Differential Expression of Calcineurin A Isoforms in the Diabetic Kidney
Jennifer L. Gooch,
Pablo E. Pèrgola,
Rebecca L. Guler,
Hanna E. Abboud and
Jeffrey L. Barnes
Department of Medicine, Division of Nephrology, University of Texas Health Science Center, San Antonio; and South Texas Veterans Health Care System, Audie Murphy Division, San Antonio, Texas.
Correspondence to Dr. Jennifer L. Gooch, Medicine/Nephrology, University of Texas Health Science Center, San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78229-3900. Phone: 210-567-4700; Fax: 210-567-4412; E-mail: Gooch{at}uthscsa.edu
ABSTRACT. Calcineurin is an important signaling molecule inmesangial cells in vitro and is involved in some manifestationsof diabetic nephropathy in vivo. However, calcineurin acts ina cell-specific and tissue-specific manner in the kidney, andmechanisms of specificity are unknown. Three closely relatedisoforms of the calcineurin A (CnA) subunit are expressed ina tissue-specific manner. This study was undertaken to determineif specificity of calcineurin action is linked to regulationof CnA isoforms in the diabetic kidney. After induction of diabeteswith streptozotocin, expression of all three CnA isoforms rapidlyincreased, primarily in the thick ascending limb of Henle (TAL).After prolonged diabetes, increase specifically of the isoformwas observed in collecting ducts (CD) and in endothelial cellsof glomeruli. Aquaporin 2 (AQP2), a putative substrate of calcineurinphosphatase in the kidney, is also involved in diabetic nephropathy.Co-localization of CnA isoforms with AQP2 revealed that CnA-is the predominant isoform that associates with AQP2 in thediabetic kidney. Furthermore, inhibition of calcineurin withcyclosporin A (CsA) alters AQP2 localization and phosphorylationin principal cells of CD. Alterations in subcellular localizationof AQP2 were parallel with CnA-. Similarly, CsA treatment resultsin a further increase in urine output compared with diabetesalone, suggesting a functional consequence of inhibiting calcineurin-mediatedregulation of AQP2. In conclusion, all three isoforms of CnAare upregulated in the diabetic kidney. Increased expressionof CnA-, in particular, is observed in glomeruli and CD andparticipates in regulation of AQP2 expression, phosphorylation,and function.
Calcineurin is an important signaling molecule in many tissues,including the kidney. We recently reported that calcineurinA (CnA) subunit expression is upregulated in the diabetic kidney.Concomitant with increased protein, calcineurin activity isincreased and expression of the calcineurin substrate NFATcis upregulated, and its nuclear localization is increased. Inhibitionof calcineurin with cyclosporin A (CsA) reduces diabetes-associatedglomerular hypertrophy and extracellular matrix (ECM) accumulation(1). However, the role of calcineurin in the kidney is tissue-specificas CsA increases ECM accumulation in the tubulointerstitiumof normal kidneys but has no effect on diabetes-induced ECMaccumulation. This is consistent with a role for calcineurinin downregulation of ECM proteins and TGF in tubule epithelia(2–5), and it is distinct from our findings that calcineurinis required for both IGF-I-mediated and TGF-mediated ECM accumulationin vitro in cultured mesangial cells (6,7). Specificity of calcineurinaction in the kidney is therefore an area of great interest.One potential level of calcineurin signaling specificity isin differential expression and/or action of calcineurin isoforms.
Calcineurin is a serine/threonine phosphatase comprised of twosubunits: the catalytic subunit A, which contains the phosphatasedomain, and the regulatory subunit B, which binds calcium andthe A subunit. There are three closely related isoforms of theA subunit: , , and . The and isoforms are widely expressed,whereas the is expressed predominantly in the testes (8). Inthe kidney, expression of both the and isoforms have beendetected in proximal tubules, collecting ducts (CD), and themedulla (9,10). Areas of highest calcineurin activity are theproximal and distal tubules and correspond to expression ofpredominantly the isoform (10,11).
