Calcineurin is activated in diabetes and is required forglomerular hypertrophy - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Jennifer L.Gooch, Jeffrey L.Barnes, SergioGarcia,  Hanna E.Abboud,作者单位：1 South Texas Veterans Health Care Administration,Audie Murphy Memorial Hospital, and RenalDivision, University of Texas Health Science Center, San Antonio, Texas78284-3900 , c5 {$ `4 u. _. p) d) L5 i6 ]5 e1 E
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      【摘要】" E0 B& w0 @; Q+ H
   Diabetic nephropathy ischaracterized by the rapid onset of hypertrophy and ECM expansion.Previously, we showed that calcineurin phosphatase is required forhypertrophy and ECM synthesis in cultured mesangial cells. Therefore,we examined the effect of calcineurin inhibition on renal hypertrophyand ECM accumulation in streptozotocin-induced diabetic rats. After 2 wk of diabetes, calcineurin protein was increased in whole cortex andglomeruli in conjunction with increased phosphatase activity. Dailyadministration of cyclosporin A blocked accumulation of bothcalcineurin protein and calcineurin activity. Also associated withcalcineurin upregulation was nuclear localization of the calcineurinsubstrate NFATc1. Inhibition of calcineurin reduced whole kidneyhypertrophy and abolished glomerular hypertrophy in diabetic rats.Furthermore, calcineurin inhibition substantially reduced ECMaccumulation in diabetic glomeruli but not in cortical tissue,suggesting a differential effect of calcineurin inhibition inglomerular vs. extraglomerular tissue. Corresponding increases infibronectin mRNA and transforming growth factor- mRNA were observedin tubulointerstitium but not in glomeruli. In summary, calcineurinplays an important role in glomerular hypertrophy and ECM accumulationin diabetic nephropathy. , b5 N( L# p6 I/ k5 u
      【关键词】 extracellular matrix kidney cyclosporin A mesangialcells" e* D) L6 w( d8 I
   INTRODUCTION

RENAL CELL HYPERTROPHY AND ECM expansion are controlled by a variety ofhormones, cytokines, and peptide growth factors. In diabetes, elevatedblood glucose concentration and factors such as transforming growthfactor- 1 (TGF- 1), angiotensin II, and insulin-like growthfactor-I (IGF-I) contribute to the development and maintenance of renalhypertrophy and matrix expansion ( 1, 39 ). Our laboratoryand others have shown that IGF-I mediates hypertrophy and induces ECMaccumulation in cultured cells ( 15, 28, 30 ). In addition,we have shown that although IGF-I activates well-known signalingpathways such as Erk1/Erk2 MAPK and PI-3 kinase, neither of thesesignaling mechanisms appears to be required for hypertrophy oraccumulation of ECM proteins ( 15 ). Instead, IGF-I-mediatedhypertrophy and ECM synthesis requires activation of thecalcium-dependent, serine/threonine phosphatase calcineurin. Inaddition, we found that calcineurin is also required for ECM synthesisby TGF- ( 14 ), suggesting that calcineurin may be acommon signaling mechanism for multiple stimuli that regulate hypertrophy and/or ECM accumulation in glomeruli.
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Calcineurin is a serine/threonine phosphatase whose activation requiresincreased availability of intracellular calcium. In response to avariety of stimuli, calcineurin binds calmodulin and calcium, resultingin enhanced phosphatase activity ( 29 ). Targets ofcalcineurin dephosphorylation include transcription factors, such asmembers of the nuclear factors of activated T cell (NFAT) family,myocyte enhancer-binding factors, and GATA proteins. In particular,NFATc1 and NFATc2 are activated by IGF-I and implicated in thehypertrophic effects of calcineurin ( 15, 27, 31 ). Afterdephosphorylation, these factors undergo translocation from thecytoplasm to the nucleus, bind other transcription factors includingAP-1 components fos and jun, and modulate transcription of target genes( 4, 6 ). Calcineurin has been best characterized as amediator of T cell signal transduction, and inhibition of calcineurinwith the drug cyclosporin A (CsA) successfully suppresses Tcell-mediated immunity.
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There are indications that calcineurin is expressed in the kidney andmay be important in certain aspects of renal physiology. Severalinvestigators have reported expression of calcineurin mRNA, primarilyin the medulla but also in the cortex of the kidney ( 5, 25 ). Furthermore, calcineurin protein has been detected in themedulla as well as in dissected proximal tubule epithelial cells andcortical collecting ducts ( 36 ). Calcineurin phosphatase activity corresponds to expression of calcineurin protein in renal structures ( 36 ). In addition to calcineurin expression,there is evidence that calcineurin is involved in kidney function.Aperia et al. ( 2 ) report that calcineurin is involved inthe regulation of Na-K-ATPase activity in response to angiotensin II incultured proximal tubule cells. Adding to potential functions ofcalcineurin in the kidney, data from our laboratory implicatecalcineurin and NFATc1 in IGF-I signaling and mesangial cellhypertrophy in vitro ( 15 ). However, the role ofcalcineurin and calcineurin substrates such as NFAT in diabetic renaland specifically glomerular cell hypertrophy remains unclear.0 h3 V8 d: E% a  i( }# ?  l5 o
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Our laboratory has previously shown that activation of theserine-threonine phosphatase calcineurin is required for IGF-I-induced hypertrophy of mesangial cells in vitro ( 15 ). Becausehypertrophy, ECM expansion, and increased expression of renal IGF-I areearly consequences of hyperglycemia, we were interested in the role ofcalcineurin in an in vivo model of diabetes. Therefore, the purpose ofthis study was to examine calcineurin phosphatase activity in thekidney and the effect of calcineurin inhibition on the development ofrenal and glomerular hypertrophy and ECM accumulation in streptozotocin(STZ)-induced diabetic rats.

