help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1285
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reddy, G. R.
Right arrow Articles by Menon, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reddy, G. R.
Right arrow Articles by Menon, R. K.
Endocrinology Vol. 148, No. 5 2045-2055
Copyright © 2007 by The Endocrine Society

Identification of the Glomerular Podocyte as a Target for Growth 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 of Biomedical Sciences (E.O.L., J.J.K.), College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701; and Department of Internal Medicine (S.J.F.), University of Alabama, Birmingham, Alabama 35294

Address all correspondence and requests for reprints to: Ram K. Menon, M.D., University of Michigan Medical School, 1205 MPB Box 0718, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718. E-mail: rammenon{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH excess in both the human and transgenic animal models is characterized by significant changes in blood pressure and renal function. The GH/GH receptor (GHR) axis is also implicated in the development of diabetic nephropathy. However, it is not clear whether GH’s actions on renal function are due to indirect actions mediated via changes in blood pressure and vascular tone or due to direct action of GH on the kidney. We hypothesized that functional GHRs are expressed on the glomerular podocyte enabling direct actions of GH on glomerular function. Real-time PCR, immunohistochemistry, and Western blot analysis of murine podocyte cells (MPC-5) and kidney glomeruli demonstrated expression of GHR mRNA and protein. Exposure of both murine and human podocytes to GH (50–500 ng/ml) resulted in an increase in abundance of phosphorylated signal transducer and activator of transcription-5, Janus kinase-2, and ERK1/2 proteins. Exposure of podocytes to GH also caused changes in the intracellular distribution of the Janus kinase-2 adapter protein Src homology 2-Bß, stimulation of focal adhesion kinase, increase in reactive oxygen species, and GH-dependent changes in the actin cytoskeleton. We conclude that glomerular podocytes express functional GHRs and that GH increases levels of reactive oxygen species and induces reorganization of the actin cytoskeleton in these cells. These results provide a novel mechanistic link between GH’s actions and glomerular dysfunction in disorders such as acromegaly and diabetic glomerulosclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PLEIOTROPIC ACTIONS of pituitary GH include effects on the blood pressure and fluid homeostasis. GH excess in both the human (acromegaly) and transgenic animal models is characterized by significant structural and functional changes in the kidney (1, 2). The GH/GH receptor (GHR) axis is implicated as a causative factor in the development of diabetic nephropathy (3, 4, 5, 6). Involvement of the GH/IGF-I axis in the pathogenesis of diabetic nephropathy is supported by studies of the impact of GH excess (7, 8) and deficiency (9) on kidney growth and function in various animal models, both nondiabetic and diabetic. In the human a direct relationship has been noted between the activity of the GH/IGF-I axis and renal hypertrophy, microalbuminuria, and glomerulosclerosis (3, 10, 11). Elevated mean 24-h concentrations of circulating GH and an exaggerated GH response to several physiological and pharmacological provocative stimuli are characteristic features of patients with poorly controlled type 1 diabetes mellitus (4, 5, 12, 13, 14, 15, 16, 17). In a rodent model of type 1 diabetes mellitus, GHR signaling pathways were determined to be more active in the kidneys of diabetic (DM) animals, compared with control animals (18). A more direct role for GH/GHR in the pathogenesis of DM nephropathy is supported by studies using GH and GH antagonist transgenic mice, total GHR knockout mice, and pharmacological blockade of the GHR using pegvisomant (7, 19, 20, 21). These studies demonstrate that absence of functional GHR confers a protective effect against DM nephropathy in murine models of type 1 diabetes mellitus. However, at the present time, it is unclear whether the role of GH in the pathogenesis of acromegalic and DM nephropathy is due to direct action(s) of GH on the kidney or indirect due to effects of GH on systemic vascular tone and/or blood pressure (22).

The aim of the current study was to investigate the hypothesis that GH affects glomerular function via direct action on the glomerular podocyte. Our results support the conclusion that functional GHRs are expressed on the glomerular podocyte and GH alters functioning of the podocyte.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Anti-GHR antibody AL47 (23, 24) was used in 1:1000 dilution for Western blot analyses, and 1:100 dilution for immunohistochemistry studies. In certain experiments the AL47 antibody was preadsorbed by incubation for 60 min at room temperature with recombinant glutathione-S-transferase (GST)-epitope tagged GHR protein or GST (as control) before using the antibody in either Western blot or immunohistochemistry experiments. Rabbit polyclonal anti-Src homology 2 (SH2)-Bß (25) (gift from Christin Carter-Su, University of Michigan) and mouse anti-synaptopodin monoclonal antibody (gift from Peter Mundel, Mount Sinai School of Medicine, New York, NY) (26) were used in immunohistochemistry 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) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Erk1/2 and -pErk1/2 (both used in 1:10,000 dilution) antibodies were purchased from Promega (Madison, WI); anti-focal adhesion kinase (FAK) (1:2,500 dilution) antibodies was purchased from Transduction Laboratories (San Diego, CA). The secondary antibodies used for Western blot analysis were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), and the secondary antibodies used for immunofluorescence studies were rhodamine-labeled goat antirabbit IgG (Molecular Probes, Eugene, OR), green-labeled goat antimouse IgG (Molecular Probes), green-labeled donkey antichicken IgG (Molecular Probes), 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, Wilmington, MA) were used for the present study. The GHR null [knockout (K/O)] mice (genetic background, C57BL/6J) with targeted deletion of the GHR have been described before (27). The intact kidney was harvested and processed for histological analysis. A portion of the kidney was fixed in 10% phosphate-buffered formalin before paraffin embedding. Another portion was immediately frozen in Tissue-Tek optimum cutting temperature compound before cryosectioning. The remaining portion of the kidney was snap frozen in liquid nitrogen and stored at –80 C for protein extraction. In other instances the mice were perfused with ferrous solution and glomeruli isolated from the kidneys of the perfused mice using established methods (28). All animal protocols were approved by the University of Michigan Animal Care and Use Committee.