This study was undertaken to identify the specific isoformsof CnA that are regulated in the diabetic kidney and to characterizethe tissue distribution of calcineurin expression. Furthermore,we evaluated the role of calcineurin in the regulation of onepotential substrate, aquaporin 2 (AQP2). AQP2 is important indiabetes-induced changes in urinary concentration and urineflow rates in vitro, AQP2 has been identified in subcellularvesicles that also contain calcineurin (12). Our findings indicatethat CnA isoforms are differentially regulated in the diabetickidney. Understanding the tissue-specific action of calcineurinisoforms may be useful in developing methods of targeted calcineurininhibition.
Materials
Antibodies recognizing specific isoforms of CnA include PP2B-A(SC-20), PP2BA- (C-20), and PP2BA- (M-17), which were purchasedfrom Santa Cruz (Santa Cruz, CA). Similar results were obtainedusing antibodies that recognize CnA- (AB1696) and CnA- (AB1697)from Chemicon (Temecula, CA). Anti-AQP2 and platelet endothelialcell adhesion molecule (PECAM) antibodies were also obtainedfrom Chemicon, and phospho-APQ2 antibody was a gift of SorenNielsen (University of Aarhus, Aarhus, Denmark). Anti-Tamm-Horsfallglycoprotein was purchased from ICM Biochemicals, Inc. (Aurora,OH).
Animal Model
All animal experiments were carried out with the approval ofthe Institutional Animal Care and Use Committee at the Universityof Texas Health Science Center, San Antonio. Male Sprague-Dawleyrats weighing between 200 and 250 g were divided into four groupsof 4 to 6 rats per group. Group 1 was injected intravenouslyvia the tail vein with 65 mg/kg body wt streptozotocin (STZ)in sodium citrate buffer (pH. 4.0) to induce diabetes; group2 was similarly injected with sodium citrate buffer alone; ratsin group 3 were injected with STZ and were also given dailysubcutaneous injections of CsA 5 mg/kg body wt beginning atthe time of STZ injection; and group 4 was injected with citratebuffer alone and also received daily subcutaneous injectionsof vehicle (10% ethanol). Groups 1 to 4 were studied at timepoints up to 14 d. In addition, one subset of STZ-induced diabeticrats was followed up to 6 wk before kidneys were examined. Bloodglucose concentrations were monitored using a LifeScan One Touchglucometer (Johnson & Johnson) 24 h later to verify hyperglycemiaand periodically thereafter. All rats had unrestricted accessto food and water. To inhibit calcineurin activity, 5 mg/kgbody wt CsA was utilized. CsA is a chemical inhibitor with highspecificity for calcineurin. CsA doses have been carefully examinedin the rat with regard to nephrotoxicity and pharmacokinetics(13,14). In the rat, signs of chronic nephrotoxicity have beenobserved at doses of CsA only greater than 10 mg/kg body wtadministered for at least 2 wk. No evidence of nephrotoxicitywas observed in this study in rats given 5 mg/kg body wt CsAfor 2 wk.
To measure urine output, animals were housed individually inmetabolic cages (Nalgene) with access to food and water. Urinewas collected over 24 h, and urine output (ml/min) was determined.
Immunohistochemistry Indirect.
Paraffin-embedded tissue sections (5 µm thick) were preparedby dewaxing followed by quenching of endogenous peroxidasesby incubation in 0.3% H2O2 in methanol for 30 min. Antigenswere unmasked by incubation in 10 mmol/L citrate buffer at 80°Cfor 10 min. Sections were then rehydrated in PBS-0.1% BSA for15 min before addition of appropriate blocking serum for 15min. Sections were incubated with primary antibodies (dilutionsof 1:100 to 1:250) overnight in a humidified chamber at 4°C.The next day, sections were washed three times in PBS-0.1% BSAand then incubated with biotinylated secondary antibodies (dilutionsof 1:100) for 1 h at room temperature. Bound antibody was identifiedby immunoperoxidase ABC staining following the manufacturer’sinstructions (Vector Laboratories, Burlingame, CA). In addition,sections were counterstained with hematoxylin. Finally, coverslipswere mounted with Permount (Sigma), and sections were viewedby bright-field microscopy.
Direct.
