Identification of the Glomerular Podocyte as a Target forGrowth Hormone Action

Gaddameedi R. Reddy, Mary J. Pushpanathan, Richard F. Ransom, Lawrence B. Holzman,Frank C. Brosius III, Maria Diakonova, Peter Mathieson, Moin A. Saleem, Edward O. List,John J. Kopchick, Stuart J. Frank, and Ram K. Menon

Departments of Pediatrics and Communicable Diseases (G.R.R., M.J.P., R.F.R., R.K.M.), Internal Medicine (L.B.H., F.C.B.),and Molecular and Integrative Physiology (F.C.B., R.K.M.), University of Michigan, Ann Arbor, Michigan 48109-0718;Department of Biological Sciences (M.D.), University of Toledo, Toledo, Ohio 43606; Academic and Children’s Renal Unit(P.M., M.A.S.), University of Bristol, Bristol BS1 3NY, United Kingdom; Edison Biotechnology Institute and Department ofBiomedical Sciences (E.O.L., J.J.K.), College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701; and Departmentof Internal Medicine (S.J.F.), University of Alabama, Birmingham, Alabama 35294

GH excess in both the human and transgenic animal modelsis characterized by significant changes in blood pressure andrenal function. The GH/GH receptor (GHR) axis is also impli-cated in the development of diabetic nephropathy. However,it is not clear whether GH’s actions on renal function are dueto indirect actions mediated via changes in blood pressureand vascular tone or due to direct action of GH on the kidney.We hypothesized that functional GHRs are expressed on theglomerular podocyte enabling direct actions of GH on glo-merular function. Real-time PCR, immunohistochemistry,and Western blot analysis of murine podocyte cells (MPC-5)and kidney glomeruli demonstrated expression of GHR mRNAand protein. Exposure of both murine and human podocytesto GH (50–500 ng/ml) resulted in an increase in abundance of

phosphorylated signal transducer and activator of transcrip-tion-5, Janus kinase-2, and ERK1/2 proteins. Exposure ofpodocytes to GH also caused changes in the intracellular dis-tribution of the Janus kinase-2 adapter protein Src homology2-B�, stimulation of focal adhesion kinase, increase in reac-tive oxygen species, and GH-dependent changes in the actincytoskeleton. We conclude that glomerular podocytes expressfunctional GHRs and that GH increases levels of reactive ox-ygen species and induces reorganization of the actin cytoskel-eton in these cells. These results provide a novel mechanisticlink between GH’s actions and glomerular dysfunction in dis-orders such as acromegaly and diabetic glomerulosclerosis.(Endocrinology 148: 2045–2055, 2007)

THE PLEIOTROPIC ACTIONS of pituitary GH includeeffects on the blood pressure and fluid homeostasis.

GH excess in both the human (acromegaly) and transgenicanimal models is characterized by significant structural andfunctional changes in the kidney (1, 2). The GH/GH receptor(GHR) axis is implicated as a causative factor in the devel-opment of diabetic nephropathy (3–6). Involvement of theGH/IGF-I axis in the pathogenesis of diabetic nephropathyis supported by studies of the impact of GH excess (7, 8) anddeficiency (9) on kidney growth and function in variousanimal models, both nondiabetic and diabetic. In the humana direct relationship has been noted between the activity ofthe GH/IGF-I axis and renal hypertrophy, microalbumin-uria, and glomerulosclerosis (3, 10, 11). Elevated mean 24-hconcentrations of circulating GH and an exaggerated GHresponse to several physiological and pharmacological pro-vocative stimuli are characteristic features of patients with

poorly controlled type 1 diabetes mellitus (4, 5, 12–17). In arodent model of type 1 diabetes mellitus, GHR signalingpathways were determined to be more active in the kidneysof diabetic (DM) animals, compared with control animals(18). A more direct role for GH/GHR in the pathogenesis ofDM nephropathy is supported by studies using GH and GHantagonist transgenic mice, total GHR knockout mice, andpharmacological blockade of the GHR using pegvisomant (7,19–21). These studies demonstrate that absence of functionalGHR confers a protective effect against DM nephropathy inmurine models of type 1 diabetes mellitus. However, at thepresent time, it is unclear whether the role of GH in thepathogenesis of acromegalic and DM nephropathy is due todirect action(s) of GH on the kidney or indirect due to effectsof GH on systemic vascular tone and/or blood pressure (22).

The aim of the current study was to investigate the hy-pothesis that GH affects glomerular function via direct actionon the glomerular podocyte. Our results support the con-clusion that functional GHRs are expressed on the glomer-ular podocyte and GH alters functioning of the podocyte.

Materials and MethodsAntibodies

Anti-GHR antibody AL47 (23, 24) was used in 1:1000 dilution forWestern blot analyses, and 1:100 dilution for immunohistochemistrystudies. In certain experiments the AL47 antibody was preadsorbed byincubation for 60 min at room temperature with recombinant glutathi-

First Published Online February 1, 2007Abbreviations: DAPI, 4�,6�-Diamino-2-phenylindole; DM, diabetes mel-

litus; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHR, GH receptor;GST, glutathione-S-transferase; JAK, Janus kinase; K/O, knockout; p, phos-phorylated; ROS, reactive oxygen species; SH2, Src homology 2; Stat, signaltransducer and activator of transcription; Wt1, Wilms tumor 1.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/07/$15.00/0 Endocrinology 148(5):2045–2055Printed in U.S.A. Copyright © 2007 by The Endocrine Society

