1 South Texas Veterans Health Care Administration, Audie Murphy Memorial Hospital, and 2 Renal Division, University of Texas Health Science Center, San Antonio, Texas 78284-3900
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Diabetic nephropathy is
characterized by the rapid onset of hypertrophy and ECM expansion.
Previously, we showed that calcineurin phosphatase is required for
hypertrophy and ECM synthesis in cultured mesangial cells. Therefore,
we examined the effect of calcineurin inhibition on renal hypertrophy
and ECM accumulation in streptozotocin-induced diabetic rats. After 2 wk of diabetes, calcineurin protein was increased in whole cortex and
glomeruli in conjunction with increased phosphatase activity. Daily
administration of cyclosporin A blocked accumulation of both
calcineurin protein and calcineurin activity. Also associated with
calcineurin upregulation was nuclear localization of the calcineurin
substrate NFATc1. Inhibition of calcineurin reduced whole kidney
hypertrophy and abolished glomerular hypertrophy in diabetic rats.
Furthermore, calcineurin inhibition substantially reduced ECM
accumulation in diabetic glomeruli but not in cortical tissue,
suggesting a differential effect of calcineurin inhibition in
glomerular vs. extraglomerular tissue. Corresponding increases in
fibronectin mRNA and transforming growth factor- mRNA were observed
in tubulointerstitium but not in glomeruli. In summary, calcineurin
plays an important role in glomerular hypertrophy and ECM accumulation
in diabetic nephropathy.
extracellular matrix; kidney; cyclosporin A; mesangial cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RENAL CELL
HYPERTROPHY AND ECM expansion are controlled by a variety of
hormones, cytokines, and peptide growth factors. In diabetes, elevated
blood glucose concentration and factors such as transforming growth
factor-1 (TGF-
1), angiotensin II, and insulin-like growth
factor-I (IGF-I) contribute to the development and maintenance of renal
hypertrophy and matrix expansion (1, 39). Our laboratory
and others have shown that IGF-I mediates hypertrophy and induces ECM
accumulation in cultured cells (15, 28, 30). In addition,
we have shown that although IGF-I activates well-known signaling
pathways such as Erk1/Erk2 MAPK and PI-3 kinase, neither of these
signaling mechanisms appears to be required for hypertrophy or
accumulation of ECM proteins (15). Instead, IGF-I-mediated
hypertrophy and ECM synthesis requires activation of the
calcium-dependent, serine/threonine phosphatase calcineurin. In
addition, we found that calcineurin is also required for ECM synthesis
by TGF-
(14), suggesting that calcineurin may be a
common signaling mechanism for multiple stimuli that regulate hypertrophy and/or ECM accumulation in glomeruli.
Calcineurin is a serine/threonine phosphatase whose activation requires increased availability of intracellular calcium. In response to a variety of stimuli, calcineurin binds calmodulin and calcium, resulting in enhanced phosphatase activity (29). Targets of calcineurin dephosphorylation include transcription factors, such as members 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 the hypertrophic effects of calcineurin (15, 27, 31). After dephosphorylation, these factors undergo translocation from the cytoplasm to the nucleus, bind other transcription factors including AP-1 components fos and jun, and modulate transcription of target genes (4, 6). Calcineurin has been best characterized as a mediator of T cell signal transduction, and inhibition of calcineurin with the drug cyclosporin A (CsA) successfully suppresses T cell-mediated immunity.
There are indications that calcineurin is expressed in the kidney and may be important in certain aspects of renal physiology. Several investigators have reported expression of calcineurin mRNA, primarily in the medulla but also in the cortex of the kidney (5, 25). Furthermore, calcineurin protein has been detected in the medulla as well as in dissected proximal tubule epithelial cells and cortical 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 in the regulation of Na-K-ATPase activity in response to angiotensin II in cultured proximal tubule cells. Adding to potential functions of calcineurin in the kidney, data from our laboratory implicate calcineurin and NFATc1 in IGF-I signaling and mesangial cell hypertrophy in vitro (15). However, the role of calcineurin and calcineurin substrates such as NFAT in diabetic renal and specifically glomerular cell hypertrophy remains unclear.