Frozen cortical sections (5 µm thick) were fixed and permeabilizedin acetone for 15 min and then rehydrated in PBS-0.1% BSA for15 min. Sections were then blocked with donkey IgG for 15 min,and then primary antibodies were added at a concentration of15 µg/ml for 30 min. Sections were then washed three timesfor 5 min with PBS-0.1% BSA before addition of FITC- or Cy3-conjugatedsecondary antibodies at dilutions of 1:100 or 1:50. Finally,sections were washed three times for 5 min with PBS-0.1% BSAand coverslips mounted with Crystal Mount (Biomeda, Foster City,CA). Sections were viewed by fluorescence microscopy, and dualchannel images created by using Image-Pro Plus software (MediaCybernetics, Inc.). For some experiments, 5-µm-thick paraffin-embeddedsections were prepared for staining by dewaxing and unmasking.Sections were then blocked in 50 mM NH4Cl for 30 min followedby incubation in PBS plus 1% BSA, 0.2% gelatin, and 0.05% saponinbefore addition of primary antibodies. Sections were then examinedas described above.
Western Blot
Dissected renal medullae were homogenized in TNESV lysis buffer(50 mM Tris -HCl pH 7.4, 2 mM EDTA, 1% NP-40, 100 mM NaCl, 100mM Na orthovanadate, 100 µg/ml leupeptin, 20 µg/mlaprotonin, and 10–7M phenylmethylsulfonyl [PMSF]). Twenty-fivemicrograms of protein were analyzed by 12% SDS-PAGE. After transferof the proteins to nitrocellulose, the membrane was incubatedin 5% milk-TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween20) and then immunoblotted with 1:500 dilution of total AQP2or 1:2000 dilution of anti-phospho-AQP2 antibodies. HRP-conjugateddonkey anti-rabbit secondary antibodies were added at 1:2000,and proteins were visualized by enhanced chemiluminescence (Pierce,Rockford, IL). Also, lysates were collected from primary culturedrenal cells harvested from CnA- wildtype or CnA- knockout mice(described in reference 15 and in Gooch JL, Toro JJ, Guler RL,et al, submitted for publication) for detection of CnA- isoform.
CnA-, , and Are Upregulated in the Diabetic Kidney
We previously reported that calcineurin expression and activityare increased in the cortex of diabetic rats. We therefore examinedexpression of calcineurin isoforms over time in STZ-induceddiabetic rat kidneys using specific antibodies to the (CnA-), (CnA-), and (CnA-) isoforms. Detection of isoform expressionby immunohistochemistry in serial sections revealed that allthree isoforms are progressively upregulated in the same regionsof the cortex over the course of 14 d (Figure 1). Stained kidneycross-sections were examined to determine calcineurin expressionin various regions. Table 1 summarizes the relative expressionof each isoform in the major regions of the kidney. The predominantsite of expression was confirmed to be the thick ascending limb(TAL) based on identification of expression in the macula densa(Figure 2A) and on co-localization of calcineurin isoforms withTamm-Horsfall glycoprotein expression (Figure 2B).
Figure 1. Time course of expression of calcineurin isoforms. Expression of calcineurin isoforms was determined by immunohistochemistry using antibodies specific to calcineurin A (CnA)- (a-d), CnA- (e–h), and CnA- (i–l) in serial sections of rat kidney 0, 3, 10, and 14 d after induction of diabetes with STZ. CnA isoform expression was detected using the DAB chromogen, and sections were counterstained with hematoxylin. Results shown are representative of three animals from each treatment group.
Figure 2. Thick ascending limb of Henle’s loop (TAL) is the principal region of increased CnA-, CnA-, and CnA- expression in the diabetic kidney. (A) Serial cortical sections from diabetic kidneys were examined for expression of CnA- (panel i), CnA- (panel ii), and CnA- (panel iii) by IHC, and representative macula densa adjacent to cortical glomeruli were identified. As a control, one serial section was stained with IgG alone (panel iv). Protein expression was visualized with the DAB chromagen, and sections were counterstained with hematoxylin. Results shown are representative of three animals from each treatment group. (B) Serial sections of diabetic rat kidneys were stained for CnA- (panel i), CnA- (panel ii), CnA- (panel iii.), and Tamm-Horsfall glycoprotein (panel iv). Protein expression was detected using the DAB chromogen, and sections were counterstained with hematoxylin. Results shown are representative of three animals from each treatment group.
