosphatase composed of a catalytic subunit , cna , and a regulatory subunit , cnb [ 43 , 44 ] .
calcineurin has a wide range of roles in organ development and cellular functions [ 45 , 46 ] , including the regulation of transcription in various renal cells [ 43 , 44 ] .
clinically , csa is used to reduce proteinuria in kidney diseases , such as idiopathic nephrotic syndrome ( especially fsgs ) .
the immunosuppressive effect of csa results from inhibition of signaling by the transcription factor nfat in t cells , and this action has also been believed to mediate csa 's antiproteinuric effect . although the efficacy of csa was thought to derive from its suppression of nfat activation in t cells through the inhibition of calcineurin , not all drugs suppressing t - cell activation have protective effects on glomeruli .
the study of faul et al .   suggested a novel role for csa in inhibiting the dephosphorylation of synaptopodin by calcineurin .
.   found that activation of calcineurin in the podocyte is sufficient to cause proteinuria via the degradation of synaptopodin and that csa blocks the calcineurin - mediated dephosphorylation of synaptopodin , thereby preserving the phosphorylation - dependent synaptopodin-14 - 3 - 3b interaction .
csa and e64 ameliorate lps - induced proteinuria by blocking the catl - mediated degradation of synaptopodin [ 4851 ] .
these data unveiled a calcineurin signaling pathway , which is operative in podocytes and contributes to the maintenance of kidney filter function .
although faul et al . argued against an antiproteinuric role of csa through the suppression of t cells , it is possible for the involvement of nfat proteins downstream of calcineurin in podocyte injury , since the nfat transcription factors are the most extensively studied calcineurin substrates and the major regulators of transcription in response to ca / calcineurin signals [ 47 , 52 ] .
ca signaling through ion channels has recently emerged as a potential modulator of podocyte function , and several ca - permeable channels have been identified inpodocytes [ 53 , 54 ] .
generally , upon activation by increased intracellular ca , calcineurin dephosphorylates the nfat proteins that reside in the cytoplasm in resting cells .
this dephosphorylation exposes the concealed nuclear localization signals of the nfat proteins , leading to the cytoplasm to nucleus translocation of these proteins . in the nucleus ,
the nfatc proteins form nfat transcription complex with their nuclear partners to control the transcription of target genes .
trpc6 mutations were found in families with hereditary fsgs , and trpc5 and trpc6 channels are now known as the ca influx pathways for the nonselective , cationic current in podocytes .
mutations in one of these channels , trpc6 , lead to aberrant ca signaling , podocyte dysfunction [ 21 , 22 ] , and nephrin and neph1 have been shown to interact with several ca channels , including trpc6 [ 11 , 56 ] .
inhibition of calcium channels and chelation of extracellular calcium reduced protamine sulfate - induced damage , suggesting that calcium signaling plays a critical role in the initial stages of glomerular injury .
calcineurin inhibitors ( fk506 and csa ) inhibited protamine sulfate - mediated barrier changes , indicating that calcium signaling acts , in part , through calcineurin - dependent cleavage of synaptopodin . meanwhile , mutations in trpc6 enhance the amplitude and duration of the ca channel current which cause nfat activation , indicating the activation of the calcineurin - nfat pathway as a potential mediator of fsgs .
importantly , the study of wang et al .   demonstrated that , in parallel to synaptopodin regulation , there may be an additional pathway from calcineurin to podocyte injury and proteinuria that involves nfat - mediated regulation of known and novel factors important for podocyte function . to study the role of nfat signaling in glomerular podocytes , wang et al .  
created a system for inducible activation of nfat signaling in podocytes , in which a podocin - cre transgene was used to induce the removal of the transcriptional stop cassette in a rosa26-rtta allele only in podocytes [ 59 , 60 ] . and when the cassette is deleted , the rosa26 promoter drives the production of reverse tetracycline - controlled transactivator ( rtta ) in podocytes . when treated with doxycycline , the doxycycline - rtta complex binds to the teto sequence of the teto - nfatc1 transgene .
the mice carrying three alleles ( podocin - cre , ro - sa26-rtta , and teto - nfatc1 ) were referred as mutants .
thus , results from wang et al .   provided in vivo evidence that nfat activation , either in utero or postdevelopmentally , can lead to podocyte injury and proteinuria , which suggest that activation of nfat signaling may be a key pathogenic molecular change in podocyte injury and the development of proteinuria . using a similar model for conditional nfat activation in podocytes , nijenhuis et al .  