doi: 10.1210/en.2006-1285

2045

one-S-transferase (GST)-epitope tagged GHR protein or GST (as control)before using the antibody in either Western blot or immunohistochem-istry experiments. Rabbit polyclonal anti-Src homology 2 (SH2)-B� (25)(gift from Christin Carter-Su, University of Michigan) and mouse anti-synaptopodin monoclonal antibody (gift from Peter Mundel, MountSinai School of Medicine, New York, NY) (26) were used in immuno-histochemistry studies at 1:200 and 1:2 dilutions, respectively. Anti-Janus kinase (JAK)-2 (1:1,000), -phosphorylated Janus kinase (pJAK)-2(1:750), -signal transducer and activator of transcription (Stat)-5a/b (1:1,000), -pStat5a/b (1 in 750), and -Wilms tumor 1 (Wt1; 1:1,000) werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Erk1/2 and -pErk1/2 (both used in 1:10,000 dilution) antibodies werepurchased from Promega (Madison, WI); anti-focal adhesion kinase(FAK) (1:2,500 dilution) antibodies was purchased from TransductionLaboratories (San Diego, CA). The secondary antibodies used for West-ern blot analysis were purchased from Jackson ImmunoResearch Lab-oratories (West Grove, PA), and the secondary antibodies used forimmunofluorescence studies were rhodamine-labeled goat antirabbitIgG (Molecular Probes, Eugene, OR), green-labeled goat antimouse IgG(Molecular Probes), green-labeled donkey antichicken IgG (MolecularProbes), or fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG(Sigma, St. Louis, MO).

Animals and tissues

Adult Swiss Webster male mice (�8 wk old; Charles River, Wilming-ton, MA) were used for the present study. The GHR null [knockout(K/O)] mice (genetic background, C57BL/6J) with targeted deletion ofthe GHR have been described before (27). The intact kidney was har-vested and processed for histological analysis. A portion of the kidneywas fixed in 10% phosphate-buffered formalin before paraffin embed-ding. Another portion was immediately frozen in Tissue-Tek optimumcutting temperature compound before cryosectioning. The remainingportion of the kidney was snap frozen in liquid nitrogen and stored at�80 C for protein extraction. In other instances the mice were perfusedwith ferrous solution and glomeruli isolated from the kidneys of theperfused mice using established methods (28). All animal protocols wereapproved by the University of Michigan Animal Care and UseCommittee.

Cell culture

Conditionally immortalized mouse podocyte cells (MPC-5) (29) andthose described by Endlich et al. (30) isolated from immortmouse, weregrown according to the protocol described by Mundel et al. (29). Briefly,cells were cultured under growth-permissive conditions on rat tail col-lagen type I-coated plastic dishes (BD Bioscience, Franklin Lakes, NJ) at33 C with 100% relative humidity and 5% CO2, in RPMI 1640 medium(Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (LifeTechnologies, Inc.-BRL, Grand Island, NY), 100 U/ml penicillin, 100�g/ml streptomycin (Life Technologies, Inc.-BRL), and 10 U/ml mouserecombinant �-interferon (Sigma). To induce differentiation, podocyteswere maintained in nonpermissive conditions at 37 C without �-inter-feron for 14 d before experimentation. Under these culture conditions,cells stopped proliferating and were identified as differentiated podo-cytes by their arborized morphology. Cells were routinely maintainedfor 24 h in serum-free medium before experimentation. Human podo-cytes (31) were cultured in RPMI 1640 medium with glutamine (Sigma)supplemented with 10% fetal calf serum (Life Technologies) and insulintransferrin sodium selenite (1 ml per 100 ml; Sigma). These cells wereinduced to differentiate using a protocol similar to that outlined abovefor the murine podocytes.

Immunohistochemistry and immunocytochemistry

Antigen was retrieved from deparaffinized kidney sections using anantigen unmasking solution (Vector Laboratories, Burlingame, CA). En-dogenous peroxidase activity was blocked by incubating the slides atroom temperature in 80% methanol and 30% hydrogen peroxide for 20min on a shaker. Nonspecific binding was blocked with diluted normalhorse serum (Vectastain Universal Elite ABC kit; Vector) for 20 min atroom temperature. The sections were then incubated overnight at 4 Cwith anti-GHR AL47 antibody. After thorough washing with PBS, the

sections were blotted with biotinylated secondary antibody and devel-oped with Nova Red and counterstained with hematoxylin.

Differentiated MPC-5 cells cultured on chambered slides were fixedin 4% paraformaldehyde for 15 min at room temperature. The cells werethen washed 3 � 5 min with PBS to remove the paraformaldehyde andtreated with 0.2% Triton X-100 for 15 min to permeabilize the membrane.The cells were exposed to 5% nonfat dry milk for 30 min at roomtemperature and then incubated with primary antibodies GHR AL47 orrabbit anti-SH2B� and Alexa Fluor 594 phalloidin in PBS containing 5%nonfat dry milk. After three washes in PBS, cells were incubated withFITC-conjugated antirabbit (1:100; Sigma) secondary antibodies for 30min at room temperature. The cells were mounted with Prolong GoldAnti-Fade with 4�,6�-diamino-2-phenylindole (DAPI; Molecular Probes)and examined by fluorescence microscopy.

Fluorescence microscopy

For immunofluorescence staining, kidney sections were blocked withthe normal serum and incubated with the primary antibody overnightat 4 C. Sections were then washed with PBS for 3 � 5 min and incubatedwith secondary antibodies (rhodamine-labeled antirabbit and FITC-la-beled antimouse antibodies) for 90 min at room temperature andmounted with Prolong Gold Anti-Fade with DAPI (Molecular Probes).Immunofluorescence microscopy was performed using an EclipseTE200 fluorescent microscope (Nikon, Tokyo, Japan).

RNA isolation and real-time semiquantitative PCR

Total RNA was prepared from glomeruli and liver samples usingTriZol-reagent as specified by the manufacturer (Molecular ResearchCenter, Cincinnati, OH) and quantitated by the absorbance at 260 nm.Quantitation of GHR and IGF-I mRNA expression was determined byfluorescent 5�-nuclease (TaqMan) real-time RT-PCR using the followingprimers: GHR forward, 5�-CAGTTCCAAAGATTAAAGGGATTGA-3�,GHR reverse, 5�-TTATCATGAATGCCTAAGATGGTGTT-3�; GHRprobe, 5�-ACCTCCTCCAACTTCCCTCCCTTGAGAA-3�; IGF-I for-ward, 5�-GCT CTG CTT GCT CAC CTT CAC-3�, IGF-I reverse, 5�-CACACG AAC TGA AGA GCA TCC A-3�; and IGF-I probe, 5�-AGC TCCACC ACA GCT GGA CCA GAG A-3�.