In addition to increased calcineurin expression in the corticaltubulointerstitium, calcineurin protein is also increased inglomeruli over time with diabetes (1). Therefore, we examinedexpression of specific calcineurin isoforms in glomeruli ofSTZ-induced diabetic rats. Figure 3A shows that unlike simultaneousupregulation of all three CnA isoforms in the cortex, expressionof only CnA- is increased in glomeruli (panel i). Expressionof CnA- (panel ii) and CnA- (panel iii) are evident in the maculadensa (indicated with an asterisk) but no evidence of stainingof these two isoforms is seen in the glomerulus. One serialsection was also stained with IgG alone as a negative control(panel iv). The pattern of CnA- expression is consistent withendothelial cells. Figure 3B shows the result of double immunofluorescentlabeling with CnA- and PECAM, used as a marker of endothelialcells. CnA- co-localizes with PECAM (bottom panel, yellow stainingin merged image). In addition, CnA- appears to stain regionswithin the glomerulus distinct from PECAM that are consistentwith mesangial localization (identified with arrows). To verifythe specificity of the CnA- antibodies used in immunohistochemistry,protein lysates were prepared from primary renal cells grownfrom CnA- wildtype or knockout mice (see Materials and Methods).Figure 3C shows that CnA- is readily detectable in the culturedwildtype cells and absent in the knockout cells.
Figure 3. CnA- is upregulated in diabetic glomeruli. (A) CnA isoform expression was determined in glomeruli of diabetic animals by immunohistochemistry. Panel i, CnA- expression; panel ii, CnA-; panel iii, CnA-; and panel iv is IgG control. Protein expression was detected using the DAB chromogen, and sections were counterstained with hematoxylin. Results shown are representative of three animals from each treatment group. (B) Co-localization of CnA- and the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM) was determined by double immunofluorescence using specific antibodies. CnA- was detected using a CY3-conjugated secondary antibody (red), and PECAM was detected using a FITC-conjugated secondary antibody (green). Co-localization is evident as yellow fluorescence on the merged image. Arrows indicate red staining that is consistent with mesangial cells. Results shown are representative of three animals from each treatment group.
One potential target of calcineurin that is particularly relevantto the diabetic state is the water channel AQP2, which is expressedin collecting duct principal cells and is dynamically regulatedby arginine vasopressin-induced phosphorylation (16). In thediabetic kidney, AQP2 expression is increased compared withcontrol levels of expression (Figure 4, panel b compared withpanel a). In particular, expression of phosphorylated AQP2 isincreased at the apical plasma membrane (Figure 4, panel d comparedwith panel c). This is consistent with previous reports thatAQP2 expression and phosphorylation is altered with diabetes(17).
Figure 4. Changes in aquaporin 2 (AQP2) expression and phosphorylation with diabetes. Expression and phosphorylation of AQP2 was determined in control (a and c) and diabetic (b and d) animals by IHC using specific antibodies to total (a and b) and phospho-AQP2 (c and d). Protein expression was visualized using the DAB chromogen, and sections are counterstained with hematoxylin. Arrows indicate increased apical staining of phospho-AQP2. Results shown are representative of three animals from each treatment group.
AQP2 localization in the apical plasma membrane may be dependenton phosphorylation of AQP2 at the C-terminus (18–20).In vitro, calcineurin is able to dephosphorylate AQP2; as aresult, it may be involved in regulation of AQP2 (12). Therefore,we examined co-localization of AQP2 and calcineurin isoformsin the diabetic kidney. Figure 5A shows that in serial sections,AQP2 expression in cortical CD (panel i) co-localizes with CnA-(panel ii) and, to a lesser extent, CnA- (panel iii) expression.No CnA- expression was observed in conjunction with AQP2 (paneliv). A similar pattern of co-localization was observed in medullaryCD (data not shown). Cellular expression of AQP2 and CnA- wasexamined by double immunofluorescence (Figure 5B). AQP2 (redsignal) and CnA- (green signal) co-localize to the apical surfaceof medullary CD in the diabetic rat kidney (indicated by yellowsignal in the merged image).