Cell culture
Conditionally immortalized mouse podocyte cells (MPC-5) (29) and those described by Endlich et al. (30) isolated from immortmouse, were grown according to the protocol described by Mundel et al. (29). Briefly, cells were cultured under growth-permissive conditions on rat tail collagen type I-coated plastic dishes (BD Bioscience, Franklin Lakes, NJ) at 33 C with 100% relative humidity and 5% CO2, in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Life Technologies, Inc.-BRL, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Inc.-BRL), and 10 U/ml mouse recombinant {gamma}-interferon (Sigma). To induce differentiation, podocytes were maintained in nonpermissive conditions at 37 C without {gamma}-interferon for 14 d before experimentation. Under these culture conditions, cells stopped proliferating and were identified as differentiated podocytes by their arborized morphology. Cells were routinely maintained for 24 h in serum-free medium before experimentation. Human podocytes (31) were cultured in RPMI 1640 medium with glutamine (Sigma) supplemented with 10% fetal calf serum (Life Technologies) and insulin transferrin sodium selenite (1 ml per 100 ml; Sigma). These cells were induced to differentiate using a protocol similar to that outlined above for the murine podocytes.

Immunohistochemistry and immunocytochemistry
Antigen was retrieved from deparaffinized kidney sections using an antigen unmasking solution (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was blocked by incubating the slides at room temperature in 80% methanol and 30% hydrogen peroxide for 20 min on a shaker. Nonspecific binding was blocked with diluted normal horse serum (Vectastain Universal Elite ABC kit; Vector) for 20 min at room temperature. The sections were then incubated overnight at 4 C with anti-GHR AL47 antibody. After thorough washing with PBS, the sections were blotted with biotinylated secondary antibody and developed with Nova Red and counterstained with hematoxylin.

Differentiated MPC-5 cells cultured on chambered slides were fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were then washed 3 x 5 min with PBS to remove the paraformaldehyde and treated 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 room temperature and then incubated with primary antibodies GHR AL47 or rabbit anti-SH2Bß and Alexa Fluor 594 phalloidin in PBS containing 5% nonfat dry milk. After three washes in PBS, cells were incubated with FITC-conjugated antirabbit (1:100; Sigma) secondary antibodies for 30 min at room temperature. The cells were mounted with Prolong Gold Anti-Fade with 4',6'-diamino-2-phenylindole (DAPI; Molecular Probes) and examined by fluorescence microscopy.

Fluorescence microscopy
For immunofluorescence staining, kidney sections were blocked with the normal serum and incubated with the primary antibody overnight at 4 C. Sections were then washed with PBS for 3 x 5 min and incubated with secondary antibodies (rhodamine-labeled antirabbit and FITC-labeled antimouse antibodies) for 90 min at room temperature and mounted with Prolong Gold Anti-Fade with DAPI (Molecular Probes). Immunofluorescence microscopy was performed using an Eclipse TE200 fluorescent microscope (Nikon, Tokyo, Japan).

RNA isolation and real-time semiquantitative PCR
Total RNA was prepared from glomeruli and liver samples using TriZol-reagent as specified by the manufacturer (Molecular Research Center, Cincinnati, OH) and quantitated by the absorbance at 260 nm. Quantitation of GHR and IGF-I mRNA expression was determined by fluorescent 5'-nuclease (TaqMan) real-time RT-PCR using the following primers: GHR forward, 5'-CAGTTCCAAAGATTAAAGGGATTGA-3', GHR reverse, 5'-TTATCATGAATGCCTAAGATGGTGTT-3'; GHR probe, 5'-ACCTCCTCCAACTTCCCTCCCTTGAGAA-3'; IGF-I forward, 5'-GCT CTG CTT GCT CAC CTT CAC-3', IGF-I reverse, 5'-CAC ACG AAC TGA AGA GCA TCC A-3'; and IGF-I probe, 5'-AGC TCC ACC ACA GCT GGA CCA GAG A-3'.

The primers and TaqMan probes for quantitation of the GHR and IGF-I transcripts were designed using the primer design software Primer Express (PE Biosystems, Foster City, CA); GHR primers were specific for the intracytoplasmic portion of the GHR. The primers and TaqMan probe for rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from a commercial vendor (PE Biosystems). The GHR and IGF-I probes were labeled with fluorescent reporter dye VIC and the GAPDH probe labeled with FAM. Normalization and validation of the data were carried out using GAPDH as the housekeeping control. The comparative Ct values method was used to calculate the relative quantity 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, the samples were subjected to PCR analysis using cycling parameters: 95 C x 10 min; 95 C x 15 sec->60 C x 1 min for 40 cycles. Each sample was analyzed in triplicate in individual assays performed on two or more occasions.

Western blot analysis
Isolated glomeruli and MPC-5 cells were homogenized in 100 µl of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, 1 mmol/liter sodium pyrophosphate, 10 mmol/liter sodium fluoride, and 1 mmol/liter sodium orthovanadate. The protein concentration was quantitated using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) and equal amounts of protein resolved on 10 or 4–16% gradient SDS-polyacrylamide gels as indicated and blotted onto nitrocellulose membrane for Western blot analysis. The blot was probed with suitable primary and secondary antibodies and was developed by the ECL chemiluminescent method (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. Where indicated immunoprecipitation was performed by incubating lysates with the respective antiserum and immunoprecipitates collected using protein A/G agarose beads. Immunoprecipitates were subjected to SDS-PAGE and Western blotting as described above.

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

Quantitative measurement of F-actin levels
F-actin was quantitated using a previously described phalloidin binding 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, fixed with treatment with 3.7% formalin for 20 min, and then transferred into buffer of 60 mM 1,4-piperazine diethane sulfonic acid, 25 mM HEPES, 10 mM EGTA, 2 mM Mg SO4, and 0.5% Triton X-100. The fixed cells were then stained for F-actin with Alexa Fluor 594 phalloidin (0.6 U/ml), and to normalize for cell number, the nuclei were stained with DAPI (2 µg/ml). The cells were stained for 30 min and then washed 3 x 5 min with PBS. Bound phalloidin was extracted by treatment with 100 µl methanol for 1 h in the dark. Alexa Fluor 594 phalloidin fluorescence (excitation 585 nm, emission 608 nm) and DAPI fluorescence (excitation 358 nm, emission 461 nm) were measured simultaneously using a spectrofluorimeter (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), overnight serum starved, and then incubated with 5 µM CM-H2DCFDA (Molecular Probes) for 30 min at 37 C followed by exposure to either vehicle or GH (500 ng/ml) in the absence or presence of N-acetylcysteine (25 mM) for varying time periods. CM-H2CDFDA is a nonfluorescent molecule that becomes fluorescent after oxidation in the cytoplasm of cells. Fluorescence was quantitated via a dual-scanning microplate spectrofluorometer (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 were performed to analyze statistical significance of the difference between the 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GHR mRNA and protein expression in the murine glomerular podocyte
Real-time RT PCR analysis was used to quantify expression of GHR mRNA in murine podocytes, glomeruli, and liver. To avoid detecting the alternatively spliced transcript encoding the GH binding protein, the primers used were specific for the intracytoplasmic portion of the GHR. These results demonstrated that the relative expression of GHR mRNA, normalized to GAPDH expression, was comparable in the glomeruli and liver, with less expression in differentiated MPC-5 cells (Fig. 1AGo).