MATERIALS AND METHODS, }' B" f) m0 ]8 P6 N8 @

Materials. STZ and actin and fibronectin antibodies were obtained from Sigma, CsAfrom Novartis, calcineurin and collagen type IV antibodies fromChemicon, and NFATc1 antibody from Santa Cruz.) O% o& Q6 l: \1 b8 B

Animal protocol. Male Sprague-Dawley rats weighing between 200 and 250 g weredivided into four groups of 4-6 rats/group. Group 1 wasinjected intravenously via the tail vein with 65 mg/kg body wt STZ insodium citrate buffer (pH 4.0) to induce diabetes. Group 2 was similarly injected with sodium citrate buffer alone. Rats in group 3 were injected with STZ and were also given dailysubcutaneous injections of CsA (5 mg/kg body wt) beginning at the timeof STZ injection. Group 4 received daily subcutaneousinjections of vehicle alone (10% ethanol). Blood glucoseconcentrations were monitored by using a LifeScan One Touch glucometer(Johnson & Johnson) 24 h later to verify hyperglycemia andperiodically thereafter. All rats had unrestricted access to food andwater and were maintained in accordance with Institutional Animal Careand Use Committee procedures.( m) O0 g5 N4 I& l$ F

To inhibit calcineurin activity, CsA was utilized. CsA is a chemicalinhibitor with high specificity for calcineurin. CsA doses have beencarefully examined in rats with regard to nephrotoxicity and serumconcentrations ( 26, 33 ). Signs of chronic nephrotoxicity 10 mg/kg body wt administered for atleast 2 wk. Therefore, for our experiments, we chose a dose of 5 mg/kgbody wt CsA to minimize possible nephrotoxicity.' U+ g# I; a  w
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Rats were euthanasized, and both kidneys were removed and weighed. Aslice of kidney cortex at the pole was embedded in paraffin orflash frozen in liquid nitrogen for preparation of sections for lightmicroscopy and image analyses. In addition, cortical sections from twokidneys from different animals within the treatment groups were pooledfor isolation of glomeruli by differential sieving as described( 32 ), and samples of cortical tissue were frozen forbiochemical analyses.; T( m* _) m5 Z: K
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Calcineurin phosphatase assay. Calcineurin phosphatase activity in renal cortex was determinedfollowing a protocol published by Fruman et al. ( 13 ).Briefly, the calcineurin-specific substrate RII was phosphorylated invitro with 250 U recombinant PKA, 50 mM ATP, 50 µCi[ 32 -P]ATP, 0.15 mM RII peptide, and 500 µl of 2×reaction buffer [(in mM) 40 MOPS, 4 MgCl 2, 0.1 CaCl 2, 0.4 EDTA, 0.8 EGTA, and 0.5 DTT, as well as 0.1 mg/ml BSA and 0.2 µg/µl recombinant calmodulin]. Lysates wereprepared by homogenizing cortex sections in a hypotonic lysis buffer[(in mM) 50 Tris, pH 7.5, 1 EDTA, 1 EGTA, and 0.5 DTT, and (inµg/ml) 50 PMSF, 10 leupeptin, 10 aprotinin] followed by four cyclesof freeze-thawing in liquid nitrogen and a 30°C water bath.Calcineurin activity in each sample was determined by incubating equalparts lysate, 3× reaction buffer [(in mM) 40 Tris, pH 7.5, 6 MgCl 2, 0.1 CaCl 2, and 0.5 DTT, (in nM) 500 okadaic acid and 100 calmodulin, as well as 0.1 mg/ml BSA and 0.1 MNaCl] and labeled RII peptide at 30°C for 10 min. The reaction wasstopped by addition of 0.1 M KPO 4 in 5% TCA. To determine the amount of phosphate released by calcineurin in each sample, reactions were then added to PolyPrep columns (Bio-Rad, Hercules, CA)containing AG-50× Dowex ion exchange resin (Bio-Rad) prepared asdescribed ( 13 ). Finally, 5 ml scintillation fluid wereadded to the flow-through from each column, and the released phosphate was measured in a scintillation counter.
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Western blotting. Homogenized renal cortex or isolated glomeruli were resuspended inhypotonic lysis buffer [(in mM) 50 Tris, pH 7.5, 1 EDTA, 1 EGTA, and0.5 DTT, and (in µg/ml) 50 PMSF, 10 leupeptin, and 10 aprotinin],and then protein lysates were collected by four rounds offreeze-thawing in liquid nitrogen and a 30°C water bath followed bycentrifugation at 14,000 g for 30 min at 4°C. Twenty-five micrograms of protein were analyzed by SDS-PAGE. After transfer of theproteins to nitrocellulose, the membrane was incubated in 5% milk-TBST(20 mM Tris · HCl, pH 7.6, 137 mM NaCl, 0.1%Tween 20) and then immunoblotted with 1:2,000 dilutions ofanti-fibronectin or actin antibodies and 1:1,000 dilutions ofanti-collagen type IV or calcineurin antibodies. Horseradishperoxidase-conjugated secondary antibodies were added at 1:2,000, andproteins were visualized by enhanced chemiluminescence (Pierce,Rockford, IL).