Figure 5. Co-localization of CnA-, CnA-, and CnA- with AQP2 in collecting duct principal cells. (A) Expression of CnA isoforms and AQP2 was determined in diabetic kidneys by IHC in serial sections. Panel i, total AQP2; panel ii,- CnA-; panel iii, CnA-; and panel iv, CnA-. Proteins are visualized using the DAB chromogen, and sections are counterstained with hematoxylin. Results shown are representative of three animals from each treatment group. (B) Expression of both total AQP2 and CnA- were determined in the diabetic kidney by double immunofluorescence using specific antibodies and fluorochrome-tagged secondary antibodies. Total AQP2 is detected with a CY3-conjugated secondary antibody (red), and CnA- was detected with a FITC-conjugated secondary antibody (green). Areas of co-localization are apparent as yellow fluorescence in the merged image (bottom panel). Results shown are representative of 3 animals from each treatment group.
Calcineurin activity was inhibited in diabetic rats by dailyadministration of low doses of CsA as described previously (1).In Figure 6A, AQP2 localization and phosphorylation in medullaryCD were examined by immunohistochemistry in control, diabetic,and diabetic animals treated with CsA. As expected, expressionof total and phosphorylated AQP2 were increased in collectingduct principal cells of diabetic animals compared with controlanimals (panels ii and v compared with i and iv). Interestingly,inhibition of calcineurin with CsA in diabetic animals resultedin a shift from apical to intracellular localization and a coordinatedecrease in expression of phosphorylated AQP2 at the apicalmembrane compared with diabetic animals (panels iii and vi).AQP2 expression and phosphorylation were also examined in medullarylysates from control, diabetic, and diabetic + CsA-treated animalsby Western blot. Figure 6B shows that both total and phosphorylatedAQP2 are increased in medullary lysates from diabetic animalscompared with control animals consistent with both chronic regulationof AQP2 at the protein level and acute regulation of phosphorylationstatus. Inhibition of calcineurin with CsA resulted in greaterAQP2 protein expression in CD but slightly less phosphorylation.Amounts of total and phosphorylated AQP2 as well as the relativeproportion of phosphorylated to total protein were determinedand are shown in Figure 6C. Diabetic animals treated with CsAshow a further increase in total AQP2 protein (P < 0.05)compared with diabetic animals and a slight (but NS) drop inphosphorylation. When phosphorylated AQP2 is then normalizedby total APQ2 protein, a net decrease in the proportion of phosphorylatedAQP2 is apparent, suggesting that inhibition of calcineurinhas no effect on the chronic regulation of AQP2 protein butdecreases acute phosphorylation of the protein.
Figure 6. Inhibition of calcineurin alters AQP2 localization and phosphorylation. (A) Total APQ2 (panels i–iii) and p-AQP2 (panels iv–vi) were examined by IHC in serial sections of kidneys from control (panels i and iv), diabetic (panels ii and v), and diabetic + CsA (panels iii and vi) animals. Arrow indicates broad cellular distribution of total AQP2 (panel iii). Results shown are representative of three animals from each treatment group. (B) Total and phospho-AQP2 were examined in medullary homogenates from control, diabetic, and diabetic + CsA animals by immunoblotting with specific antibodies. Expression of actin was determined using a specific antibody to verify equal loading. Each lane contains total protein from a different animal. Data shown are representative of at least two independent experiments. (C) Total and phospho-AQP2 levels were semi-quantitated to determine relative fold increase over control. The ratio of phosphorylated to total AQP2 was determined by dividing values of phospho-AQP2 by total AQP2 for each sample. Data shown are the mean ± SEM of three animals per treatment group. *P < 0.05 and **P < 0.01.
Next, co-localization of AQP2 and CnA- were examined in control,diabetic, and diabetic animals treated with CsA. Figure 7 showsthat co-expression of AQP2 and CnA- at the apical plasma membraneincreases in the diabetic kidney. When calcineurin activityis inhibited by CsA administration, both AQP2 and CnA- are localizedin the cytoplasm rather than the apical membrane. Again, consistentwith findings presented in Figure 6, total amounts of AQP2 appearto be increased in the diabetic kidneys, and levels of AQP2remain elevated after inhibition of calcineurin.