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 1. Expression of GHR in podocytes. A, GHR mRNA expression in podocytes. Total RNA was isolated from liver, glomeruli, and differentiated MPC-5 cells and GHR mRNA abundance measured by fluorescent 5'-nuclease (TaqMan) real-time RT-PCR analysis. Expression of the 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 (left panel) and differentiated MPC-5 cells (right panel). Left (top) panel, Western blot analysis for GHR protein in whole-cell lysates from liver (lanes 1 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 positive control 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 lanes 4–6 to demonstrate expression of GHR in isolated glomeruli. Right panel, Immunoprecipitate of differentiated MPC-5 cell lysates with AL47 antibody (lane 8) or differentiated MPC-5 cell lysates (lane 9) were size fractionated by SDS-PAGE and Western blotted with anti-GHR AL47 antibody. Results are representative of two independent experiments. C, Validation of specificity of AL47 anti-GHR antibody. Equal amounts of 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 blotted with anti-GHR AL47 antibody either not preadsorbed (lanes 1 and 2) or preadsorbed with either GST-GHR recombinant protein (lanes 3 and 4) or GST (lanes 5 and 6). The position of the specific GHR (~115 kDa) and nonspecific (NS; ~82 kDa) bands are indicated.

 
To detect expression of GHR protein, extracts from glomeruli and differentiated MPC-5 cells were analyzed by Western blot analysis using anti-GHR AL47 antibody, an antibody directed against the cytoplasmic domain of the GHR. A protein band migrating at approximately 115 kDa was detected by this antibody in both glomerular and MPC-5 cell lysates (Fig. 1BGo), a result consistent with the presence of GHR protein in these samples. The detection of a protein band of a similar molecular weight in MPC-5 cell lysate immunoprecipitates obtained with the AL47 antibody (Fig. 1BGo) provided further evidence of GHR expression in cultured podocytes. In Western blot analysis, the AL47 antibody cross-reacts with a non-GHR (nonspecific) 82-kDa protein band (Fig. 1BGo and Frank, S., personal communication). To verify the specificity of the AL47 antibody, we performed Western blot analysis using the AL47 antibody preadsorbed with recombinant GHR protein. These experiments revealed that the preadsorbed AL47 antibody recognized the nonspecific 82-kDa protein but failed to detect the specific GHR band (Fig. 1CGo), thus verifying the specificity of the AL47 anti-GHR antibody. The specificity of the antibody was also verified by demonstrating absence of the approximately 115-kDa putative GHR band in tissues from the GHR K/O mouse (Fig. 1Go, B and C).

The cellular localization of GHR expression in the renal glomerulus was investigated by immunohistochemical analysis. GHR expression in kidney section from mice (8 wk old) was detected using anti-GHR AL47 antibody. The binding of the primary antibody was visualized by light microscopy using the avidin-biotin HRP methodology and the slide counterstained with hematoxylin. The specific staining of glomerular cells is indicated by red arrowheads (Fig. 2AGo). The sections stained in the absence of the primary antibody or with the AL47 antibody preadsorbed with recombinant GHR protein did not elicit a signal (data not shown). Furthermore, staining of tissues from the GHR K/O mouse failed to elicit a specific signal (Fig. 2AGo). To determine the precise cellular localization of GHR in the glomerulus, we next ascertained the localization of GHR immunoreactivity with respect to podocyte proteins synaptopodin and Wt1. Indirect immunohistochemical analysis of mouse kidney sections revealed that a significant portion of the GHR immunoreactivity colocalized with synaptopodin (Fig. 2BGo) and Wt1 (Fig. 2CGo) expression in the glomerulus, supporting the conclusion that GHR is expressed in the glomerular podocyte. We also used immunocytochemical analysis to ascertain the expression and localization of GHR protein in MPC-5 podocyte cells. These results indicate that GHR immunoreactivity was predominantly localized to the cytoplasmic and nuclear regions of differentiated MPC-5 cells (Fig. 3Go).


Figure 2
View larger version (54K):
[in this window]
[in a new window]

  		FIG. 2. Immunohistochemical detection of GHR expression in renal glomerulus and podocytes. A, Immunohistochemical detection of GHR expression 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 antibody was 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 renal glomerulus of wild-type (upper panel) or GHR-K/O (middle panel). Kidney sections from mice (8 wk old) were stained with primary antibodies directed against the cytoplasmic domain of GHR or synaptopodin. The specific binding of the primary antibodies was detected by immunofluorescence using either FITC-labeled goat antirabbit IgG (GHR) or rhodamine-labeled goat antimouse IgG (synaptopodin). Colocalization of GHR 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 antibodies directed against the cytoplasmic domain of GHR or Wt1. The specific binding of the primary antibodies was detected by immunofluorescence using either rhodamine-labeled goat antirabbit IgG (GHR) or Oregon Green-labeled goat antimouse IgG (Wt-1). Colocalization of GHR and Wt-1 is illustrated in the merged image (Merged). Negative controls incubated only with secondary antibodies are also shown.

 

Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 3. Immunocytochemical detection of GHR expression in MPC-5 podocytes. Differentiated MPC-5 cells were stained for GHR with the AL47 antibody and counterstained with DAPI and the images visualized using a fluorescence microscope. The merged image (Merged) and the negative control stained only with the secondary antibody are indicated.