Our laboratory has previously shown that activation of the serine-threonine phosphatase calcineurin is required for IGF-I-induced hypertrophy of mesangial cells in vitro (15). Because hypertrophy, ECM expansion, and increased expression of renal IGF-I are early consequences of hyperglycemia, we were interested in the role of calcineurin in an in vivo model of diabetes. Therefore, the purpose of this study was to examine calcineurin phosphatase activity in the kidney and the effect of calcineurin inhibition on the development of renal and glomerular hypertrophy and ECM accumulation in streptozotocin (STZ)-induced diabetic rats.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. STZ and actin and fibronectin antibodies were obtained from Sigma, CsA from Novartis, calcineurin and collagen type IV antibodies from Chemicon, and NFATc1 antibody from Santa Cruz.
Animal protocol. Male Sprague-Dawley rats weighing between 200 and 250 g were divided into four groups of 4-6 rats/group. Group 1 was injected intravenously via the tail vein with 65 mg/kg body wt STZ in sodium 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 daily subcutaneous injections of CsA (5 mg/kg body wt) beginning at the time of STZ injection. Group 4 received daily subcutaneous injections of vehicle alone (10% ethanol). Blood glucose concentrations were monitored by using a LifeScan One Touch glucometer (Johnson & Johnson) 24 h later to verify hyperglycemia and periodically thereafter. All rats had unrestricted access to food and water and were maintained in accordance with Institutional Animal Care and Use Committee procedures.
To inhibit calcineurin activity, CsA was utilized. CsA is a chemical inhibitor with high specificity for calcineurin. CsA doses have been carefully examined in rats with regard to nephrotoxicity and serum concentrations (26, 33). Signs of chronic nephrotoxicity were observed at doses of CsA >10 mg/kg body wt administered for at least 2 wk. Therefore, for our experiments, we chose a dose of 5 mg/kg body wt CsA to minimize possible nephrotoxicity. Rats were euthanasized, and both kidneys were removed and weighed. A slice of kidney cortex at the pole was embedded in paraffin or flash frozen in liquid nitrogen for preparation of sections for light microscopy and image analyses. In addition, cortical sections from two kidneys from different animals within the treatment groups were pooled for isolation of glomeruli by differential sieving as described (32), and samples of cortical tissue were frozen for biochemical analyses.Calcineurin phosphatase assay.
Calcineurin phosphatase activity in renal cortex was determined
following a protocol published by Fruman et al. (13).
Briefly, the calcineurin-specific substrate RII was phosphorylated in
vitro 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 MgCl2, 0.1 CaCl2, 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 were
prepared 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 (in
µg/ml) 50 PMSF, 10 leupeptin, 10 aprotinin] followed by four cycles
of freeze-thawing in liquid nitrogen and a 30°C water bath.
Calcineurin activity in each sample was determined by incubating equal
parts lysate, 3× reaction buffer [(in mM) 40 Tris, pH 7.5, 6 MgCl2, 0.1 CaCl2, and 0.5 DTT, (in nM) 500 okadaic acid and 100 calmodulin, as well as 0.1 mg/ml BSA and 0.1 M
NaCl] and labeled RII peptide at 30°C for 10 min. The reaction was
stopped by addition of 0.1 M KPO4 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 as
described (13). Finally, 5 ml scintillation fluid were
added to the flow-through from each column, and the released phosphate was measured in a scintillation counter.
Western blotting. Homogenized renal cortex or isolated glomeruli were resuspended in hypotonic lysis buffer [(in mM) 50 Tris, pH 7.5, 1 EDTA, 1 EGTA, and 0.5 DTT, and (in µg/ml) 50 PMSF, 10 leupeptin, and 10 aprotinin], and then protein lysates were collected by four rounds of freeze-thawing in liquid nitrogen and a 30°C water bath followed by centrifugation at 14,000 g for 30 min at 4°C. Twenty-five micrograms of protein were analyzed by SDS-PAGE. After transfer of the proteins 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 of anti-fibronectin or actin antibodies and 1:1,000 dilutions of anti-collagen type IV or calcineurin antibodies. Horseradish peroxidase-conjugated secondary antibodies were added at 1:2,000, and proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL).
Determination of glomerular surface area. Light microscopy of hematoxylin- and eosin-stained sections from the different treatment groups was used for morphometric studies. The surface area (µm) of a minimum of 50 glomerular sections from each animal was determined by using Image-Pro Plus software.