Figure 7. CnA- and AQP2 co-localize in the apical membrane of CD with diabetes and in the cytoplasm after CsA treatment. Expression of total AQP2 and CnA- in CD was examined by double immunofluorescence using specific antibodies in control (panels a, d, and g), diabetic (panels b, e, and h), and diabetic + CsA (panels c, f, and i) animals. AQP2 was visualized using a CY3-conjugated secondary antibody (red) and CnA- using a FITC-conjugated secondary antibody (green). Top row, total AQP2; middle row, CnA-; bottom row, merged images. Results shown are representative of three animals from each treatment group.
AQP2 transports water in CD and is therefore closely involvedin the control of urine concentration and urine flow rates.The diabetic state induces alterations in urine output. Ourdata demonstrate that CnA- is involved in regulation of AQP2phosphorylation and cellular localization. We therefore examinedthe effect of calcineurin inhibition on urine output in normaland diabetic rats. Table 2 shows that diabetic rats have significantlyincreased rates of urine output at both 10 and 14 d after inductionof diabetes. CsA alone had no effect on urine volume at 10 d,and a small decrease was observed at 14 d. On the other hand,CsA administration to diabetic rats resulted in a further increasein urine output that was significantly increased compared withdiabetes alone at both 10 and 14 d.
The role of calcineurin in the kidney is an area of major interestand importance, but mechanisms of tissue-specific calcineurinaction are poorly understood. This study contributes severalsignificant findings to the area of calcineurin specificity.
First, our study adds to the knowledge of calcineurin isoformexpression and regulation in the kidney. We show that all threecalcineurin isoforms are upregulated in the diabetic kidney,particularly in the TAL, and CnA- is upregulated in CD and inglomeruli. There are a limited number of studies describingexpression of calcineurin isoforms in the kidney. Butinni etal. (9) and Tumlin et al. (10) both report that CnA- is thepredominant isoform expressed in the kidney, with the greatestexpression in proximal tubules. CnA- is also expressed, primarilyin the medulla. Evidence of both CnA- and CnA- expression inCD has been reported; however, CnA- was not detected in anystudy. Our findings support those of Butinni and Tumlin in thatCnA- is the predominant isoform detected in the diabetic kidney.In addition, this study is the first to identify calcineurinisoform expression in the glomerulus. The present study alsoexpands on their observations to include tissue-specific regulationof calcineurin isoforms in a disease state, and we also reportCnA- expression in the kidney. To our knowledge, this is thefirst report of CnA- expression in the kidney and the firstto describe CnA- expression in association with a disease state.However, it should be noted that further characterization ofCnA- expression in diabetes, as well as possibly other diseasestates, is warranted to confirm expression of this isoform inthe kidney.
Next, we provide evidence that calcineurin isoforms are differentiallyregulated in the kidney. We previously reported that inhibitionof calcineurin in a rat model of diabetes resulted in tissue-specificeffects. In glomeruli, inhibition of calcineurin is associatedwith decreased accumulation of ECM proteins and reduced glomerularhypertrophy (1,7). In contrast, calcineurin inhibition aloneinduced ECM accumulation in the tubulointerstitium in controlanimals and failed to prevent ECM accumulation in diabetic animals(1). This result is consistent with what is known about nephrotoxicresponses to calcineurin inhibitors in humans and animals (2–5).In this study, we find that upregulation of only CnA- is specificto the glomerulus, and it appears that both endothelial cellsand mesangial cells express CnA- in the diabetic state. As previouswork has identified a role for calcineurin in IGF-I-mediatedand TGF-mediated effects in cultured mesangial cells, it isinteresting to speculate that the beneficial effects of CsAadministration on diabetic glomeruli were the result of inhibitionof CnA-. Alternatively, pro-fibrotic effects of CsA administrationmay be the result of inhibition of both and CnA- and CnA- (aswell as possibly CnA-) isoforms in the tubulointerstitium.