demonstrated that podocyte - specific inducible expression of a constitutively active nfat mutant increased trpc6 expression and induced severe proteinuria , and that calcineurin inhibition by csa downregulated trpc6 expression and reduced proteinuria .
importantly , this study showed that a deleterious feed - forward mechanism , in which trpc6-mediated ca influx stimulates nfat - dependent trpc6 expression , is involved in angiotensin ii ( ang ii)-associated podocyte injury . in vitro and in vivo models , ang ii ,
a key contributor to the pathogenesis of glomerular disease , increases trpc6 expression in podocytes .
the regulation of trpc6 expression by angii is dependent on trpc6-mediated ca influx and the activation of the ca - dependent calcineurin / nfat signaling .
tian et al .   showed that ang ii resulted in significant reduction in the abundance of synaptopodin , and gene silencing of trpc6 resulted in loss of synaptopodin in podocytes in contrast , gene silencing of trpc5 did not affect synaptopodin abundance .
trpc6-depleted cells treated with csa restored synaptopodin abundance , suggesting an association between ca influx through trpc5 and trpc6 channels and synaptopodin signaling in podocytes .
.   showed that that all three trpc6 mutations ( p112q , r895c , and e897k ) to enhance channel activity lead to enhanced basal nfat - mediated transcription in cultured podocytes , which are dependent on channel activity and are dominant when mutants are coexpressed with wild - type trpc6 .
activation of nfat by trpc6 mutants is blocked by inhibitors of calcineurin , calmodulin - dependent kinase ii , and phosphatidylinositol 3-kinase .
in addition , another study showed that calcineurin induces podocyte apoptosis in a genetic model of type 1 diabetes mellitus ( akita mice ) . in cultured podocytes , activated nfat promotes podocyte apoptosis in a calcineurin - dependent fashion , and induction of apoptosis by either angiotensin ii or endothelin-1
in other cell populations , nfatc1 activation leads to distinctive changes in transcription and cellular behavior [ 6365 ] .
interestingly , rcan1 , wnt6 , and fzd9 were shown to be upregulated in glomeruli isolated from nfatc1 
 nuc 
  transgenic mice , making them potentially direct targets of nfatc1 .
the upregulation of rcan1 , a known target of nfat [ 66 , 67 ] , may reflect a potential regulation axis of calcineurin , nfat , and rcan1 in podocyte .
moreover , upregulation of wnt signaling was found to be detrimental to podocytes , and wnt signaling was also shown to be upregulated in nfatc1 
 nuc 
  transgenic mice , indicating that the upregulation of wnt signaling seems to be the pathogenesis of nfat activation - induced podocyte injury and fsgs .
however , recently published researches showed that the effect of csa on proteinuria is not dependent on nfat inhibition in t cells , but rather results from its effects on kidney podocytes , including the stabilization of synaptopodin .
moreover , in vivo evidence from nfatc1 
 nuc 
  transgenic mice and akita mice showed nfat activation - induced podocyte apoptosis and injury , and nfat - dependent trpc6 expression , the mutations of which have been shown to be associated with proteinuria and glomerulosclerosis in human patients . in summary , recently , there is the possibility that csa might have a coupled nonimmunological antiproteinuric effect : one as demonstrated by faul et al .
on synaptopodin and the podocyte cytoskeleton , another separate one through the inhibition of nfat as nfat induces proteinuria and podocyte apoptosis ( figure 1 ) .
 the antiproteinuric effect of cyclosporine a(csa ) has been believed to result from its immunosuppressive effect on the transcription factor nfat in t cells . 
 however , current evidences supporting this hypothesis are missing . 
 a recent study showed that csa has a direct antiproteinuric effect on podocytes , suggesting a novel non - immunosuppressive mechanism for csa 's antiproteinuric effect . 
 conditional nfatc1 activation in podoyctes per se is sufficient to induce proteinuria in mice , indicating that nfat activation in podocytes is a critical pathogenic molecular event leading to podocyte injury and proteinuria . 
 meanwhile , evidence showed that trpc6-mediated ca2 + influx stimulates nfat - dependent trpc6 expression . 
 altogether , these advances in podocyte research indicate that calcineurin - nfat signal or calcineurin - synaptopodin axis has a direct proteinuric effect on podocytes which raises the possibility of developing specific antiproteinuric drugs that lack the unwanted effects of calcineurin or nfat inhibition . 
1. Introduction
2. Proteinuria and Its Molecular Mechanisms
3. The Role of Calcineurin Signal in Podocyte
4. Conclusion