The primers and TaqMan probes for quantitation of the GHR andIGF-I transcripts were designed using the primer design software PrimerExpress (PE Biosystems, Foster City, CA); GHR primers were specific forthe intracytoplasmic portion of the GHR. The primers and TaqManprobe for rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH)were purchased from a commercial vendor (PE Biosystems). The GHRand IGF-I probes were labeled with fluorescent reporter dye VIC and theGAPDH probe labeled with FAM. Normalization and validation ofthe data were carried out using GAPDH as the housekeeping control.The comparative Ct values method was used to calculate the relativequantity of GHR and IGF-I expression as previously described (32).Briefly, 2-ng aliquots of total RNA were analyzed using the Separate-Tube RT-PCR protocol (PE Biosystems). After RT at 48 C for 30 min, thesamples were subjected to PCR analysis using cycling parameters: 95C � 10 min; 95 C � 15 sec360 C � 1 min for 40 cycles. Each sample wasanalyzed in triplicate in individual assays performed on two or moreoccasions.

Western blot analysis

Isolated glomeruli and MPC-5 cells were homogenized in 100 �l oflysis buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 2 mmEGTA, 0.1% Triton X-100, 1 mmol/liter sodium pyrophosphate, 10mmol/liter sodium fluoride, and 1 mmol/liter sodium orthovanadate.The protein concentration was quantitated using the Bio-Rad proteinassay (Bio-Rad Laboratories, Hercules, CA) and equal amounts of pro-tein resolved on 10 or 4–16% gradient SDS-polyacrylamide gels as in-dicated and blotted onto nitrocellulose membrane for Western blot anal-ysis. The blot was probed with suitable primary and secondaryantibodies and was developed by the ECL chemiluminescent method(Amersham Biosciences, Piscataway, NJ) according to the manufactur-er’s instructions. Where indicated immunoprecipitation was performedby incubating lysates with the respective antiserum and immunopre-cipitates collected using protein A/G agarose beads. Immunoprecipi-

2046 Endocrinology, May 2007, 148(5):2045–2055 Reddy et al. • GH Receptor and Glomerular Podocyte

tates were subjected to SDS-PAGE and Western blotting as describedabove.

Quantification of membrane ruffling

To measure the effect of GH on membrane ruffling, MPC-5 cells weredeprived of serum for 24 h, treated with GH (500 ng/ml) for the indi-cated time periods, and then fixed and permeabilized as describedabove. Filamentous actin was labeled with Alexa Fluor 594 phalloidinand imaged by epifluorescence microscopy (Carl Zeiss, New York, NY).The number of ruffles per cell was determined by a single observerblinded to the identity of the sample.

Quantitative measurement of F-actin levels

F-actin was quantitated using a previously described phalloidin bind-ing assay (33) with minor modifications. MPC-5 cells (104 cells/well)were plated and allowed to differentiate over 14 d. After differentiation,the cells were exposed to GH or vehicle for varying time periods, fixedwith treatment with 3.7% formalin for 20 min, and then transferred intobuffer of 60 mm 1,4-piperazine diethane sulfonic acid, 25 mm HEPES, 10mm EGTA, 2 mm Mg SO4, and 0.5% Triton X-100. The fixed cells werethen stained for F-actin with Alexa Fluor 594 phalloidin (0.6 U/ml), andto normalize for cell number, the nuclei were stained with DAPI (2�g/ml). The cells were stained for 30 min and then washed 3 � 5 minwith PBS. Bound phalloidin was extracted by treatment with 100 �lmethanol for 1 h in the dark. Alexa Fluor 594 phalloidin fluorescence(excitation 585 nm, emission 608 nm) and DAPI fluorescence (excitation358 nm, emission 461 nm) were measured simultaneously using a spec-trofluorimeter (Spectramax GeminiEM; Molecular Devices, Sunnyvale,CA). F-actin content per cell was calculated as the ratio of Alexa Fluor-phalloidin to DAPI (nuclear) fluorescence.

Measurement of levels of reactive oxygen species (ROS)

Cells were grown in 12-well plates (�8000 cells/well), overnightserum starved, and then incubated with 5 �m CM-H2DCFDA (Molec-ular Probes) for 30 min at 37 C followed by exposure to either vehicleor GH (500 ng/ml) in the absence or presence of N-acetylcysteine (25mm) for varying time periods. CM-H2CDFDA is a nonfluorescent mol-ecule that becomes fluorescent after oxidation in the cytoplasm of cells.Fluorescence was quantitated via a dual-scanning microplate spectroflu-orometer (Spectra MAX Gemini EM; Molecular Devices) at 480-nm(excitation) and 530-nm (emission) wavelengths.

Statistical analysis

The Mann-Whitney and Kruskal-Wallis nonparametric tests wereperformed to analyze statistical significance of the difference betweenthe distributions of two or multiple independent samples, respectively,using SPSS software (version 11.5 for Windows; SPSS, Inc., Chicago, IL).P � 0.05 was considered significant.

ResultsGHR mRNA and protein expression in the murineglomerular podocyte

Real-time RT PCR analysis was used to quantify expres-sion of GHR mRNA in murine podocytes, glomeruli, andliver. To avoid detecting the alternatively spliced transcriptencoding the GH binding protein, the primers used werespecific for the intracytoplasmic portion of the GHR. Theseresults demonstrated that the relative expression of GHRmRNA, normalized to GAPDH expression, was comparablein the glomeruli and liver, with less expression in differen-tiated MPC-5 cells (Fig. 1A).