Immunohistochemistry. Paraffin-embedded tissue sections (5-µM thick) were prepared by dewaxing and unmasking. After incubation with specific primary and biotin-conjugated secondary antibodies, calcineurin and NFATc1 were identified by immunoperoxidase ABC staining following the manufacturer's instructions (Vector Laboratories). Coverslips were mounted with Crystal Mount (Biomeda, Foster City, CA), and sections were viewed by brightfield microscopy.
In situ hybridization.
Synthesis of riboprobe, tissue preparation, in situ
hybridization, and autoradiography were identical to methods previously described (3). Briefly, fragments of fibronectin and
TGF- cDNAs cloned into pGEM vector were used for generation of
35S-labeled riboprobes to detect cellular localization of
mRNA in sections of renal tissue. All experiments were performed
simultaneously 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 a
representative interstitial field of each animal was determined.
Renal function. Two weeks after induction of diabetes, animals were placed overnight in metabolic cages, with unrestricted access to food and water. Urine was collected, urine flow rate was calculated (µl/min), and nitrogen and creatinine levels were determined. At the time of death, whole blood was collected and serum blood urea nitrogen and serum creatinine levels were measured. Renal function was determined as the average of nitrogen clearance and creatinine clearance.
Statistics. As indicated, two-way ANOVA or Student's t-test was used to determine statistical significance for experiments with multiple or single variables, respectively. For all Student's t-test analyses, a paired t-test was used and a result was considered significant if P < 0.05. For all analyses using two-way ANOVA, treatment vs. time was used and a result was considered significant if P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 single
intravenous injection of STZ (65 mg/kg body wt). Control and diabetic
rats were treated with CsA (daily subcutaneous injection of 5 mg/kg
body wt) to inhibit calcineurin activity or vehicle alone. Glucose
levels, weight, kidney mass, and kidney/body mass ratio are shown in
Table 1.
|
|
|
|
Inhibition of renal hypertrophy by CsA.
Our data show that calcineurin is activated in the diabetic kidney and
that calcineurin phosphatase activity (and corresponding upregulation
of calcineurin protein and NFATc1 nuclear localization) can be
inhibited by daily administration of CsA. In vitro, calcineurin is
involved in hypertrophy and ECM accumulation (15);
therefore, we examined the effect of calcineurin inhibition in vivo on
hypertrophy and ECM accumulation in the diabetic kidney. Whole kidney
hypertrophy (total kidney wt/body mass) was assessed at time points up
to 14 days after STZ-induced diabetes. Figure
4 shows that as early as 3 days, there is
a statistically significant increase in kidney/body mass ratio in the
STZ-treated animals. Over the course of the experiment, there is a
significant decrease in whole kidney hypertrophy in CsA-treated
diabetic animals (CsA + STZ) compared with diabetic animals (STZ alone)
(P < 0.01, two-way ANOVA). In addition, at both 10 and
14 days, CsA treatment significantly reduced whole kidney hypertrophy
(P < 0.05, Student's t-test). There is no
difference in kidney/body mass ratio between control and CsA-treated
control animals. As summarized in Table 1, the effect of CsA treatment on STZ-induced whole kidney hypertrophy is seen in a trend toward a
decrease in total kidney weight as well as an improvement in weight
loss at 14 days. The result is a significant decrease in the
kidney/body mass ratio due to calcineurin inhibition with CsA.
|
|
|
|
|
Effect of CsA on STZ-induced diabetic rats. To verify that the dose of CsA administered daily was sufficient to achieve therapeutic and nontoxic circulating levels, serum CsA levels were measured in animals receiving only CsA and in diabetic animals receiving CsA at multiple time points from 1 to 14 days. Administration of CsA alone resulted in a mean level of 797.9 ± 98.3 ng/ml over a course of 2 wk, whereas CsA + STZ animals had serum levels of 445.6 ± 50.1 ng/ml, a statistically significant difference (P < 0.005, two-way ANOVA). Despite the difference in mean circulating levels, both treatment groups had CsA levels considerably below what has been described to induce adverse side-effects, including nephrotoxicity (16, 26, 33). Furthermore, we did not find evidence of nephrotoxicity in any of the groups when cortical sections were examined by light microscopy.