Determination of glomerular surface area. Light microscopy of hematoxylin- and eosin-stained sections fromthe different treatment groups was used for morphometric studies. Thesurface area (µm) of a minimum of 50 glomerular sections from eachanimal was determined by using Image-Pro Plus software.
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Immunohistochemistry. Paraffin-embedded tissue sections (5-µM thick) were prepared bydewaxing and unmasking. After incubation with specific primary andbiotin-conjugated secondary antibodies, calcineurin and NFATc1 wereidentified by immunoperoxidase ABC staining following the manufacturer's instructions (Vector Laboratories). Coverslips weremounted with Crystal Mount (Biomeda, Foster City, CA), and sectionswere viewed by brightfield microscopy.
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In situ hybridization. Synthesis of riboprobe, tissue preparation, in situhybridization, and autoradiography were identical to methods previously described ( 3 ). Briefly, fragments of fibronectin andTGF- cDNAs cloned into pGEM vector were used for generation of 35 S-labeled riboprobes to detect cellular localization ofmRNA in sections of renal tissue. All experiments were performedsimultaneously with the sense riboprobe as a negative control.Semiquantitative analyses were made by using Image-Pro Plus software.The relative intensity of signal from a minimum of 10 glomeruli from 3 animals/group was determined. Also, relative intensity of arepresentative interstitial field of each animal was determined.0 z2 s' q2 s4 R3 h' G0 V

Renal function. Two weeks after induction of diabetes, animals were placed overnight inmetabolic cages, with unrestricted access to food and water. Urine wascollected, urine flow rate was calculated (µl/min), and nitrogen andcreatinine levels were determined. At the time of death, whole bloodwas collected and serum blood urea nitrogen and serum creatinine levelswere measured. Renal function was determined as the average of nitrogenclearance and creatinine clearance.- [* u! Z( |, ^. `0 [8 z3 \. y1 Z. s
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Statistics. As indicated, two-way ANOVA or Student's t -test was used todetermine statistical significance for experiments with multiple orsingle variables, respectively. For all Student's t -testanalyses, a paired t -test was used and a result wasconsidered significant if P time was used and a result wasconsidered significant if P+ f  T8 w9 s1 ~: E# M: x7 B