To detect expression of GHR protein, extracts from glo-meruli and differentiated MPC-5 cells were analyzed byWestern blot analysis using anti-GHR AL47 antibody, anantibody directed against the cytoplasmic domain of the

GHR. A protein band migrating at approximately 115 kDawas detected by this antibody in both glomerular and MPC-5cell lysates (Fig. 1B), a result consistent with the presence ofGHR protein in these samples. The detection of a proteinband of a similar molecular weight in MPC-5 cell lysateimmunoprecipitates obtained with the AL47 antibody (Fig.1B) provided further evidence of GHR expression in culturedpodocytes. In Western blot analysis, the AL47 antibodycross-reacts with a non-GHR (nonspecific) 82-kDa proteinband (Fig. 1B and Frank, S., personal communication). Toverify the specificity of the AL47 antibody, we performedWestern blot analysis using the AL47 antibody preadsorbedwith recombinant GHR protein. These experiments revealedthat the preadsorbed AL47 antibody recognized the nonspe-cific 82-kDa protein but failed to detect the specific GHRband (Fig. 1C), thus verifying the specificity of the AL47anti-GHR antibody. The specificity of the antibody was alsoverified by demonstrating absence of the approximately 115-kDa putative GHR band in tissues from the GHR K/O mouse(Fig. 1, B and C).

The cellular localization of GHR expression in the renalglomerulus was investigated by immunohistochemical anal-ysis. GHR expression in kidney section from mice (8 wk old)was detected using anti-GHR AL47 antibody. The binding ofthe primary antibody was visualized by light microscopyusing the avidin-biotin HRP methodology and the slidecounterstained with hematoxylin. The specific staining ofglomerular cells is indicated by red arrowheads (Fig. 2A). Thesections stained in the absence of the primary antibody orwith the AL47 antibody preadsorbed with recombinant GHRprotein did not elicit a signal (data not shown). Furthermore,staining of tissues from the GHR K/O mouse failed to elicita specific signal (Fig. 2A). To determine the precise cellularlocalization of GHR in the glomerulus, we next ascertainedthe localization of GHR immunoreactivity with respect topodocyte proteins synaptopodin and Wt1. Indirect immu-nohistochemical analysis of mouse kidney sections revealedthat a significant portion of the GHR immunoreactivity co-localized with synaptopodin (Fig. 2B) and Wt1 (Fig. 2C)expression in the glomerulus, supporting the conclusion thatGHR is expressed in the glomerular podocyte. We also usedimmunocytochemical analysis to ascertain the expressionand localization of GHR protein in MPC-5 podocyte cells.These results indicate that GHR immunoreactivity was pre-dominantly localized to the cytoplasmic and nuclear regionsof differentiated MPC-5 cells (Fig. 3).

Functional integrity of GHR expressed in the glomerularpodocyte

To verify the functional integrity of GHR expressed inpodocytes, MPC-5 cells were exposed to GH and the acti-vation of canonical GHR signaling pathways ascertained.Western blot analysis of MPC-5 whole-cell lysates estab-lished that GH increased steady-state levels of tyrosyl phos-phorylated JAK2, Stat5a/b, and ERK1/2 (Fig. 4, A and B).Dose-response studies indicated that the maximum responsewas observed at 250–500 ng/ml (Fig. 4A). To exclude thepossibility that these results may be unique to the MPC-5 cellline, we surveyed two disparate podocyte cell lines, one

Reddy et al. • GH Receptor and Glomerular Podocyte Endocrinology, May 2007, 148(5):2045–2055 2047

murine (34) and the other human (31), for activation of GH-dependent signaling cascades. These results reveal that sim-ilar to results obtained with the MPC-5 cells, GH stimulatedcanonical GHR pathways in murine Endlich (Fig. 4C) andhuman podocyte (Fig. 4D) cells.

Effect of GH on the podocyte actin cytoskeleton

GH stimulates actin reorganization and microtubule poly-merization (35, 36). Alteration in the actin cytoskeleton plays acritical role in the functioning of the podocyte (37–39). Wehypothesized that one of the roles of GH/GHR axis in thepodocyte is to regulate podocyte function via alterations in theactin cytoskeleton. To test this hypothesis, MPC-5 cells wereexposed to GH and the formation of ruffles visualized andquantitated using fluorescence microscopy. These results indi-cate that there was a GH-dependent increase in the number of

ruffles (Fig. 5A). To quantitate GH-induced actin polymeriza-tion in MPC-cells, we measured the content of F-actin in thesecells via binding of Alexa Fluor 594 phalloidin. These resultsestablish that levels of total F-actin increased within 5 min ofexposure of MPC-5 cells to GH (Fig. 5B).

GH-dependent changes in mediators of GH’s actions onactin reorganization and polymerization

Having established that GH stimulates actin reorganiza-tion and polymerization in podocytes, we next investigatedthe role of two mediators of GH’s actions on actin polymer-ization, FAK and SH2-B�. FAK is a cytoplasmic nonreceptortyrosine kinase that serves as an effector molecule linkingactin reorganization with transcriptional events (40, 41). Pre-vious studies demonstrated that stimuli such as albumin canstimulate FAK activation in glomerular podocytes (41). Fur-