 
Functional integrity of GHR expressed in the glomerular podocyte
To verify the functional integrity of GHR expressed in podocytes, MPC-5 cells were exposed to GH and the activation of canonical GHR signaling pathways ascertained. Western blot analysis of MPC-5 whole-cell lysates established that GH increased steady-state levels of tyrosyl phosphorylated JAK2, Stat5a/b, and ERK1/2 (Fig. 4Go, A and B). Dose-response studies indicated that the maximum response was observed at 250–500 ng/ml (Fig. 4AGo). To exclude the possibility that these results may be unique to the MPC-5 cell line, we surveyed two disparate podocyte cell lines, one murine (34) and the other human (31), for activation of GH-dependent signaling cascades. These results reveal that similar to results obtained with the MPC-5 cells, GH stimulated canonical GHR pathways in murine Endlich (Fig. 4CGo) and human podocyte (Fig. 4DGo) cells.


Figure 4
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 4. GH stimulates canonical GHR signaling pathways in podocytes. A, Differentiated MPC-5 cells were serum starved for 12–16 h and then exposed to indicated doses of GH for 10–15 min. The cells were washed with PBS; lysed in radioimmunoprecipitation assay (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 enhanced chemiluminescence (ECL) system. B, Differentiated MPC-5 cells were serum starved for 12–16 h and then exposed to 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 molecular weights of the specific bands are indicated. C, Differentiated Endlich podocyte cells were deprived 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-specific antibody. D, Differentiated human podocytes were deprived 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-specific antibody. Results are representative of three independent experiments.

 
Effect of GH on the podocyte actin cytoskeleton
GH stimulates actin reorganization and microtubule polymerization (35, 36). Alteration in the actin cytoskeleton plays a critical role in the functioning of the podocyte (37, 38, 39). We hypothesized that one of the roles of GH/GHR axis in the podocyte is to regulate podocyte function via alterations in the actin cytoskeleton. To test this hypothesis, MPC-5 cells were exposed to GH and the formation of ruffles visualized and quantitated using fluorescence microscopy. These results indicate that there was a GH-dependent increase in the number of ruffles (Fig. 5AGo). To quantitate GH-induced actin polymerization in MPC-cells, we measured the content of F-actin in these cells via binding of Alexa Fluor 594 phalloidin. These results establish that levels of total F-actin increased within 5 min of exposure of MPC-5 cells to GH (Fig. 5BGo).


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		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 (500 ng/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 the staining visualized (magnification, x60) by immunofluorescence for detection of stress fiber formation and membrane ruffling. In the illustration, 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. Differentiated MPC-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 ratio of phalloidin/DAPI fluorescence to normalize for cell number. Results (mean ± SE; n = 5) are depicted as relative to F-actin content with exposure to vehicle. *, P < 0.05 (Kruskal-Wallis test), compared with exposure to vehicle only. C, Time course of FAK tyrosine phosphorylation induced by 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 lysates prepared and immunoprecipitated with polyclonal antiserum against FAK. Immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine antibody to detect phosphorylated FAK. The membrane was then stripped and reblotted with anti-FAK antiserum to detect total FAK. These results shown are the representatives of two independent experiments.

 
GH-dependent changes in mediators of GH’s actions on actin reorganization and polymerization
Having established that GH stimulates actin reorganization and polymerization in podocytes, we next investigated the role of two mediators of GH’s actions on actin polymerization, FAK and SH2-Bß. FAK is a cytoplasmic nonreceptor tyrosine kinase that serves as an effector molecule linking actin reorganization with transcriptional events (40, 41). Previous studies demonstrated that stimuli such as albumin can stimulate FAK activation in glomerular podocytes (41). Furthermore, GH has been shown to stimulate activation of FAK in CHO fibroblasts and osteoblast-like Saos2 cells (42). To investigate the role of FAK in GH’s actions on the podocyte, differentiated MPC-5 podocytes were stimulated with GH and the cell lysates analyzed by Western blot analysis for abundance of tyrosine phosphorylated FAK. These results indicate that GH increases levels of tyrosine phosphorylated FAK in podocytes with the maximal activation being observed within 5–10 min of exposure to GH (Fig. 5CGo).

Prior studies established the essential role of the JAK2-adapter protein SH2-Bß in mediating GH’s actions on actin reorganization and cell motility (36, 43). To investigate the role of SH2-Bß in GH’s actions on the podocyte, differentiated MPC-5 podocytes were stimulated with GH (500 ng/ml) and the cells stained for SH2-Bß and actin. In the absence of exposure to GH, SH2-Bß is predominantly localized to nucleus (Fig. 6Go, B and C). Stimulation of the podocyte with GH results in formation of membrane ruffles and migration of SH2-Bß to the leading edge of the ruffles (Fig. 6Go, E and F). These results indicate that canonical GHR signaling pathways implicated in GH-stimulated actin reorganization are active in the podocyte.


Figure 6
View larger version (35K):
[in this window]
[in a new window]

  		FIG. 6. GH-dependent localization of SH2-Bß to membrane ruffles in podocytes. MPC-5 immortalized podocytes were stained with Alexa Fluor 594 phalloidin and anti-SH2-Bß antibody in the presence (D–F) or absence (A–C) of GH (500 ng/ml) and the specific binding of the SH2-Bß antibody detected by immunofluorescence using FITC-labeled goat antirabbit IgG. Colocalization of actin and SH2-Bß is illustrated in the merged images (C and F). Negative controls include staining for SH2-Bß in presence of SH2-Bß 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-Bß to ruffles (F, white arrows). Results shown are representative of three independent experiments.

 
GH increases levels of ROS in the glomerular podocyte
The podocyte has been implicated in the pathogenesis of diabetic nephropathy. GH has also been shown to play a critical permissive role in diabetic nephropathy. Increase in oxidative stress is a factor that has been implicated in the pathogenesis 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 GH could play a role in diabetic nephropathy is via increase in oxidative stress in podocytes. To test this hypothesis, we exposed MPC-5 podocytes to GH and measured ROS levels using the immunofluorescence dye CM-H2DCFDA. The increase in ROS was quantitated via both spectrofluorometric measurements of immunofluorescence (Fig. 7AGo) and by direct visualization of cells staining positive for immunofluorescence (Fig. 7BGo). These results demonstrate that GH stimulated a time-dependent increase in ROS levels in these cells (Fig. 7Go). The increase was maximal at 15–30 min, and the effect was abrogated in the presence of the antioxidant N-acetylcysteine (Fig. 7AGo).