RESULTS
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Regulation of calcineurin in the diabetic kidney. To study the role of calcineurin in diabetic renal hypertrophy, type 1 diabetes was induced in male Sprague-Dawley rats by a singleintravenous injection of STZ (65 mg/kg body wt). Control and diabeticrats were treated with CsA (daily subcutaneous injection of 5 mg/kgbody wt) to inhibit calcineurin activity or vehicle alone. Glucoselevels, weight, kidney mass, and kidney/body mass ratio are shown inTable 1.
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Table 1. Glucose level, weight, kidney mass, and kidney/body mass ratio after 14 days of diabetes  _/ O$ [- Y! c# N  Z. q* }- R
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Several studies, including our own, have demonstrated that induction ofcalcineurin phosphatase activity coincides with increased calcineurinprotein levels ( 20, 21, 34 ). In fact, increased proteinlevels may be a reliable indicator of phosphatase activity ( 24 ). Therefore, we examined expression of calcineurin incortical homogenates and isolated glomeruli from control and STZ-,CsA-, and CsA STZ-treated animals. Figure 1, A and B, showsthat there is an increase in calcineurin protein over time withdiabetes in both isolated glomeruli and whole cortex.
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Fig. 1. Calcineurin protein levels are increased over time withdiabetes. Protein levels of calcineurin were determined by directimmunoblotting with specific antibodies. Actin was included as acontrol for loading and the specificity of changes in proteinexpression. A : isolated glomeruli; each lane representsglomerular lysates pooled from 2 animals and is representative of atleast 2 independent experiments. B : same as in A except that each lane represents protein lysates of homogenized cortexfrom a single animal.0 k" \& _+ w/ U* S1 C$ U5 C2 i
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Next, we examined the effect of CsA on protein expression and cellularlocalization of calcineurin. In glomeruli, daily CsA administrationdecreases accumulation of calcineurin associated with diabetes (Fig. 2 A ). In contrast, in corticalhomogenates, increased calcineurin protein associated with STZ diabeteswas not inhibited by CsA treatment (Fig. 2 B ). Byimmunohistochemistry, calcineurin expression was determined to berelatively low in extraglomerular cortex and generally localized to thebasolateral surface of tubules. Calcineurin appears to be expressed ata much lower level in glomeruli (Fig. 2 C, a ). Twoweeks after induction of diabetes, calcineurin expression is markedlyincreased in both extraglomerular cortex and glomeruli, inagreement with calcineurin expression determined by immunoblotting inFig. 1, A and B. Interestingly, increasedcalcineurin is detected in extraglomerular cortex of animals treatedwith CsA alone (Fig. 2 C, c ). Consistent withprotein levels determined by immunoblotting, increased calcineurinexpression associated with diabetes in glomeruli is inhibited by CsAtreatment (Fig. 2 C, d ), whereas expression inextraglomerular cortex remains high compared with control, again inagreement with data in Fig. 2 A.) Z9 ^( @1 k* C) E' c7 K6 ^/ H/ d3 t

Fig. 2. Streptozotocin (STZ)-induced calcineurin protein upregulation isreversed by cyclosporin A (CsA) treatment. A : protein levelsof calcineurin in samples from isolated glomeruli of control, STZ, andSTZ CsA animals after 3 and 14 days of treatment. B :protein levels of calcineurin in samples from cortex homogenates ofcontrol, STZ, and STZ CsA animals after 3 and 14 days of treatment. C : calcineurin expression and cellular localization weredetected by immunoperoxidase staining of kidney sections from control( a ), STZ ( b ), CsA ( c ), and CsA STZ( d ) animals. *, glomeruli.
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Protein extracts were collected from homogenized cortical sections, andcalcineurin activity was determined by an in vitro phosphatase assay( 13 ). After 14 days of diabetes, calcineurin activity isincreased in the renal cortex of diabetic animals compared withcontrol, 539.8 ± 30.1 vs. 3,168 ± 1,214 released counts · min 1 · µgprotein 1 ( P diabetes was blocked by daily CsAinjection, and calcineurin activity in the cortex of control, normalrats was not changed with CsA treatment 626.3 ± 69.9 and543.0 ± 26.8 releasedcounts · min 1 · µgprotein 1, respectively.
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NFATs are one group of proteins that are targets of calcineurindephosphorylation. Our laboratory's previous work showed that CsAtreatment of cultured mesangial cells inhibited IGF-I-mediated hypertrophy and ECM accumulation as well as NFATc1 nuclearlocalization, suggesting that NFATc1 is involved in glomerularhypertrophy and/or ECM regulation ( 15 ). Figures 1 and 2 and our in vitro phosphatase assay demonstrate that calcineurinactivity is increased in the diabetic kidney. Therefore, we examinedNFATc1 localization in the renal cortex. Immunoperoxidase staining incontrol and STZ-, CsA-, and CsA STZ-treated animals demonstrated adramatic increase in NFATc1 protein expression in the renal cortex ofdiabetic animals (Fig. 3 ). In addition,although NFATc1 appears to be localized to the basolateral surface oftubular cells in control animals (Fig. 3, A and E ), NFATc1 can be found in the nucleus in tubules ofdiabetic rat kidneys, consistent with activation of calcineurin phosphatase (compare with Fig. 3, B and F ). Alsoconsistent with inhibition of calcineurin activity, NFATc1 expressionand localization are normalized in diabetic animals treated with CsA.
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Fig. 3. NFATc1 expression and nuclear localization is associatedwith calcineurin activation in the diabetic kidney. NFATc1 expressionand cellular localization were determined in control ( A ) andSTZ- ( B ), CsA- ( C ), and CsA STZ-treated( D ) animals by immunoperoxidase staining with an NFATc1antibody. E and F : increased magnification ofareas outlined in A and B, respectively. *, glomeruli; arrows, nuclear localization of NFATc1.