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FIG. 1. Expression of GHR in podocytes. A, GHR mRNA expression in podocytes. Total RNA was isolated from liver, glomeruli, and differ-entiated MPC-5 cells and GHR mRNA abundance measured by fluorescent 5�-nuclease (TaqMan) real-time RT-PCR analysis. Expression ofthe housekeeping gene GAPDH was used as an internal control to normalize results. The results (n � 3–5) are depicted as mean and range.The relative amounts of the transcript in the glomerulus and the MPC-5 cells are depicted relative to the expression in liver. *, P � 0.05(Kruskal-Wallis test), compared with liver and glomerulus. B, Detection of GHR protein in mouse tissues and isolated murine glomeruli (leftpanel) and differentiated MPC-5 cells (right panel). Left (top) panel, Western blot analysis for GHR protein in whole-cell lysates from liver (lanes1 and 2), kidney (lanes 3 and 6), or glomerular isolate (two individual samples are shown in lanes 4 and 5) of either wild-type (lanes 1 and 3–5)or GHR-K/O mice (lanes 2 and 6) are shown. Whole-cell lysates from 3T3-F442A preadipocyte cells (lane 7) were also analyzed as a positivecontrol for GHR expression. Equal amounts of protein were size fractionated by SDS-PAGE and Western blotted with anti-GHR AL47 antibody.The position of the specific GHR (�115 kDa) and nonspecific (NS; �82 kDa) bands are indicated. Left (bottom) panel, Overexposure of lanes4–6 to demonstrate expression of GHR in isolated glomeruli. Right panel, Immunoprecipitate of differentiated MPC-5 cell lysates with AL47antibody (lane 8) or differentiated MPC-5 cell lysates (lane 9) were size fractionated by SDS-PAGE and Western blotted with anti-GHR AL47antibody. Results are representative of two independent experiments. C, Validation of specificity of AL47 anti-GHR antibody. Equal amountsof protein form either GHR-K/O (lanes 1, 3, and 5) or wild-type mice (lanes 2, 4, and 6) were size fractionated by SDS-PAGE and Western blottedwith anti-GHR AL47 antibody either not preadsorbed (lanes 1 and 2) or preadsorbed with either GST-GHR recombinant protein (lanes 3 and4) or GST (lanes 5 and 6). The position of the specific GHR (�115 kDa) and nonspecific (NS; �82 kDa) bands are indicated.

2048 Endocrinology, May 2007, 148(5):2045–2055 Reddy et al. • GH Receptor and Glomerular Podocyte

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FIG. 2. Immunohistochemical detection of GHR expression in renal glomerulus and podocytes. A, Immunohistochemical detection of GHRexpression in the renal glomerulus of wild-type (upper panel) or GHR-K/O (lower panel) mouse. GHR expression in kidney section from mice(8 wk old) was detected using anti-GHR antibody AL47 directed against the cytoplasmic domain of the GHR. The binding of the primary antibodywas visualized by light microscopy using the avidin-biotin horseradish peroxidase methodology and the slide counterstained with hematoxylin.The specific staining of glomerular cells is indicated by red arrowheads. B, Colocalization of GHR and synaptopodin (Synpo) in the renalglomerulus of wild-type (upper panel) or GHR-K/O (middle panel). Kidney sections from mice (8 wk old) were stained with primary antibodiesdirected against the cytoplasmic domain of GHR or synaptopodin. The specific binding of the primary antibodies was detected by immuno-fluorescence using either FITC-labeled goat antirabbit IgG (GHR) or rhodamine-labeled goat antimouse IgG (synaptopodin). Colocalization ofGHR and synaptopodin is illustrated in the merged images (Merged). A negative control, incubated only with secondary antibody, is also shown(Control). C, Colocalization of GHR and Wt1 in the mouse glomerulus. Kidney sections from mice (8 wk old) were stained with primary antibodiesdirected against the cytoplasmic domain of GHR or Wt1. The specific binding of the primary antibodies was detected by immunofluorescenceusing either rhodamine-labeled goat antirabbit IgG (GHR) or Oregon Green-labeled goat antimouse IgG (Wt-1). Colocalization of GHR and Wt-1is illustrated in the merged image (Merged). Negative controls incubated only with secondary antibodies are also shown.

GHR API

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FIG. 3. Immunocytochemical detection of GHR expressionin MPC-5 podocytes. Differentiated MPC-5 cells werestained for GHR with the AL47 antibody and counter-stained with DAPI and the images visualized using a flu-orescence microscope. The merged image (Merged) and thenegative control stained only with the secondary antibodyare indicated.

Reddy et al. • GH Receptor and Glomerular Podocyte Endocrinology, May 2007, 148(5):2045–2055 2049

thermore, GH has been shown to stimulate activation of FAKin CHO fibroblasts and osteoblast-like Saos2 cells (42). Toinvestigate the role of FAK in GH’s actions on the podocyte,differentiated MPC-5 podocytes were stimulated with GHand the cell lysates analyzed by Western blot analysis forabundance of tyrosine phosphorylated FAK. These resultsindicate that GH increases levels of tyrosine phosphorylatedFAK in podocytes with the maximal activation being ob-served within 5–10 min of exposure to GH (Fig. 5C).

Prior studies established the essential role of the JAK2-adapter protein SH2-B� in mediating GH’s actions on actinreorganization and cell motility (36, 43). To investigate therole of SH2-B� in GH’s actions on the podocyte, differenti-ated MPC-5 podocytes were stimulated with GH (500 ng/ml)and the cells stained for SH2-B� and actin. In the absence ofexposure to GH, SH2-B� is predominantly localized to nu-cleus (Fig. 6, B and C). Stimulation of the podocyte with GHresults in formation of membrane ruffles and migration ofSH2-B� to the leading edge of the ruffles (Fig. 6, E and F).These results indicate that canonical GHR signaling path-ways implicated in GH-stimulated actin reorganization areactive in the podocyte.

GH increases levels of ROS in the glomerular podocyte

The podocyte has been implicated in the pathogenesis ofdiabetic nephropathy. GH has also been shown to play a

critical permissive role in diabetic nephropathy. Increase inoxidative stress is a factor that has been implicated in thepathogenesis of diabetic nephropathy. Activation of the GH/IGF-I axis is associated with increased oxidative stress (44).We hypothesized that one of the mechanisms whereby GHcould play a role in diabetic nephropathy is via increase inoxidative stress in podocytes. To test this hypothesis, weexposed MPC-5 podocytes to GH and measured ROS levelsusing the immunofluorescence dye CM-H2DCFDA. The in-crease in ROS was quantitated via both spectrofluorometricmeasurements of immunofluorescence (Fig. 7A) and by di-rect visualization of cells staining positive for immunofluo-rescence (Fig. 7B). These results demonstrate that GH stim-ulated a time-dependent increase in ROS levels in these cells(Fig. 7). The increase was maximal at 15–30 min, and theeffect was abrogated in the presence of the antioxidant N-ace-tylcysteine (Fig. 7A).