Finally, glucose concentrations, body weight, and glomerular filtration rates (GFRs) were examined. STZ-treated animals exhibited an increase in serum glucose over 14 days, with a combined mean of 371.6 ± 15.4 mg/dl (Fig. 9A). Diabetic animals treated with CsA had a slightly lower mean glucose level of 343.3 ± 10.4, which is significantly different over time from STZ alone (P < 0.001, two-way ANOVA). In addition, there is a statistically significant difference in the glucose levels of CsA + STZ animals at day 14 compared with STZ alone (P < 0.05, Student's t-test). There is no significant difference in the mean glucose levels between controls and control animals treated with CsA alone (69.4 ± 1.4 and 72.0 ± 1.4 mg/dl, respectively), indicating that CsA does not reduce blood glucose concentration in the absence of diabetes.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whole kidney, including glomerular cell, hypertrophy are early manifestations of diabetic nephropathy. Previous work in our laboratory has demonstrated that calcineurin is an important signaling mediator of mesangial cell hypertrophy and ECM accumulation in vitro (14). However, calcineurin activity has never been examined in the diabetic kidney, and the role of calcineurin in diabetic glomerular hypertrophy is unknown. In this study, we show for the first time that calcineurin phosphatase is activated in the renal cortex of diabetic rats. Furthermore, we show that increased expression of calcineurin is associated with nuclear localization of a target transcription factor, NFATc1, suggesting a possible transcriptional mechanism for calcineurin action. Our work demonstrates that inhibition of calcineurin with CsA reduces whole kidney hypertrophy and completely blocks glomerular hypertrophy and ECM accumulation. These results suggest that calcineurin is a mediator of diabetic hypertrophy and ECM regulation in glomeruli in vivo.
We show that calcineurin protein levels are increased 3 days after
induction of diabetes and that calcineurin is activated in the renal
cortex of diabetic rats after 2 wk of hyperglycemia. Coincident with
this early time frame of disease is the increase in expression of
polypeptide growth factors such as TGF- and IGF-I in the kidney
(1, 11, 12). We have shown in vitro that IGF-I induces
hypertrophy and ECM accumulation in mesangial cells via calcineurin
(15). Therefore, the increase in calcineurin protein and
the stimulation of calcineurin activity may be due to increased
availability of these, or other, peptide factors. The increased protein
levels of calcineurin in the glomerulus and activation of calcineurin
in the renal cortex are inhibited by treatment with CsA. Additional
evidence of calcineurin activation in vivo is provided by increased
expression and nuclear localization of the calcineurin substrate NFATc1
in the renal cortex of diabetic animals. NFATc1 regulation is clearly
dependent on calcineurin activity, because treatment of animals with
CsA abolished changes in both protein expression and nuclear localization.
Of major interest is that unlike its effects in glomeruli to decrease
ECM expansion, CsA treatment alone increased both calcineurin protein
levels and ECM accumulation in extraglomerular cortex. It is possible
that binding of CsA/cyclophilin complexes to calcineurin inhibits
normal turnover of the protein, leading to a subsequent accumulation of
calcineurin in addition to inhibition of phosphatase activity. However,
the mechanism of this differential effect of CsA on calcineurin
expression is unclear. CsA-mediated increases in ECM proteins, however,
have been described in several cell types, including cortical
fibroblasts (19, 37), human endothelial and epithelial
cells (9), and proximal tubule cells (37). In
addition, CsA treatment has been associated with increased TGF-
expression (18, 19, 38), leading to the hypothesis that
TGF-
mediates the subsequent increase in ECM proteins. Our data
would support this model because CsA increased TGF-
and fibronectin
mRNA levels in the tubulointerstitium. Of considerable interest is the
finding that a similar event does not occur in the glomeruli. The data
show no increase in fibronectin mRNA or protein and no increase in
TGF-
mRNA with CsA treatment in glomeruli. Furthermore, we have
previously shown that in cultured mesangial cells, there was no
increase in fibronectin or collagen type IV protein expression in
response to CsA treatment (15).