Inhibition of renal hypertrophy by CsA. Our data show that calcineurin is activated in the diabetic kidney andthat calcineurin phosphatase activity (and corresponding upregulationof calcineurin protein and NFATc1 nuclear localization) can beinhibited by daily administration of CsA. In vitro, calcineurin isinvolved in hypertrophy and ECM accumulation ( 15 );therefore, we examined the effect of calcineurin inhibition in vivo onhypertrophy and ECM accumulation in the diabetic kidney. Whole kidneyhypertrophy (total kidney wt/body mass) was assessed at time points upto 14 days after STZ-induced diabetes. Figure 4 shows that as early as 3 days, there isa statistically significant increase in kidney/body mass ratio in theSTZ-treated animals. Over the course of the experiment, there is asignificant decrease in whole kidney hypertrophy in CsA-treateddiabetic animals (CsA STZ) compared with diabetic animals (STZ alone)( P both 10 and14 days, CsA treatment significantly reduced whole kidney hypertrophy( P t -test). There is nodifference in kidney/body mass ratio between control and CsA-treatedcontrol animals. As summarized in Table 1, the effect of CsA treatment on STZ-induced whole kidney hypertrophy is seen in a trend toward adecrease in total kidney weight as well as an improvement in weightloss at 14 days. The result is a significant decrease in thekidney/body mass ratio due to calcineurin inhibition with CsA.5 B2 |% J4 G3 _8 F' z, u
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Fig. 4. Inhibition of calcineurin with CsA decreases whole kidneyhypertrophy over time. After 1, 3, 10, or 14 days of treatment, animalswere killed and kidneys were removed and weighed. Kidney mass wasdivided by body weight and is expressed as percent body weight. Valuesare means ± SE of 4-8 animals. ** P P t -test.0 j5 V, K6 n+ [) `

The effect of calcineurin inhibition was also determined ondiabetes-induced glomerular hypertrophy. Figure 5 shows that after 14 days of treatment,glomeruli of STZ animals are significantly larger than control orCsA-treated control animals ( P Student's t -test). Glomeruli of CsA STZ animals are significantly smaller than STZ animals ( P t -test) and are not different from glomeruli of controlanimals. CsA alone had no effect on the size of glomeruli of normalrats. No evidence of ischemia was observed in either group ofanimals treated with CsA, and normal arterioles were identified in allgroups.

Fig. 5. Inhibition of calcineurin blocks glomerular hypertrophy.Glomerular area was determined by microscopic examination ofhematoxylin- and eosin-stained paraffin-embedded cortex sections. Theareas of a minimum of 50 glomeruli/section were determined by usingImage-Pro software. A : representative glomeruli from eachtreatment group. Arrows, normal arterioles in CsA and CsA STZanimals. * P t -test). ** P t -test). B : each bar represents themean ± SE of glomerular area from 4-6 animals." H  e) m- R( Z/ x' H8 z
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Next, we determined the effect of calcineurin inhibition on ECMaccumulation in glomeruli and whole cortex. Isolated glomeruli fromSTZ-treated animals demonstrated increased accumulation of the ECMproteins fibronectin and collagen type IV compared with controlanimals (Fig. 6 A ). Inhibitionof calcineurin resulted in decreased accumulation of these proteins inglomeruli of CsA STZ animals compared with STZ animals. CsA treatmentalone has no effect on expression of ECM proteins compared withcontrols. Figure 6 B shows that ECM proteins are alsoincreased with STZ treatment in whole cortex. In contrast to thefinding in glomeruli, expression of fibronectin and collagen type IV isincreased in cortex homogenates of animals treated with CsA alone andinhibition of calcineurin with CsA does not decrease diabetes-inducedECM accumulation., G& Q& I  d$ u* M- i0 e
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Fig. 6. Inhibition of calcineurin decreases accumulation of ECMin glomeruli but increases ECM accumulation in the cortex. A : total protein was collected from isolated glomeruli fromcontrol, STZ, CsA, and CsA STZ animals. Fibronectin and collagen typeIV proteins were detected by direct immunoblotting. Each lane containsprotein from isolated glomeruli pooled from 2 animals. Lanes1 and 2 : control animals. Lanes 3 and 4 : STZ-treated animals. Lanes 5-7 :CsA-treated animals. Lanes 8-10 : CsA STZ animals. B : total protein was collected from homogenized corticalsections from STZ, CsA, and CsA STZ animals. Fibronectin and collagentype IV proteins were detected by direct immunoblotting. Lanes1 and 2 : control animals. Lanes 3 and 4 : STZ-treated animals. Lanes 5-7 :CsA-treated animals. Lanes 8-10 : CsA STZ animals.( C% A7 Y6 s; E! ^  T# w3 t9 @