Discussion

The major findings of this study are that GHRs are ex-pressed on the glomerular podocyte and that podocytes dis-play GH-dependent activation of canonical GHR signalingpathways. Furthermore, our studies indicate that GH stim-ulates alterations in the organization of the podocyte actincytoskeleton and GH increases levels of ROS in these cells.

Our studies support the conclusion that functional GHRs

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MPC-5 mouse podocyte cells

Endlich mouse podocyte cells

FIG. 4. GH stimulates canonical GHR signaling pathways in podocytes. A, Differentiated MPC-5 cells were serum starved for 12–16 h and thenexposed to indicated doses of GH for 10–15 min. The cells were washed with PBS; lysed in radioimmunoprecipitation assay (RIPA) buffer; equalamounts of protein size fractionated by SDS-PAGE; and then immunoblotted with the indicated state-specific antibody. Specific signals werevisualized using the enhanced chemiluminescence (ECL) system. B, Differentiated MPC-5 cells were serum starved for 12–16 h and then exposedto GH (500 ng/ml) for 10–15 min. The cells were washed with PBS, lysed in RIPA buffer, equal amounts of protein size fractionated by SDS-PAGE,and then immunoblotted with the indicated state-specific antibody. Specific signals were visualized using the ECL system. The molecularweights of the specific bands are indicated. C, Differentiated Endlich podocyte cells were deprived of serum for 16 h and treated with or withoutGH (500 ng/ml) for 10–15 min and cell lysates immunoblotted with indicated state-specific antibody. D, Differentiated human podocytes weredeprived of serum for 16 h and treated with or without GH (500 ng/ml) for 10–15 min and cell lysates immunoblotted with indicated state-specificantibody. Results are representative of three independent experiments.

2050 Endocrinology, May 2007, 148(5):2045–2055 Reddy et al. • GH Receptor and Glomerular Podocyte

are expressed in the podocyte. To the best of our knowledge,this is the first report to establish expression of GHRs inpodocytes and verify the functional integrity of these recep-tors. Multiple lines of evidence support the conclusion thatGHRs are expressed on podocytes. These include detectionof GHR mRNA and protein in whole-cell lysates from intactmouse glomeruli and MPC-5 podocyte cells and colocaliza-tion of GHR immunoreactivity with that of canonical podo-cyte proteins Wt1 and synaptopodin. The demonstration ofactivation of canonical GHR signaling pathways includingGH-dependent increase in the abundance of tyrosine phos-phorylated JAK2, STAT5b, and ERK1/2, and changes in theintracellular distribution of the adapter molecule SH2-B�,verifies the functional integrity of these receptors. We ex-cluded the possibility that our results represent detection ofthe alternatively spliced product, GH binding protein be-cause the primers used in the real-time PCR assay and theantibody used in the immunodetection studies (immunohis-tochemistry and Western blot analysis) were both directed atthe intracytoplasmic portion of the GHR unique to the intactreceptor. A previous study that examined the expression ofthe GHR/IGF-I system in the kidney concluded that GHRmRNA was expressed exclusively in the proximal straight

tubule (45). More recently Doi et al. (46) used RT-PCR todetect the presence of GHR mRNA in glomeruli.

The present study extends these findings with the dem-onstration of GHR expression in the glomerular podocyte.Our studies, however, do not exclude the possibility thatGHR may also be expressed in other components of theglomeruli has been previously demonstrated for culturedmesangial cells (46). Indeed our immunohistochemical anal-ysis (Fig. 2) indicates that the GHR signal within the glo-merulus did not exclusively colocalize with podocyte mark-ers, suggesting that GHR may also be expressed inextrapodocyte components of the glomerulus. Our immu-nohistochemistry results indicate that the GHR is present inthe podocyte cytoplasm and, based on the observed colo-calization with the nuclear protein Wt1, also in the podocytenucleus. Whereas the canonical location of GHR is on theplasma membrane, previous reports established that GHRcan also be detected in the cytoplasm and nucleus (47, 48).GHR undergoes extensive recycling within the cytoplasm,and GHR within this compartment could contribute to theimmunoreactivity observed in the cytoplasm. The explana-tion and physiological significance for the nuclear localiza-tion of GHR in cells remains unclear. Whereas a significant

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FIG. 5. GH induces rearrangement of actin cytoskeleton and stimulates tyrosine phosphorylation of FAK in podocytes. A, MPC-5 cells (104

cells/well) were allowed to differentiate for 14 d. After differentiation, the cells were serum starved for 24 h and exposed to vehicle or GH (500ng/ml) for 10–15 min. The fixed cells were then stained for F-actin and nuclei with Alexa Fluor 594 phalloidin and DAPI, respectively, and thestaining visualized (magnification, �60) by immunofluorescence for detection of stress fiber formation and membrane ruffling. In the illus-tration, membrane ruffles are indicated by white arrows. Bars represent mean � SE (n � 3) number of ruffles per 100 cells counted. *, P � 0.01(Mann-Whitney test), compared with exposure to vehicle only (Control). B, Quantitation of GH-induced increase in F-actin levels. DifferentiatedMPC-5 podocytes cells were exposed to GH (100 ng/ml) or vehicle (solid bar) for varying time periods, fixed with formalin, and stained for F-actin(Alexa Fluor 594-phalloidin) and nuclei (DAPI). Bound phalloidin was extracted and the F-actin content per cell was calculated as the ratioof phalloidin/DAPI fluorescence to normalize for cell number. Results (mean � SE; n � 5) are depicted as relative to F-actin content with exposureto vehicle. *, P � 0.05 (Kruskal-Wallis test), compared with exposure to vehicle only. C, Time course of FAK tyrosine phosphorylation inducedby GH in MPC-5 cells. Differentiated MPC-5 cells were exposed to GH (100 ng/ml) or vehicle for varying time periods and whole-cell lysatesprepared and immunoprecipitated with polyclonal antiserum against FAK. Immunoprecipitates were analyzed by SDS-PAGE followed byWestern blotting with antiphosphotyrosine antibody to detect phosphorylated FAK. The membrane was then stripped and reblotted withanti-FAK antiserum to detect total FAK. These results shown are the representatives of two independent experiments.