Also of interest is the fact that although animals in our experiments
were administered daily subcutaneous injections of 5 mg/kg body wt CsA,
there was a significant difference in the serum levels of CsA achieved
in CsA alone and CsA + STZ animals. Diabetic animals were found to have
roughly 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 in
CsA + STZ- vs. CsA alone-treated animals is responsible for decreased
circulating levels. It is also possible that STZ or the diabetic state
could activate metabolic pathways, leading to an increased rate of
breakdown of CsA, and it could accelerate metabolism of the drug within
the liver (the site of the majority of CsA metabolism). In either case,
no toxicity was observed in animals treated with CsA alone, consistent
with reports that nephrotoxicity of this drug within the glomeruli is
not 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 and
slight increase in body weight contributed to the changes that were
observed. However, the relatively small differences in glucose levels
and body weight are not sufficient to explain the complete
normalization of glomerular hypertrophy. This result suggests a direct
role for calcineurin in hypertrophy of cells within the glomeruli.
Indeed, our laboratory's previous finding using cultured mesangial
cells lends support to this interpretation (15).
Furthermore, a slight decrease in hyperglycemia cannot account for the
disparity in ECM regulation and TGF- production in glomeruli and
extraglomerular 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 as
expected, inhibition of calcineurin resulted in a further increase in
GFR in diabetic animals that was associated with improvements in both
hypertrophy and ECM accumulation.
Our finding that calcineurin is important in tissue hypertrophy is in agreement with several other reports in the literature. First, several groups have reported that calcineurin is activated in in vitro models of 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 both induce 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 in vivo (10, 21, 24). Inhibition of calcineurin activity with CsA blocked development of cardiac hypertrophy in some models of hypertrophy (10, 20, 34). Therefore, in addition to its role in cardiac hypertrophy, we show that calcineurin is also important for renal cell hypertrophy and ECM expansion.
In conclusion, we show that inhibition of calcineurin with CsA results in marked attenuation of glomerular hypertrophy and ECM accumulation, supporting our in vitro finding that calcineurin may be an important mediator of hypertrophy and/or ECM regulation in mesangial cells. In addition, CsA treatment results in a decrease in whole kidney hypertrophy, suggesting that calcineurin may also play a role in hypertrophy of other renal cells, such as tubular epithelial cells. As calcineurin is activated by IGF-I and possibly by other factors such as angiotensin II (2) in renal cells, this finding could be due to inhibition of multiple pathways. More needs to be learned about the complex role of calcineurin in the kidney. In addition, inhibition of calcineurin to improve or preserve kidney function in the diabetic state is an important area to be examined.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors acknowledge Drs. Dan Riley and Robert Kunau for helpful discussion and analyses, Shuko Lee for statistical interpretation of the data, and Maria Bunega and Yuping Tang for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by the Research Enhancement Award Program (H. E. Abboud and J. L. Barnes) and Associate Investigator award (J. L. Gooch) from the Department of Veterans Affairs and by the Southern Arizona Foundation (J. L. Gooch).
Address for reprint requests and other correspondence: J. L. Gooch, Renal Div., Univ. of Texas Health Science Ctr.-San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-3900 (E-mail: Gooch{at}UTHSCSA.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 11, 2002;10.1152/ajprenal.00158.2002
Received 25 April 2002; accepted in final form 28 August 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abboud, HE.
Growth factors and diabetic nephropathy: an overview.
Kidney Int
52:
S3-S6,
1997.
2.
Aperia, A,
Holtback U,
Syren M,
Svensson L,
Fryckstedt J,
and
Greengard P.
Activation/deactivation of renal Na/K- ATPase: a final common pathway for regulation of natriuresis.
FASEB J
8:
436-439,
1994
3.
Barnes, V,
Musa J,
Mitchell R,
and
Branes J.
Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.
J Histochem Cytochem
47:
787-798,
1999
4.
Blaser, F,
Ho N,
Prywes R,
and
Chatila T.
Calcium-dependent gene expression mediated by MEF2 transcription factors.
J Biol Chem
275:
197-209,
2000
5.
Buttini, M,
Limonta S,
Luyten M,
and
Boddeke H.
Distribution of calcineurin A isoenzyme mRNAs in rat thymus and kidney.
Histochem J
27:
291-299,
1995[ISI][Medline].
6.
Crabtree, GR.
Calcium, calcineurin, and the control of transcription.
J Biol Chem
276:
2313-2316,
2000
7.
Delling, U,
Tureckova J,
Lim HW,
De Windt L,
Rotwein P,
and
Molkentin JD.
A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and slow myosin heavy-chain expression.
Mol Cell Biol
20:
6600-6611,
2000
8.
De Windt, J,
Lim H,
Taigen T,
Wencker D,
Condorelli G,
Dorn GN,
Kitsis R,
and
Molkentin J.
Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: an apoptosis-independent model of dilated heart failure.
Circ Res
86:
255-263,
2000
9.
Esposito, C,
Fornoni A,
Cornacchia F,
Bellotti N,
Fasoli G,
Foschi A,
Mazzucchelli I,
Mazzullo T,
Semeraro L,
and
Dal Canton A.
Cyclosporine induces different responses in human epithelial, endothelial and fibroblast cell cultures.
Kidney Int
58:
123-130,
2000[ISI][Medline].
10.
Eto, Y,
Yonekura K,
Sonoda M,
Arai N,
Sata M,
Sugiura S,
Takenaka K,
Gualberto A,
Hixon ML,
Wagner MW,
and
Aoyagi T.
Calcineurin is activated in rat hearts with physiological left ventricular hypertrophy induced by voluntary exercise training.
Circulation
101:
2134-2137,
2000
11.
Flvybjerg, A,
Hill C,
Nielson B,
Gronbaek H,
Bak M,
Christiansen T,
Logan A,
and
Orskov H.
The role of growth hormone, insulin-like growth factors, epidermal growth factor and transforming growth factor beta in diabetic kidney disease: an update.
In: The Kidney and Hypertension in Diabetes Mellitus, edited by Mogensen CE.. Boston, MA: Kluwer, 1997, p. 307-319.
12.
Flyvbjerg, A,
Kessler U,
Dorka B,
Funk B,
Orskov H,
and
Kiess W.
Transient increase in renal insulin-like growth factor binding proteins during initial kidney hypertrophy in experimental diabetes in rats.
Diabetologia
35:
589-593,
1992[ISI][Medline].
13.
Fruman, DA,
Pai SY,
Klee CN,
Burakoff SJ,
and
Bierer BE.
Measurement of calcineurin phosphatase activity in cell extracts.
Methods Enzymol
9:
146-154,
1996.
14.
Gooch, J,
Gorin Y,
Tang YP,
and
Abboud H.
TGFb-mediated activation of Erk1/Erk2 and induction of ECM synthesis is associated with activation of calcineurin phosphatase and generation of reactive oxygen species (ROS) (Abstract).
J Am Soc Nephrol
12:
591A,
2001.
15.
Gooch, JL,
Tang Y,
Ricono JM,
and
Abboud HE.
Insulin-like growth factor-I (IGF-I) induces renal hypertrophy via a calcineurin-dependent mechanism.
J Biol Chem
276:
42492-42500,
2001
16.
Halloran, PF,
Helms LM,
Kung L,
and
Noujaim J.
The temporal profile of calcineurin inhibition by cyclosporine in vivo.
Transplantation
68:
1356-1361,
1999[ISI][Medline].
17.
Hayashida, W,
Kihara Y,
Yasaka A,
and
Sasayama S.
Cardiac calcineurin during transition from hypertrophy to heart failure in rats.
Biochem Biophys Res Commun
273:
347-351,
2000[ISI][Medline].
18.
Islam, M,
Burke JFJ,
McGowan TA,
Zhu Y,
Dun SR,
McCue P,
Kanalas J,
and
Sharma K.
Effect of transforming growth factor beta antibodies in cyclosporine-induced renal dysfunction.
Kidney Int
59:
498-506,
2001[ISI][Medline].
19.
Johnson, D,
Saunders H,
Johnson F,
Huq S,
Field M,
and
Pollock C.
Cyclosporin exerts a direct fibrogenic effect on human tubulointerstitial cells: roles of insulin-like growth factor I, transforming growth factor beta1, and platlet-derived growth factor.
J Pharmacol Exp Ther
289:
535-542,
1999
20.
Lim, HW,
De Windt LJ,
Mante J,
Kimball TR,
Witt SA,
Sussman MA,
and
Molkentin JD.
Reversal of cardiac hypertrophy in transgenic disease models by calcineurin inhibition.
J Mol Cell Cardiol
32:
697-709,
2000[ISI][Medline].
21.
Lim, HW,
De Windt LJ,
Steinberg L,
Taigen T,
Witt SA,
Kimball TR,
and
Molkentin JD.
Calcineurin expression, activation, and function in cardiac pressure-overload hypertrophy.
Circulation
101:
2431-2437,
1999
22.
Lindholm, A.
Factors influencing the pharmacokinetics of cyclosporine in man.