CsA increases transcription of fibronectin in cultured proximal tubulecells ( 37 ). However, it is not clear what effect CsA hason fibronectin mRNA in glomerular cells or what role calcineurin mayplay in regulation of fibronectin associated with diabetes. Therefore,we examined fibronectin mRNA in both glomeruli and cortex of controland STZ-, CsA-, and CsA STZ-treated animals by in situ hybridization.Fibronectin mRNA was upregulated by diabetes in both glomeruli andcortex (Fig. 7, b and h compared with a and g ). Animalstreated with CsA showed increased fibronectin mRNA in the cortex butnot in glomeruli (Fig. 7, i and c, respectively). Finally, diabetes-induced fibronectin mRNA was reduced in glomeruli butnot in the cortex (Fig. 7, d and j,respectively).7 \5 `( u$ v( z& w( A5 [

Fig. 7. Fibronectin mRNA is upregulated in both glomeruli andcortex of STZ animals and in cortex of CsA animals. A :fibronectin mRNA was detected in glomeruli of control ( a ),STZ ( b ), CsA ( c ), and CsA STZ ( d )animals and in cortex of control ( g ), STZ ( h ),CsA ( i ), and CsA STZ ( j ) animals by in situhybridization. Hybridization with sense probe is shown as a negativecontrol in glomeruli ( e ) and in cortex( f ). B : semiquantitation of datashown in A. Values are representative of 10 glomeruli and 10 cortical fields from 3 animals/group. * P' o" z' Z. n6 y2 N
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One mechanism for regulation of fibronectin in renal cells isupregulation of TGF-. Therefore, in Fig. 8, we examined TGF- mRNA expression byin situ hybrizidation. Similar to fibronectin regulation in diabeticanimals, TGF- mRNA was increased in both glomeruli and cortex ofSTZ-treated animals (Fig. 8, b and h compared with a and g ). Also consistent with increasedfibronectin mRNA, TGF- was upregulated in the cortex but notglomeruli of animals treated with CsA alone. Finally, TGF- mRNA wasreduced by calcineurin inhibition in glomeruli but was furtherincreased in the cortex of diabetic animals (Fig. 8, d vs. h ).( M# X: P* s& J$ ~. |; e

Fig. 8. Transforming growth factor- (TGF- ) mRNA isupregulated in both glomeruli and cortex of STZ animals and in cortexof CsA animals. A : TGF- mRNA was detected in glomeruli ofcontrol ( a ), STZ ( b ), CsA ( c ), andCsA STZ ( d ) animals and in cortex of control( g ), STZ ( h ), CsA ( i ), and CsA STZ( j ) animals by in situ hybridization. Hybridization withsense probe is shown as a negative control in glomeruli ( e )and in cortex ( f ). B :semiquantitation of data shown in A. Data are representativeof 10 glomeruli and 10 cortical fields from 3 animals/group.* P

Effect of CsA on STZ-induced diabetic rats. To verify that the dose of CsA administered daily was sufficient toachieve therapeutic and nontoxic circulating levels, serum CsA levelswere measured in animals receiving only CsA and in diabetic animalsreceiving CsA at multiple time points from 1 to 14 days. Administrationof CsA alone resulted in a mean level of 797.9 ± 98.3 ng/ml overa course of 2 wk, whereas CsA STZ animals had serum levels of445.6 ± 50.1 ng/ml, a statistically significant difference( P difference inmean circulating levels, both treatment groups had CsA levelsconsiderably below what has been described to induce adverseside-effects, including nephrotoxicity ( 16, 26, 33 ). Furthermore, we did not find evidence of nephrotoxicity in any of thegroups when cortical sections were examined by light microscopy.