Finally, our data indicate that one function of calcineurinmay be to contribute to the regulation of AQP2. In vitro workby Jo et al. demonstrated that calcineurin can be found in associationwith AQP2 in isolated vesicles (12). Furthermore, the authorsreported that calcineurin is able to dephosphorylate AQP2 inan in vitro assay. Therefore, it is reasonable to propose thatcalcineurin is involved in regulation of AQP2, although a mechanismfor such regulation is lacking. We report that calcineurin isco-expressed in CD with AQP2 in the diabetic kidney. More specifically,we find that CnA- co-localizes with AQP2 at the apical plasmamembrane of principal cells. Interestingly, inhibition of calcineurinwith CsA results in alterations in intracellular localizationof both CnA- and AQP2 to the cytoplasm rather than the apicalmembrane. In addition to alterations in intracellular localization,AQP2 appears to be regulated by both chronic and acute mechanismsin the diabetic kidney. In the medulla, chronic regulation isevident as increased total AQP2 protein in diabetic comparedwith control animals. The amount of phosphorylated AQP2 is greaterthan the proportional increase in protein, demonstrating anacute mechanism of regulation at the level of phosphorylation.Inhibition of calcineurin with CsA did not interfere with chronicregulation of AQP2 protein; in fact, protein levels were increasedin both cortical and medullary lysates. Instead, calcineurinappears to be involved in acute alterations in AQP2 phosphorylation.The proportion of phosphorylated AQP2 to total APQ2 is significantlydecreased compared with diabetes alone in both cortical andmedullary lysates. Interestingly, this net decrease in phosphorylationof AQP2 is associated with alterations in subcellular localization.It is possible that the increase in AQP2 protein observed withinhibition of calcineurin may be due to lower turnover of AQP2protein, either by shedding of apical vesicles into the urinaryspace or by degradation after endocytosis, since the proteinfails to complete its normal trafficking cycle. An overall decreaseof AQP2 protein suggests that, while calcineurin may be involvedin regulation of AQP2 trafficking, it is unlikely that AQP2is an in vivo substrate for calcineurin dephosphorylation. Instead,it is possible that calcineurin is acting as a signaling intermediateand is required to facilitate phosphorylation of AQP2 by activatedkinases. In support of this, we provide functional evidencethat inhibition of calcineurin also interferes with action ofAQP2, as urine volume of diabetic rats is significantly increasedwith CsA treatment.
In conclusion, calcineurin is an important molecule in the kidneyand is involved in diverse functions. Previous reports havedescribed roles for calcineurin in glomerular hypertrophy andECM accumulation in vitro and in vivo (1,7), regulation of TGF,and ECM expression in the tubulointerstitium (2–5); wenow add regulation of AQP2. One mechanism for specificity ofcalcineurin action in the kidney may be the result of tissue-specificexpression and/or action of CnA isoforms. Understanding howcalcineurin isoforms contribute to differential actions mayprovide critical insight into how targeted inhibition of calcineurinisoforms can be accomplished. Strategies to selectively inhibitcalcineurin isoforms may provide new opportunities to inhibitthe activity of this phosphatase without associated nephrotoxicity.
Acknowledgments
This work was supported by a Research Award Enhancement Program(REAP) grant from the Department of Veterans Affairs (JLG, HEA,and JLB), the American Diabetes Association (JLG), and the GeorgeO’Brien Kidney Research Center from the NIH (JLG, PEP,HEA, and JLB).
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Received for publication August 22, 2003.
Accepted for publication March 16, 2004.
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J. L. Gooch, R. L. Guler, J. L. Barnes, and J. J. Toro Loss of calcineurin A{alpha} results in altered trafficking of AQP2 and in nephrogenic diabetes insipidus
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T. Oka, Y.-S. Dai, and J. D. Molkentin Regulation of Calcineurin through Transcriptional Induction of the calcineurin A{beta} Promoter In Vitro and In Vivo
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Y. Moz, R. Levi, V. Lavi-Moshayoff, K. B. Cox, J. D. Molkentin, J. Silver, and T. Naveh-Many Calcineurin A{beta} Is Central to the Expression of the Renal Type II Na/Pi Co-transporter Gene and to the Regulation of Renal Phosphate Transport
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J. L. Gooch, J. J. Toro, R. L. Guler, and J. L. Barnes Calcineurin A-{alpha} But Not A-{beta} Is Required for Normal Kidney Development and Function
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Abstract
Introduction
isoform was observed in collecting ducts (CD) and in endothelial cells of glomeruli. Aquaporin 2 (AQP2), a putative substrate of calcineurin phosphatase in the kidney, is also involved in diabetic nephropathy. Co-localization of CnA isoforms with AQP2 revealed that CnA-
in tubule epithelia (2–5), and it is distinct from our findings that calcineurin is required for both IGF-I-mediated and TGF
. The 