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 7. GH increases levels of ROS in podocytes. A, Differentiated MPC-5 cells were deprived of serum for 16 h, preincubated with CM-H2DCFDA (Molecular Probes) for 15 min, exposed to either vehicle (control) or GH (500 ng/ml) in the absence (open bar) or presence (hatched bar) N-acetylcysteine (NAC) (25 mM) for indicated time periods, 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, preincubated with CM-H2DCFDA (Molecular Probes) for 15 min, exposed to GH (500 ng/ml) for indicated time periods, washed with PBS, and then fixed with 3.7% paraformaldehyde. Photomicrographs of immunofluorescence are shown. Cells exposed to either vehicle (control) or H2O2 (a canonical stimulator of ROS) are also shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of this study are that GHRs are expressed on the glomerular podocyte and that podocytes display GH-dependent activation of canonical GHR signaling pathways. Furthermore, our studies indicate that GH stimulates alterations in the organization of the podocyte actin cytoskeleton and GH increases levels of ROS in these cells.

Our studies support the conclusion that functional GHRs are expressed in the podocyte. To the best of our knowledge, this is the first report to establish expression of GHRs in podocytes and verify the functional integrity of these receptors. Multiple lines of evidence support the conclusion that GHRs are expressed on podocytes. These include detection of GHR mRNA and protein in whole-cell lysates from intact mouse glomeruli and MPC-5 podocyte cells and colocalization of GHR immunoreactivity with that of canonical podocyte proteins Wt1 and synaptopodin. The demonstration of activation of canonical GHR signaling pathways including GH-dependent increase in the abundance of tyrosine phosphorylated JAK2, STAT5b, and ERK1/2, and changes in the intracellular distribution of the adapter molecule SH2-Bß, verifies the functional integrity of these receptors. We excluded the possibility that our results represent detection of the alternatively spliced product, GH binding protein because the primers used in the real-time PCR assay and the antibody used in the immunodetection studies (immunohistochemistry and Western blot analysis) were both directed at the intracytoplasmic portion of the GHR unique to the intact receptor. A previous study that examined the expression of the GHR/IGF-I system in the kidney concluded that GHR mRNA was expressed exclusively in the proximal straight tubule (45). More recently Doi et al. (46) used RT-PCR to detect the presence of GHR mRNA in glomeruli.

The present study extends these findings with the demonstration of GHR expression in the glomerular podocyte. Our studies, however, do not exclude the possibility that GHR may also be expressed in other components of the glomeruli has been previously demonstrated for cultured mesangial cells (46). Indeed our immunohistochemical analysis (Fig. 2Go) indicates that the GHR signal within the glomerulus did not exclusively colocalize with podocyte markers, suggesting that GHR may also be expressed in extrapodocyte components of the glomerulus. Our immunohistochemistry results indicate that the GHR is present in the podocyte cytoplasm and, based on the observed colocalization with the nuclear protein Wt1, also in the podocyte nucleus. Whereas the canonical location of GHR is on the plasma membrane, previous reports established that GHR can 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 the immunoreactivity observed in the cytoplasm. The explanation and physiological significance for the nuclear localization of GHR in cells remains unclear. Whereas a significant proportion of GH’s biological effects in the intact animal are mediated by IGF-I, recent studies highlighted selected tissue/cell responses to GH that are independent of IGF-I (49, 50, 51). Analysis of RNA by semiquantitative RT-PCR revealed that stimulation of the podocyte for 15–30 min with GH failed to stimulate a significant increase in the steady-state abundance of IGF-I mRNA in the podocyte (data not shown). These results suggest that in this model system, the observed effects of GH are, at least in part, independent of IGF-I.

Our studies indicate that GH stimulates reorganization of the podocyte actin cytoskeleton. Podocytes are highly differentiated visceral epithelial cells with a complex cellular morphology located inside the kidney glomerulus (12, 52). Podocyte foot processes are anchored to the glomerular basement membrane via {alpha}3ß1-integrin and {alpha}- and ß-dystroglycans. Neighboring foot processes are connected by a specialized cell-cell junction, the glomerular slit diaphragm, which represents the main size-selective filter barrier in the kidney. Podocyte dysfunction results in glomerulosclerosis and progressive kidney failure (53, 54). The actin cytoskeleton plays a critical role in the functioning of the podocyte resulting from dynamic interactions between the cytoskeleton and podocyte-specific proteins such as podocin and nephrin. The results of the current study demonstrating GH-dependent polymerization and reorganization of the podocyte actin cytoskeleton provide a mechanistic link between GH action and podocyte dysfunction. One possibility is that the GH-dependent reorganization of the actin cytoskeleton results in abnormal functioning of the slit diaphragm and hence increased permeability of the filtration barrier with ensuing proteinuria. Another consequence of GH action could be podocyte effacement and resulting glomerular dysfunction. The validity of these hypotheses will have to be tested in future studies that directly address these issues.

The GH/IGF-I axis is casually linked to the pathogenesis of diabetic nephropathy and glomerulosclerosis associated with acromegaly. Excessive GH secretion is a hallmark of poorly controlled type 1 diabetes mellitus, and GH is implicated as a causative factor in the development of microangiopathic complications of diabetes (3, 4, 5, 6). Involvement of the GH/IGF-I axis in the pathogenesis of DM nephropathy is also strongly suggested from studies of the impact of GH excess (8, 20) and deficiency (9) on kidney growth and function in various animal models, both nondiabetic and diabetic. In the human, a direct relationship has been noted between the activity of the GH/IGF-I axis and renal hypertrophy, microalbuminuria, and glomerulosclerosis (3, 10, 11). Patients with acromegaly display early markers of glomerular injury including microalbuminuria and excretion of glycosylaminoglycans. Furthermore, in the mouse overexpression of GH results in the development of glomerulosclerosis, suggesting a direct link between GH action and glomerular injury. However, at present it is not clear whether this causal role of the GH/GHR axis in the pathogenesis of glomerular injury in conditions such as DM nephropathy is due to direct actions of GH on the kidney or indirect due to effects on systemic vascular tone and /or blood pressure. The results of the current study demonstrating direct effects of GH on the podocyte support the hypothesis that GH’s role in the pathogenesis of DM nephropathy is, at least in part, due to direct actions of GH on the glomerulus. A schematic representation of the proposed model for GH’s action on the glomerular podocyte is depicted in Fig. 8Go.