Finally, glucose concentrations, body weight, and glomerular filtrationrates (GFRs) were examined. STZ-treated animals exhibited an increasein serum glucose over 14 days, with a combined mean of 371.6 ± 15.4 mg/dl (Fig. 9 A ). Diabeticanimals treated with CsA had a slightly lower mean glucose level of343.3 ± 10.4, which is significantly different over time from STZalone ( P two-way ANOVA). In addition, thereis a statistically significant difference in the glucose levels of CsA STZ animals at day 14 compared with STZ alone( P t -test). There is nosignificant difference in the mean glucose levels between controls andcontrol animals treated with CsA alone (69.4 ± 1.4 and 72.0 ± 1.4 mg/dl, respectively), indicating that CsA does not reduce bloodglucose concentration in the absence of diabetes.
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Fig. 9. Effect of CsA treatment on serum CsA level, blood glucoseconcentration, body weight, and glomerular filtration rate (GFR). A : serum CsA levels were determined at various time pointsin CsA- and CsA STZ-treated animals. Values are serum levels fromindividual animals. * P B : serum glucose levels were determined atmultiple time points up to 14 days after induction of diabetes. Valuesare means ± SE of 5-10 animals. ** P P t -test). C : body weight was determined atmultiple time points up to 14 days after induction of diabetes. Valuesare means ± SE of 5-10 animals. ** P P t -test). D : glomerular filtration rates weredetermined by calculating the mean of nitrogen clearance and creatinineclearance per gram body wt. * P

Interestingly, diabetic animals treated with CsA showed a significantimprovement in weight loss over time compared with STZ animals( P 9 B ). Thetime course of improved weight loss corresponds to a similar time frameas the reduction in glucose levels in CsA STZ-treated animals. Administration of CsA alone resulted in slower weight gain over the2-wk period than in control animals. This is consistent with otherreports that CsA administration is associated with anorexia ( 17, 21 ).0 I# {0 s+ j/ H' s3 t2 J

Prolonged CsA treatment is frequently associated with nephrotoxicity,particularly glomerular ischemia and impaired filtration. Toassess the effect of CsA treatment on renal function, GFR was determined as the mean of nitrogen clearance and creatinine clearance per gram body weight (Fig. 9 C ). As expected, diabeticanimals have a slightly increased GFR [ P (Student's t -test)]. The GFR ofanimals treated with CsA alone was not different from control.However, the mean GFR of CsA STZ-treated animals is slightly higherthan STZ-treated animals [ P t -test)].
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DISCUSSION' ]7 n* e2 E8 v# F: d

Whole kidney, including glomerular cell, hypertrophy are earlymanifestations of diabetic nephropathy. Previous work in our laboratoryhas demonstrated that calcineurin is an important signaling mediator ofmesangial cell hypertrophy and ECM accumulation in vitro( 14 ). However, calcineurin activity has never beenexamined in the diabetic kidney, and the role of calcineurin indiabetic glomerular hypertrophy is unknown. In this study, we show for the first time that calcineurin phosphatase is activated in the renalcortex of diabetic rats. Furthermore, we show that increased expressionof calcineurin is associated with nuclear localization of a targettranscription factor, NFATc1, suggesting a possible transcriptionalmechanism for calcineurin action. Our work demonstrates that inhibitionof calcineurin with CsA reduces whole kidney hypertrophy and completelyblocks glomerular hypertrophy and ECM accumulation. These resultssuggest that calcineurin is a mediator of diabetic hypertrophy and ECMregulation in glomeruli in vivo.! S) |4 Y0 N# [( D& W( M8 G

We show that calcineurin protein levels are increased 3 days afterinduction of diabetes and that calcineurin is activated in the renalcortex of diabetic rats after 2 wk of hyperglycemia. Coincident withthis early time frame of disease is the increase in expression ofpolypeptide growth factors such as TGF- and IGF-I in the kidney( 1, 11, 12 ). We have shown in vitro that IGF-I induceshypertrophy and ECM accumulation in mesangial cells via calcineurin( 15 ). Therefore, the increase in calcineurin protein andthe stimulation of calcineurin activity may be due to increasedavailability of these, or other, peptide factors. The increased proteinlevels of calcineurin in the glomerulus and activation of calcineurinin the renal cortex are inhibited by treatment with CsA. Additionalevidence of calcineurin activation in vivo is provided by increasedexpression and nuclear localization of the calcineurin substrate NFATc1in the renal cortex of diabetic animals. NFATc1 regulation is clearlydependent on calcineurin activity, because treatment of animals withCsA abolished changes in both protein expression and nuclear localization.
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Of major interest is that unlike its effects in glomeruli to decreaseECM expansion, CsA treatment alone increased both calcineurin proteinlevels and ECM accumulation in extraglomerular cortex. It is possiblethat binding of CsA/cyclophilin complexes to calcineurin inhibitsnormal turnover of the protein, leading to a subsequent accumulation ofcalcineurin in addition to inhibition of phosphatase activity. However,the mechanism of this differential effect of CsA on calcineurinexpression is unclear. CsA-mediated increases in ECM proteins, however,have been described in several cell types, including corticalfibroblasts ( 19, 37 ), human endothelial and epithelialcells ( 9 ), and proximal tubule cells ( 37 ). Inaddition, CsA treatment has been associated with increased TGF- expression ( 18, 19, 38 ), leading to the hypothesis thatTGF- mediates the subsequent increase in ECM proteins. Our datawould support this model because CsA increased TGF- and fibronectinmRNA levels in the tubulointerstitium. Of considerable interest is thefinding that a similar event does not occur in the glomeruli. The datashow no increase in fibronectin mRNA or protein and no increase inTGF- mRNA with CsA treatment in glomeruli. Furthermore, we havepreviously shown that in cultured mesangial cells, there was noincrease in fibronectin or collagen type IV protein expression inresponse to CsA treatment ( 15 ).