Reddy et al. • GH Receptor and Glomerular Podocyte Endocrinology, May 2007, 148(5):2045–2055 2051

proportion of GH’s biological effects in the intact animal aremediated by IGF-I, recent studies highlighted selected tis-sue/cell responses to GH that are independent of IGF-I (49–51). Analysis of RNA by semiquantitative RT-PCR revealedthat stimulation of the podocyte for 15–30 min with GH failedto stimulate a significant increase in the steady-state abun-dance of IGF-I mRNA in the podocyte (data not shown).These results suggest that in this model system, the observedeffects of GH are, at least in part, independent of IGF-I.

Our studies indicate that GH stimulates reorganization ofthe podocyte actin cytoskeleton. Podocytes are highly dif-ferentiated visceral epithelial cells with a complex cellularmorphology located inside the kidney glomerulus (12, 52).Podocyte foot processes are anchored to the glomerular base-ment membrane via �3�1-integrin and �- and �-dystrogly-cans. Neighboring foot processes are connected by a spe-cialized cell-cell junction, the glomerular slit diaphragm,which represents the main size-selective filter barrier in thekidney. Podocyte dysfunction results in glomerulosclerosisand progressive kidney failure (53, 54). The actin cytoskel-eton plays a critical role in the functioning of the podocyteresulting from dynamic interactions between the cytoskele-ton and podocyte-specific proteins such as podocin andnephrin. The results of the current study demonstrating GH-dependent polymerization and reorganization of the podo-cyte actin cytoskeleton provide a mechanistic link betweenGH action and podocyte dysfunction. One possibility is thatthe GH-dependent reorganization of the actin cytoskeletonresults in abnormal functioning of the slit diaphragm andhence increased permeability of the filtration barrier with

ensuing proteinuria. Another consequence of GH actioncould be podocyte effacement and resulting glomerular dys-function. The validity of these hypotheses will have to betested in future studies that directly address these issues.

The GH/IGF-I axis is casually linked to the pathogenesisof diabetic nephropathy and glomerulosclerosis associatedwith acromegaly. Excessive GH secretion is a hallmark ofpoorly controlled type 1 diabetes mellitus, and GH is impli-cated as a causative factor in the development of microan-giopathic complications of diabetes (3–6). Involvement of theGH/IGF-I axis in the pathogenesis of DM nephropathy isalso strongly suggested from studies of the impact of GHexcess (8, 20) and deficiency (9) on kidney growth and func-tion in various animal models, both nondiabetic and diabetic.In the human, a direct relationship has been noted betweenthe activity of the GH/IGF-I axis and renal hypertrophy,microalbuminuria, and glomerulosclerosis (3, 10, 11). Pa-tients with acromegaly display early markers of glomerularinjury including microalbuminuria and excretion of glyco-sylaminoglycans. Furthermore, in the mouse overexpressionof GH results in the development of glomerulosclerosis, sug-gesting a direct link between GH action and glomerularinjury. However, at present it is not clear whether this causalrole of the GH/GHR axis in the pathogenesis of glomerularinjury in conditions such as DM nephropathy is due to directactions of GH on the kidney or indirect due to effects onsystemic vascular tone and /or blood pressure. The resultsof the current study demonstrating direct effects of GH on thepodocyte support the hypothesis that GH’s role in the patho-genesis of DM nephropathy is, at least in part, due to direct

B SH2- Bββββ / GH(-)A Actin / GH(-) C Merged / GH(-)

F Merged / GH(+ )E SH2- Bββββ / GH(+)D Actin / GH(+)

G SH2- Bββββ blockingpeptide

H Secondary Ab I Normal rabbit serum

FIG. 6. GH-dependent localization of SH2-B� to membrane ruffles in podocytes. MPC-5 immortalized podocytes were stained with Alexa Fluor594 phalloidin and anti-SH2-B� antibody in the presence (D–F) or absence (A–C) of GH (500 ng/ml) and the specific binding of the SH2-B�antibody detected by immunofluorescence using FITC-labeled goat antirabbit IgG. Colocalization of actin and SH2-B� is illustrated in themerged images (C and F). Negative controls include staining for SH2-B� in presence of SH2-B� blocking peptide (G), only the secondary antibody(H), or normal rabbit serum (I). Inset, Higher magnification view of merged C and F illustrating the GH-dependent localization of SH2-B� toruffles (F, white arrows). Results shown are representative of three independent experiments.

2052 Endocrinology, May 2007, 148(5):2045–2055 Reddy et al. • GH Receptor and Glomerular Podocyte

actions of GH on the glomerulus. A schematic representationof the proposed model for GH’s action on the glomerularpodocyte is depicted in Fig. 8.

Diabetic nephropathy is characterized by excessive deposi-tion of extracellular matrix in the kidney, resulting in glomer-ular mesangial expansion and tubulointerstitial fibrosis. Clin-ical and experimental studies indicate that increased oxidativestress, enhanced generation of ROS, and consequent up-regu-lation of TGF� and fibronectin expression play important rolesin the pathogenesis of DM nephropathy (55). High glucose isone of the stimuli identified to stimulate ROS in glomerularmesangial cells (55). Our results demonstrating that GH in-creases levels of ROS in glomerular podocytes suggest that theGH/GHR axis also plays a role in the changes in ROS in thekidney in states such as diabetes mellitus. Several reports sug-gested that the loss of glomerular podocytes precedes and pre-dicts the onset of clinical nephropathy and may be an early andimportant pathological manifestation of diabetic nephropathy

(56, 57). At present the cellular mechanisms that result in podo-cyte abnormalities in DM are not known. In general, increase inROS is associated with increase in apoptosis. Based on ourresults demonstrating that GH increases levels of ROS inglomerular podocytes, we speculate that direct action of GH onthe podocytes in diabetes mellitus plays a role in the glomerularpodocyte loss in diabetic nephropathy.