Figure 8
View larger version (53K):
[in this window]
[in a new window]

  		FIG. 8. Cartoon of proposed model for GH’s action on the glomerular podocyte. GH (1 ) interacts with its receptor (GH receptor) (2 ) on the glomerular podocyte enabling dimerization of the GH receptor (3 ) and consequent activation of the intracellular signaling cascade resulting in changes in the actin cytoskeleton of the podocyte (4 ). These changes in actin cytoskeleton alter the pore size of the slit diaphragm (5 ), a size selective filtration barrier formed by fenestrated endothelium, podocytes, and glomerular basement membrane (GBM). Changes in the size and/or functioning of the slit diaphragm manifest as alterations in the filtration process including the appearance of microalbuminuria in diabetic nephropathy.

 
Diabetic nephropathy is characterized by excessive deposition of extracellular matrix in the kidney, resulting in glomerular mesangial expansion and tubulointerstitial fibrosis. Clinical and experimental studies indicate that increased oxidative stress, enhanced generation of ROS, and consequent up-regulation of TGFß and fibronectin expression play important roles in the pathogenesis of DM nephropathy (55). High glucose is one of the stimuli identified to stimulate ROS in glomerular mesangial cells (55). Our results demonstrating that GH increases levels of ROS in glomerular podocytes suggest that the GH/GHR axis also plays a role in the changes in ROS in the kidney in states such as diabetes mellitus. Several reports suggested that the loss of glomerular podocytes precedes and predicts the onset of clinical nephropathy and may be an early and important pathological manifestation of diabetic nephropathy (56, 57). At present the cellular mechanisms that result in podocyte abnormalities in DM are not known. In general, increase in ROS is associated with increase in apoptosis. Based on our results demonstrating that GH increases levels of ROS in glomerular podocytes, we speculate that direct action of GH on the podocytes in diabetes mellitus plays a role in the glomerular podocyte loss in diabetic nephropathy.

In summary, the current report establishes that functional GHRs are expressed on the glomerular podocyte and that GH has direct actions on the podocyte actin cytoskeleton and stimulates changes in levels of ROS in the podocyte. These results provide a novel mechanistic link between GH’s actions and glomerular dysfunction in diseases such as acromegaly and type 1 diabetes mellitus.


   Acknowledgments
 
The authors acknowledge the assistance and generous provision of reagents by Drs. Christin Carter-Su, Jessica Schwartz, Peter Mundel, and David Kershaw and members of their respective laboratories. The technical assistance of the Oxidative Core Laboratory (JDFR Center for the Study of Complications in Diabetes, University of Michigan) is also acknowledged.


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

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 1, 2007

Abbreviations: DAPI, 4',6'-Diamino-2-phenylindole; DM, diabetes mellitus; 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, phosphorylated; ROS, reactive oxygen species; SH2, Src homology 2; Stat, signal transducer and activator of transcription; Wt1, Wilms tumor 1.

Received September 19, 2006.