Ther Drug Monit
13:
465-477,
1991[ISI][Medline].
23.
Liu, J,
Farmer JJ,
Lane W,
Friedman J,
Weissman I,
and
Schreiber S.
Calcineurin is a common target of cyclophilin-cyclosporin A and FKBR-FK506 complexes.
Cell
66:
807-815,
1991[ISI][Medline].
24.
Molkentin, J.
Calcineurin and beyond: cardiac hypertrophic signaling.
Circ Res
87:
731-738,
2000
25.
Mukai, H,
Kuno T,
Chang C,
Lane B,
Luly J,
and
Tanaka C.
FKBP-12-FK506 complex inhibits phosphatase activity in two mammalian isoforms of calcineurin irrespective of their substrates or activation mechanisms.
J Biochem (Tokyo)
113:
292-298,
1993[Abstract].
26.
Murray, BM,
Paller MS,
and
Ferris TF.
Effect of cyclosporine administration on renal hemodynamics in conscious rats.
Kidney Int
28:
767-774,
1985[ISI][Medline].
27.
Musaro, A,
McCullagh KJ,
Naya FJ,
Olsen EN,
and
Rosenthal N.
IGF-I induces skeletal myocyte hypertrophy in association with GATA-2 and NF-ATc1.
Nature
400:
581-585,
1999[ISI][Medline].
28.
Pricci, F,
Pugliese G,
Romano G,
Romeo G,
Locuratolo N,
Pugliese F,
Mene P,
Galli G,
Casini A,
Rotella C,
and
Di Mario U.
Insulin-like growth factors I and II stimulate extracellular matrix production in human glomerular mesangial cells. Comparison with transforming growth factor-beta.
Endocrinology
137:
879-885,
1996[Abstract].
29.
Rusnak, F,
and
Mertz P.
Calcineurinform and function.
Physiol Rev
80:
1483-1521,
2000
30.
Schreiber, B,
Hughes M,
and
Groggel G.
Insulin-like growth factor-I stimulates production of mesangial cell matrix components.
Clin Nephrol
43:
368-374,
1995[ISI][Medline].
31.
Semsarian, C,
Wu MJ,
Ju YK,
Marciniec T,
Yeoh T,
Allen DG,
Harvey RP,
and
Graham RM.
Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signaling pathway.
Nature
400:
576-580,
1999[ISI][Medline].
32.
Shultz, P,
DeCorleto P,
Silver B,
and
Abboud H.
Mesangial cells express PDGF mRNAs and proliferate in response to PDGF.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F674-F684,
1988
33.
Su, Q,
Weber L,
Le Hir M,
Zenke G,
and
Ryffel B.
Nephrotoxicity of cyclosporin A and FK506: inhibition of calcineurin phosphatase.
Renal Physiol Biochem
18:
128-139,
1995[Medline].
34.
Sussman, MA,
Lim HW,
Gude N,
Taigen T,
Olsen EN,
Wieczorek DF,
and
Molkentin JD.
Prevention of cardiac hypertrophy in mice by calcineurin inhibition.
Science
281:
1690-1693,
1998
35.
Swoap, SJ,
Hunter RB,
Stevenson EJ,
Felton HM,
Kansagra NV,
Lang JM,
Esser KA,
and
Kandarian SC.
The calcineurin-NFAT pathway and muscle fiber-type gene expression.
Am J Physiol Cell Physiol
279:
C915-C924,
2000
36.
Tumlin, J.
Expression and function of calcineurin in the mammalian nephron: physiological roles, receptor signaling and ion transport.
Am J Kidney Dis
30:
884-895,
1997[ISI][Medline].
37.
Wolf, G,
Killen P,
and
Neilson E.
Cyclosporin A stimulates transcription and procollagen secretion in tubulointersititial fibroblasts and proximal tubule cells.
J Am Soc Nephrol
6:
918-922,
1990.
38.
Wolf, G,
Zahner G,
Ziyadeh F,
and
Stahl R.
Cyclosporin A induces transcription of transforming growth factor beta in a cultured murine proximal tubular cell line.
Exp Nephrol
5:
304-308,
1996.
39.
Wolf, G,
and
Ziyadeh FN.
Molecular mechanisms of diabetic renal hypertrophy.
Kidney Int
56:
393-405,
1999[ISI][Medline].