In summary, the current report establishes that functionalGHRs are expressed on the glomerular podocyte and thatGH has direct actions on the podocyte actin cytoskeleton andstimulates changes in levels of ROS in the podocyte. Theseresults provide a novel mechanistic link between GH’s ac-tions and glomerular dysfunction in diseases such as acro-megaly and type 1 diabetes mellitus.

Acknowledgments

The authors acknowledge the assistance and generous provision ofreagents by Drs. Christin Carter-Su, Jessica Schwartz, Peter Mundel, andDavid Kershaw and members of their respective laboratories. The tech-nical assistance of the Oxidative Core Laboratory (JDFR Center for theStudy of Complications in Diabetes, University of Michigan) is alsoacknowledged.

Received September 19, 2006. Accepted January 23, 2007.Address all correspondence and requests for reprints to: Ram K.

Menon, M.D., University of Michigan Medical School, 1205 MPB Box0718, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718.E-mail: rammenon@umich.edu.

This work was supported in part by Grants DK49845 (to R.K.M.),DK46395 (to S.J.F.), and P60DK-20572 (to Michigan Diabetes Researchand Training Center) from the National Institutes of Health and grantsfrom the American Diabetes Association.

Disclosure Statement: The authors have nothing to disclose.

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FIG. 7. GH increases levels of ROS in podocytes. A, DifferentiatedMPC-5 cells were deprived of serum for 16 h, preincubated withCM-H2DCFDA (Molecular Probes) for 15 min, exposed to either ve-hicle (control) or GH (500 ng/ml) in the absence (open bar) or presence(hatched bar) N-acetylcysteine (NAC) (25 mM) for indicated time pe-riods, washed with PBS, and analyzed for fluorescence at 485 nm(excitation) and 520 nm (emission). Mean � SE; n � 5. *, P � 0.01(Kruskal-Wallis test), compared with 0 time point, **, P � 0.02(Kruskal-Wallis test), compared with absence of N-acetylcysteine. B,Differentiated MPC-5 cells were deprived of serum for 16 h, prein-cubated with CM-H2DCFDA (Molecular Probes) for 15 min, exposedto GH (500 ng/ml) for indicated time periods, washed with PBS, andthen fixed with 3.7% paraformaldehyde. Photomicrographs of immu-nofluorescence are shown. Cells exposed to either vehicle (control) orH2O2 (a canonical stimulator of ROS) are also shown.

FIG. 8. Cartoon of proposed model for GH’s action on the glomerularpodocyte. GH (1) interacts with its receptor (GH receptor) (2) on theglomerular podocyte enabling dimerization of the GH receptor (3) andconsequent activation of the intracellular signaling cascade resultingin changes in the actin cytoskeleton of the podocyte (4). These changesin actin cytoskeleton alter the pore size of the slit diaphragm (5), a sizeselective filtration barrier formed by fenestrated endothelium, podo-cytes, and glomerular basement membrane (GBM). Changes in thesize and/or functioning of the slit diaphragm manifest as alterationsin the filtration process including the appearance of microalbumin-uria in diabetic nephropathy.

Reddy et al. • GH Receptor and Glomerular Podocyte Endocrinology, May 2007, 148(5):2045–2055 2053

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Erratum

In the article “Vascular Lipotoxicity: Endothelial Dysfunction via Fatty-Acid-Induced Reactive OxygenSpecies Overproduction in Obese Zucker Diabetic Fatty Rats” by Ichiro Chinen, Michio Shimabukuro, KenYamakawa, Namio Higa, Toshihiro Matsuzaki, Katsuhiko Noguchi, Shinichiro Ueda, Matao Sakanashi, andNobuyuki Takasu (Endocrinology 148:160-165), the authors note two errors. On page 161, the entire paragraphunder “Immunoprecipitations and Western Blotting” is incorrect. The correct paragraph is as follows:

Western blot analysis was performed as described previously (15). Protein samples (200 �g) were preparedfrom thoracic aortas of four groups of rats and incubated with anti-eNOS (Santa Cruz Biotechnology, SantaCruz, CA) or anti-p47phox (Upstate, Charlottesville, VA) antibodies at 4 C for 16 h on a rocking platform.Fifty microliters of protein G-agarose suspension (Roche, Germany) was then added and incubated for 2 hat 4 C. After washing the beads, according to manufacturer’s manual, the samples were centrifuged at12,000 � g for 20 sec, and were subjected to SDS-PAGE. After transfer onto polyvinylidene difluoride transfermembranes, the membranes were probed with anti-phosphoserine antibody (Chemicon International, Te-mecula, CA), and immunoreactive proteins were detected by chemiluminescence (SuperSignal West PicoChemiluminescent Substrate; Pierce, Rockford, IL). Band intensity was quantified by NIH ImageJ 1.32j, andthe ratio of anti-phosphoserine blot intensity to those of eNOS or p47phox blot intensity was used to representthe enzyme catalytic activities. Lipid peroxidation level of aorta was determined using the TBARS assay kit(ZeptoMetrix Corp., Buffalo, NY).

On page 163, above the left panel in Fig. 2, the text “tyrosine phosphorylation of eNOS” should read “serinephosphorylation of eNOS.”

The authors regret and apologize for any problems these errors may have caused.

Reddy et al. • GH Receptor and Glomerular Podocyte Endocrinology, May 2007, 148(5):2045–2055 2055