Accepted for publication January 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wanke R, Wolf E, Hermanns W, Folger S, Buchmuller T, Brem G 1992 The GH-transgenic mouse as an experimental model for growth research: clinical and pathological studies. Horm Res 37(Suppl 3):74–87
  2. Flyvbjerg A 2004 The role of growth hormone in the pathogenesis of diabetic kidney disease. Pediatr Endocrinol Rev 1(Suppl 3):525–529
  3. Blankestijn PJ, Derkx FH, Birkenhager JC, Lamberts SW, Mulder P, Verschoor L, Schalekamp MA, Weber RF 1993 Glomerular hyperfiltration in insulin-dependent diabetes mellitus is correlated with enhanced growth hormone secretion. J Clin Endocrinol Metab 77:498–502[Abstract]
  4. Edge JA, Dunger DB, Matthews DR, Gilbert JP, Smith CP 1990 Increased overnight growth hormone concentrations in diabetic compared with normal adolescents. J Clin Endocrinol Metab 69:1356–1362
  5. Lorenzi M, Karam JH, McIlroy MB, Forsham PH 1980 Increased growth hormone response to dopamine infusion in insulin-dependent diabetic subjects. J Clin Invest 65:146–153[Medline]
  6. Salardi S, Cacciari E, Ballardini D, Righetti F, Capelli M, Cicognani A, Zucchini S, Natali G, Tassinari D 1986 Relationships between growth factors (somatomedin C and growth hormone) and body development, metabolic control, and retinal changes in children and adolescents with IDDM. Diabetes 35:832–836[Abstract]
  7. Chen NY, Chen WY, Bellush L, Yang C-W, Striker LJ, Striker GE, Kopchick JJ 1995 Effects of streptozocin treatment in growth hormone (GH) and GH antagonist transgenic mice. Endocrinology 136:660–667[Abstract]
  8. Doi T, Striker LJ, Gibson CC, Agodoa LYC, Brinster R, Striker GE 1990 Glomerular lesions in mice transgenic for growth hormone and insulinlike growth factor-I. I. Relationship between increased glomerular size and mesangial sclerosis. Am J Pathol 137:541–552[Abstract]
  9. Muchaneta-Kubara EC, Sayed-Ahmed N, Besbas N, Zhang G, Cope GH, el Nahas AM 1994 Experimental diabetic renal growth: role of growth hormone and insulin-like growth factor-I. Nephrol Dial Transplant 9:1395–1401[Abstract/Free Full Text]
  10. Christiansen JS, Gammelgaard J, Orskov H, Andersen AR, Telmer S, Parving HH 1981 Kidney function and size in normal subjects before and during growth hormone administration for one week. Eur J Clin Invest 11:487–490[Medline]
  11. Cummings EA, Sochett EB, Dekker MG, Lawson ML, Daneman D 1998 Contribution of growth hormone and IGF-1 to early diabetic nephropathy in type 1 diabetes. Diabetes 47:1341–1346[Abstract]
  12. Almqvist EG, Groop LC, Manhem PJ 1999 Growth hormone response to the insulin tolerance test and clonidine tests in type 1 diabetes. Scand J Clin Lab Invest 59:375–582[CrossRef][Medline]
  13. Asplin CM, Faria ACS, Carlsen EC, Vaccaro VA, Barr RE, Iranmanesh A, Lee MM, Veldhuis JD, Evans WS 1989 Alterations in the pulsatile mode of growth hormone release in men and women with insulin-dependent diabetes mellitus. Metabolism 69:239–245
  14. Hansen AP 1970 Abnormal serum growth hormone response to exercise in juvenile diabetics. J Clin Invest 49:1467–1478[Medline]
  15. Hayford JT, Danney MM, Hendrix JA, Thompson RG 1980 Integrated concentrations of growth hormone in juvenile-onset diabetes. Diabetes 29:391–398[Abstract]
  16. Krassowski J, Felber JP, Rogala H, Jeske W, Zgliczynski S 1988 Exaggerated growth hormone response to growth hormone-releasing hormone in type I diabetes mellitus. Acta Endocrinol (Copenh) 117:225–229[Abstract/Free Full Text]
  17. Sperling MA, Wollesen F, DeLamater PV 1973 Daily production and metabolic clearance of growth hormone in juvenile diabetics. Diabetologia 9:380–383[CrossRef][Medline]
  18. Thirone AC, Scarlett JA, Gasparetti AL, Araujo EP, Lima MH, Carvalho CR, Velloso LA, Saad MJ 2002 Modulation of growth hormone signal transduction in kidneys of streptozotocin-induced diabetic animals: effect of a growth hormone receptor antagonist. Diabetes 51:2270–2281[Abstract/Free Full Text]
  19. Bellush LL, Doublier S, Holland AN, Striker LJ, Striker GE, Kopchick JJ 2000 Protection against diabetes-induced nephropathy in growth hormone receptor/binding protein gene-disrupted mice. Endocrinology 141:163–168[Abstract/Free Full Text]
  20. Chen NY, Chen WY, Kopchick JJ 1997 Liver and kidney growth hormone (GH) receptors are regulated differently in diabetic GH and GH antagonist transgenic mice. Endocrinology 138:1988–1994[Abstract/Free Full Text]
  21. Flyvbjerg A, Bennett WF, Rasch R, Kopchick JJ, Scarlett JA 1999 Inhibitory effect of a growth hormone receptor antagonist (G120K-PEG) on renal enlargement, glomerular hypertrophy, and urinary albumin excretion in experimental diabetes in mice. Diabetes 48:377–382[Abstract]
  22. Boger RH, Skamira C, Bode-Boger SM, Brabant G, von zur Muhlen A, Frolich JC 1996 Nitric oxide may mediate the hemodynamic effects of recombinant growth hormone in patients with acquired growth hormone deficiency. A double-blind, placebo-controlled study. J Clin Invest 98:2706–2713[Medline]
  23. Zhang Y, Guan R, Jiang J, Kopchick JJ, Black RA, Baumann G, Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. J Biol Chem 276:24565–24573[Abstract/Free Full Text]
  24. Jiang J, Wang X, He K, Li X, Chen C, Sayeski PP, Waters MJ, Frank SJ 2004 A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: impact on GH signaling and GHR proteolysis. Mol Endocrinol 18:2981–2996[Abstract/Free Full Text]
  25. Rui L, Mathews LS, Hotta K, Gustafson TA, Carter-Su C 1997 Identification of SH2-Bß as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. Mol Cell Biol 17:6633–6644[Abstract]
  26. Mundel P, Gilbert P, Kriz W 1991 Podocytes in glomerulus of rat kidney express a characteristic 44 KD protein. J Histochem Cytochem 39:1047–1056[Abstract]
  27. Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Cataldo L, Coschigamo K, Wagner TE, Baumann G, Kopchick JJ 1997 A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 94:13215–13220[Abstract/Free Full Text]
  28. Downer G, Phan SH, Wiggins RC 1988 Analysis of renal fibrosis in a rabbit model of crescentic nephritis. J Clin Invest 82:998–1006[Medline]
  29. Mundel P, Reiser J, Zuniga Mejia Borja A, Pavenstadt H, Davidson GR, Kriz W, Zeller R 1997 Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236:248–258[CrossRef][Medline]
  30. Endlich N, Kress KR, Reiser J, Uttenweiler D, Kriz W, Mundel P, Endlich K 2001 Podocytes respond to mechanical stress in vitro. J Am Soc Nephrol 12:413–422[Abstract/Free Full Text]
  31. Saleem M, O’Hare MJ, Reiser J, Coward RJ, Inward CD, Farren T, Xing CY, Ni L, Mathieson PW, Mundel P2002 A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. J Am Soc Nephrol 13:630–638
  32. Menon RK, Shaufl A, Yu JH, Stephan DA, Friday RP 2001 Identification and characterization of a novel transcript of the murine growth hormone receptor gene exhibiting development- and tissue-specific expression. Mol Cell Endocrinol 172:135–146[CrossRef][Medline]
  33. Howard TH, Oresajo CO 1985 The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils. J Cell Biol 101:1078–1085[Abstract/Free Full Text]
  34. Schiwek D, Endlich N, Holzman L, Holthofer H, Kriz W, Endlich K 2004 Stable expression of nephrin and localization to cell-cell contacts in novel murine podocyte cell lines. Kidney Int 66:91–101[CrossRef][Medline]
  35. Goh EL, Pircher TJ, Lobie PE 1998 Growth hormone promotion of tubulin polymerization stabilizes the microtubule network and protects against colchicine-induced apoptosis. Endocrinology 139:4364–4372[Abstract/Free Full Text]
  36. Herrington J, Diakonova M, Rui L, Gunter DR, Carter-Su C 2000 SH2-B is required for growth hormone-induced actin reorganization. J Biol Chem 275:13126–13133[Abstract/Free Full Text]
  37. Barisoni L, Mundel P 2003 Podocyte biology and the emerging understanding of podocyte diseases. Am J Nephrol 23:353–360[CrossRef][Medline]
  38. Koshikawa M, Mukoyama M, Mori K, Suganami T, Sawai K, Yoshioka T, Nagae T, Yokoi H, Kawachi H, Shimizu F, Sugawara A, Nakao K 2005 Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental Nephrotic syndrome. J Am Soc Nephrol 16:2690–2701[Abstract/Free Full Text]
  39. Oh J, Reiser J, Mundel P 2004 Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome. Pediatr Nephrol 19:130–137[CrossRef][Medline]
  40. Schratt G, Philippar U, Berger J, Schwarz H, Heidenreich O, Nordheim A 2002 Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells. J Cell Biol 156:737–750[Abstract/Free Full Text]
  41. Morigi M, Buelli S, Angioletti S, Zanchi C, Longaretti L, Zoja C, Galbusera M, Gastoldi S, Mundel P, Remuzzi G, Benigni A 2005 In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies. Am J Pathol 166:1309–1320[Abstract/Free Full Text]
  42. Takahashi MO, Takahashi Y, Iida K, Okimura Y, Kaji H, Abe H, Chihara K 1999 Growth hormone stimulates tyrosine phosphorylation of focal adhesion kinase (p125(FAK)] and actin stress fiber formation in human osteoblast-like cells, Saos2. Biochem Biophys Res Commun 263:100–106[CrossRef][Medline]
  43. Diakonova M, Gunter DR, Herrington J, Carter-Su C 2002 SH2-Bß is a Rac-binding protein that regulates cell motility. J Biol Chem 277:10669–10677[Abstract/Free Full Text]
  44. Brown-Borg HM, Rakoczy SG, Romanick MA, Kennedy MA 2002 Effects of growth hormone and insulin-like growth factor-1 on hepatocyte antioxidative enzymes. Exp Biol Med (Maywood) 227:94–104[Abstract/Free Full Text]
  45. Chin E, Zhou J, Bondy CA 1992 Renal growth hormone receptor gene expression relationship to renal insulin-like growth factor system. Endocrinology 131:3061–3066[Abstract/Free Full Text]
  46. Doi SQ, Jacot TA, Sellitti DF, Hirszel P, Hirata MH, Striker GE, Striker LJ 2000 Growth hormone increases inducible nitric oxide synthase expression in mesangial cells. J Am Soc Nephrol 11:1419–1425[Abstract/Free Full Text]
  47. Cunha KS, Barboza EP, Da Fonseca EC 2003 Identification of growth hormone receptor in localised neurofibromas of patients with neurofibromatosis type 1. J Clin Pathol 56:758–763[Abstract/Free Full Text]
  48. Landsman T, Waxman DJ 2005 Role of the cytokine-induced SH2 domain-containing protein CIS in growth hormone receptor internalization. J Biol Chem 280:37471–37480[Abstract/Free Full Text]
  49. Lu C, Schwartzbauer G, Sperling MA, Devaskar SU, Thamotharan S, Robbins PD, McTiernan CF, Liu JL, Jiang J, Frank SJ, Menon RK 2001 Demonstration of direct effects of growth hormone on neonatal cardiomyocytes. J Biol Chem 276:22892–22900[Abstract/Free Full Text]
  50. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A 2001 Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 229:141–162[CrossRef][Medline]
  51. Baixeras E, Jeay S, Kelly PA, Postel-Vinay MC 2001 The proliferative and antiapoptotic actions of growth hormone and insulin-like growth factor-1 are mediated through distinct signaling pathways in the Pro-B Ba/F3 cell line. Endocrinology 142:2968–2977[Abstract/Free Full Text]
  52. Tryggvason K, Wartiovaara J 2001 Molecular basis of glomerular permselectivity. Curr Opin Nephrol Hypertens 10:543–549[CrossRef][Medline]
  53. Kim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, Holzman L, Kershaw D, Wiggins R 2001 Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int 60:957–968[CrossRef][Medline]
  54. Mundel P, Shankland SJ 2002 Podocyte biology and response to injury. J Am Soc Nephrol 13:3005–3015[Free Full Text]
  55. Lee HB, Yu MR, Yang Y, Jiang Z, Ha H 2003 Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol 14:S241–S245
  56. Meyer TW, Bennett P, H, Nelson R, G 1999 Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria. Diabetologia 42:1341–1344[CrossRef][Medline]
  57. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW 1997 Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99:342–348[Medline]