Also of interest is the fact that although animals in our experimentswere administered daily subcutaneous injections of 5 mg/kg body wt CsA,there was a significant difference in the serum levels of CsA achievedin CsA alone and CsA STZ animals. Diabetic animals were found to haveroughly one-half the levels of CsA compared with nondiabetic animals.However, because CsA is at least partially secreted in the urine( 22 ), it is possible that increased clearance observed inCsA STZ- vs. CsA alone-treated animals is responsible for decreasedcirculating levels. It is also possible that STZ or the diabetic statecould activate metabolic pathways, leading to an increased rate ofbreakdown of CsA, and it could accelerate metabolism of the drug withinthe liver (the site of the majority of CsA metabolism). In either case,no toxicity was observed in animals treated with CsA alone, consistentwith reports that nephrotoxicity of this drug within the glomeruli isnot observed when it is administered even at higher concentrations (10 mg/kg body wt) for a similar time period ( 16, 26, 33 ). Next, there was a mild reduction in glucose levels in CsA STZ-treated animals. It is possible that the mild improvement in hyperglycemia andslight increase in body weight contributed to the changes that wereobserved. However, the relatively small differences in glucose levelsand body weight are not sufficient to explain the completenormalization of glomerular hypertrophy. This result suggests a directrole for calcineurin in hypertrophy of cells within the glomeruli.Indeed, our laboratory's previous finding using cultured mesangialcells lends support to this interpretation ( 15 ).Furthermore, a slight decrease in hyperglycemia cannot account for thedisparity in ECM regulation and TGF- production in glomeruli andextraglomerular cortex observed in animals treated with CsA. Again,these data argue for a specific role of calcineurin in these processes.Finally, although diabetes was associated with an increase in GFR asexpected, inhibition of calcineurin resulted in a further increase inGFR in diabetic animals that was associated with improvements in bothhypertrophy and ECM accumulation.$ J- q5 R5 G4 V) ~0 ~8 o- @& O% C" L

Our finding that calcineurin is important in tissue hypertrophy is inagreement with several other reports in the literature. First, severalgroups have reported that calcineurin is activated in in vitro modelsof cardiomyocyte and skeletal muscle hypertrophy ( 7, 8, 35 ). Furthermore, Semsarian et al. ( 31 ) and Musaro et al. ( 27 ) showed that IGF-I alone is sufficient to bothinduce hypertrophy and activate calcineurin in cultured skeletal muscle cells and cardiomyocytes. There is considerable evidence that calcineurin is activated in several models of cardiac hypertrophy invivo ( 10, 21, 24 ). Inhibition of calcineurin activity withCsA blocked development of cardiac hypertrophy in some models ofhypertrophy ( 10, 20, 34 ). Therefore, in addition to its role in cardiac hypertrophy, we show that calcineurin is also importantfor renal cell hypertrophy and ECM expansion.! ^) X/ L3 V7 b5 Z) p- ^1 P

In conclusion, we show that inhibition of calcineurin with CsA resultsin marked attenuation of glomerular hypertrophy and ECM accumulation,supporting our in vitro finding that calcineurin may be an importantmediator of hypertrophy and/or ECM regulation in mesangial cells. Inaddition, CsA treatment results in a decrease in whole kidneyhypertrophy, suggesting that calcineurin may also play a role inhypertrophy of other renal cells, such as tubular epithelial cells. Ascalcineurin is activated by IGF-I and possibly by other factors such asangiotensin II ( 2 ) in renal cells, this finding could bedue to inhibition of multiple pathways. More needs to be learned aboutthe complex role of calcineurin in the kidney. In addition, inhibitionof calcineurin to improve or preserve kidney function in the diabeticstate is an important area to be examined.- C% g- G0 Y) x( f& s* O

ACKNOWLEDGEMENTS$ A) A8 ^6 q1 ~
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The authors acknowledge Drs. Dan Riley and Robert Kunau for helpfuldiscussion and analyses, Shuko Lee for statistical interpretation ofthe data, and Maria Bunega and Yuping Tang for technical assistance.) Q0 s; u! J% w4 W" q7 g
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