This article has been cited by other articles:


Home page
 Endocr. Rev.Home page
C. Ohlsson, S. Mohan, K. Sjogren, A. Tivesten, J. Isgaard, O. Isaksson, J.-O. Jansson, and J. Svensson
The Role of Liver-Derived Insulin-Like Growth Factor-I
Endocr. Rev., August 1, 2009; 30(5): 494 - 535.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
L. Rider, J. Tao, S. Snyder, B. Brinley, J. Lu, and M. Diakonova
Adapter Protein SH2B1{beta} Cross-Links Actin Filaments and Regulates Actin Cytoskeleton
Mol. Endocrinol., July 1, 2009; 23(7): 1065 - 1076.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
G. Kenth, J. A M. Mergelas, and C. G. Goodyer
Developmental changes in the human GH receptor and its signal transduction pathways
J. Endocrinol., July 1, 2008; 198(1): 71 - 82.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. Kamenicky, S. Viengchareun, A. Blanchard, G. Meduri, P. Zizzari, M. Imbert-Teboul, A. Doucet, P. Chanson, and M. Lombes
Epithelial Sodium Channel Is a Key Mediator of Growth Hormone-Induced Sodium Retention in Acromegaly
Endocrinology, July 1, 2008; 149(7): 3294 - 3305.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
X. Wang, J. Jiang, J. Warram, G. Baumann, Y. Gan, R. K. Menon, L. A. Denson, K. R. Zinn, and S. J. Frank
Endotoxin-Induced Proteolytic Reduction in Hepatic Growth Hormone (GH) Receptor: A Novel Mechanism for GH Insensitivity
Mol. Endocrinol., June 1, 2008; 22(6): 1427 - 1437.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reddy, G. R.
Right arrow Articles by Menon, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reddy, G. R.
Right arrow Articles by Menon, R. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals