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From the Renal-Electrolyte and Hypertension Division of the Department of Medicine and the Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to TGF-ß, the expression of the TGF-ß receptors has not been as extensively examined. Only a handful of in vitro studies have demonstrated that high ambient glucose stimulates TGF-ß type II receptor (TßRII) expression in cultured renal cells.15-17 In the streptozotocin-diabetic mouse, we previously showed that TßRII mRNA and protein, assessed by Northern and Western blots, were increased in the renal cortex.7,17
The db/db mouse (C57BL/KsJ), a genetic model of type 2
diabetes, exhibits clinical and histological features of diabetic
nephropathy that track the human disease.18,19
This
animal becomes hyperglycemic by 8 weeks of age and develops overt
proteinuria and renal insufficiency by 16 weeks of age. The kidneys
show the characteristic histological lesions of diabetic nephropathy
including mesangial matrix expansion and glomerular basement membrane
thickening. At the molecular level, mRNAs encoding 
1(IV) collagen
and fibronectin are overexpressed.20-22
We recently
reported that chronic treatment of db/db mice with a
neutralizing anti-TGF-ß antibody successfully prevented mesangial
matrix expansion and renal insufficiency,23
proving that
the TGF-ß system plays an important role in the development of
diabetic nephropathy. However, the precise details of the expression of
TGF-ß, its type II receptor, and the signaling pathway were not
delineated in that study.
The Smad family of proteins has recently been identified as a predominant signaling pathway by which TGF-ß activates the transcription of several well-known TGF-ß-inducible genes.24,25 Binding of TGF-ß to its type II receptor and subsequent recruitment of the type I receptor result in the phosphorylation and activation of two receptor-regulated Smads (R-Smads), Smad2 and Smad3. After associating with a common-Smad (co-Smad), Smad4, the Smad complex translocates into the nucleus where it cooperates with other transcription factors to coordinate the expression of target genes.26 Because diabetes results in the activation of the renal TGF-ß system, the Smad pathway is probably activated in the diabetic state, but this has not yet been shown either in vitro or in vivo.
The current study seeks to more fully characterize the intrarenal TGF-ß system in type 2 diabetes. The glomerular and tubular distribution of TGF-ß1 and its type II receptor were assessed at the mRNA and protein levels by in situ hybridization and immunohistochemistry, respectively. Translocation of Smad3 into the nucleus and nuclear binding activity to Smad-binding element (SBE) were ascertained by immunohistochemistry and Southwestern histochemistry, respectively. Our findings present a more complete picture of how the ligand, receptor, and signaling components of the renal TGF-ß system may interact to promote the progression of diabetic kidney disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The diabetic db/db and nondiabetic db/m mice were purchased from Jackson Laboratory (Bar Harbor, ME). Six animals from each group were killed at 16 weeks of age when the db/db mice have been overtly hyperglycemic and were likely to have developed diabetic nephropathy. Blood glucose was measured by the glucose oxidase technique (Sigma, St. Louis, MO), and serum creatinine was measured by a colorimetric assay (Sigma). The left kidney from each mouse was excised, bisected, and processed in fixative solutions as described below.
Each kidney was processed into frozen sections for in situ hybridization and into paraffin sections for periodic acid-Schiff staining, immunohistochemistry, and Southwestern histochemistry. Selected kidneys from each group were processed for ultrastructural examination as described below.
Histological and Ultrastructural Examination
Formalin-fixed kidneys were embedded in paraffin, sectioned at 5 µm, and stained with periodic acid-Schiff. Thirty glomeruli were randomly selected from each animal, and the extent of extracellular mesangial matrix was identified by periodic acid-Schiff-positive material in the mesangium and factored by the glomerular tuft area.21
For ultrastructural evaluation, tissue was fixed in 3% glutaraldehyde, postfixed in osmium tetroxide, and stained with uranyl acetate and lead citrate. The specimen was thin-sectioned and examined under a transmission electron microscope. Electron microscopic pictures were randomly taken at x1,000, x2,000, and x10,000 magnification in each group. With the computer program, Image-Pro Plus 3.0 (Media Cybernetics, Silver Spring, MD), the glomerular basement membrane thickness was measured on high magnification. Mesangial cell size and number were determined on low magnification.
Immunohistochemical Staining of TGF-ß1, TßRII, and Smad3
Immunohistochemical stains for TGF-ß1, TßRII, and Smad3 were performed using a streptavidin biotin-staining method (Vector Laboratories, Burlingame, CA). After removal of paraffin, endogenous peroxidase activity was quenched by a 30-minute incubation with 0.3% H2O2 in absolute methanol. The slides were placed in 10 mmol/L citrate buffer (pH 6.0), microwaved for 10 minutes, and cooled down to room temperature. Slides were then placed in phosphate-buffered saline (PBS) for 15 minutes followed by protein-blocking solution (Immunotech, Cedex, France) for 30 minutes. Sections were incubated overnight at 4°C with one of three primary antibodies as follows: a polyclonal rabbit antibody against mouse TGF-ß1, a polyclonal rabbit antibody against mouse TßRII (both Santa-Cruz Biotechnology, Santa Cruz, CA), and a polyclonal rabbit antibody against mouse Smad3 (Zymed Laboratory, South San Francisco, CA), all at 1:100 dilution. The same concentration of isotype-matched antibody of irrelevant specificity was used as a negative control. After three 5-minute washes in PBS, a secondary biotinylated anti-rabbit antibody (Vector Laboratories) was added for 45 minutes at room temperature. After three 5-minute washes in PBS, streptavidin-biotin peroxidase (Vector Laboratories) was added for 45 minutes at room temperature. The color reaction was developed with the diaminobenzidine detection kit (Vector Laboratories) and counterstained with hematoxylin.
TGF-ß1 and TßRII Riboprobe Preparation and in Situ Hybridization
The cDNA probes encoding mouse TGF-ß1 and TßRII were synthesized by polymerase chain reaction using murine kidney cDNA as template and specific oligonucleotide primers based on the published cDNA sequences as previously described.7,9,27 The polymerase chain reaction products were subcloned into pCRII TA cloning vector (Invitrogen, Carlsbad, CA). The mouse TGF-ß1 cDNA was linearized with NotI for antisense and HindIII for sense orientation, and the TßRII cDNA was linearized with HindIII for antisense and NotI for sense orientation. The transcription reaction using digoxigenin-labeling of riboprobe was performed according to the manufacturer’s instructions (Roche, Indianapolis, IN). The labeling mixture consisted of 1 µg of template cDNA (in 13 µl of water), 2 µl of NTP labeling mixture, 2 µl of transcription buffer, 1 µl of RNase inhibitor, and 2 µl of T7 or SP6 polymerase. Transcription was performed for 2 hours at 37°C, followed by digestion with 2 µl of RNase-free DNase for 15 minutes at 37°C and purification by spin columns. For in situ hybridization, frozen kidney sections (5 µm) were overlaid with 30 µl of hybridization buffer containing the labeled RNA probe and incubated at 58°C overnight in a humid chamber. After hybridization, sections were washed once in 2x standard saline citrate (SSC) at room temperature, once in 2x SSC at 65°C, and once in 0.1x SSC at 65°C. Slides were equilibrated with buffer I (100 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5) for 5 minutes. Anti-digoxigenin antibody conjugated to alkaline phosphatase (1:5,000) was applied to the slides, which were incubated in a humid chamber for 2 hours at room temperature. After two washes in buffer I for 15 minutes each, the sections were equilibrated with developing buffer (100 mmol/L Tris, 100 mmol/L NaCl, 50 mmol/L MgCl2, pH 9.5) for 5 minutes. The color reaction was developed using nitro blue tetrazolium (NBT/BCIP, Roche) according to the manufacturer’s instructions.
Southwestern Histochemistry
Southwestern histochemistry was performed according to the literature with slight modifications.28 Complementary oligonucleotides containing a Smad-binding consensus sequence29 were synthesized as follows: 5'-GGAGTATGTCTAGACTGACAATGTAC-3'. Paraffin-embedded kidney sections were dewaxed and rehydrated. The preparations were incubated with levamisole (Sigma Chemical Co., St. Louis, MO) to inhibit endogenous alkaline phosphatase activity and then fixed with 0.2% paraformaldehyde for 30 minutes at 28°C. Sections were subsequently digested with pepsin A (433 U/mg, Sigma) and washed twice in HEPES-bovine serum albumin. The sections were incubated with 0.1 mg/ml DNase I in HEPES-bovine serum albumin for 30 minutes at 30°C.
The labeled SBE probe, diluted to 100 pmol/L in HEPES-bovine serum albumin containing 0.5 µg/ml poly (dI-dC) (Amersham Pharmacia, Piscataway, NJ), was applied to each slide overnight at 37°C. A 100-fold unlabeled SBE was used as competitive inhibitor. After incubation in blocking solution (0.01x SSC, 0.01% sodium dodecyl sulfate, 0.03% Tween 20, 0.1 mol/L maleic acid, 0.15 mol/L NaCl, pH 7.5), anti-digoxigenin antibody conjugated with alkaline phosphatase (1:250 in blocking solution, Roche) was added overnight at 4°C. The color reaction was developed using nitro blue tetrazolium (NBT/BCIP, Roche) according to the manufacturer’s instructions.
Quantification of Immunohistochemistry, in Situ Hybridization, and Southwestern Histochemistry Data
Twenty glomeruli and 10 tubular areas, at x400 or x600 magnification, were randomly selected for analysis in each of the experimental groups. The mRNA expression and protein production of TGF-ß1 and TßRII were measured by the density of the in situ hybridization signal and immunohistochemical-positive staining, respectively, in the glomerular and tubular compartments using the computer program, Image-Pro Plus 3.0. The nuclear accumulation of Smad3 protein and nuclear protein binding to SBE were assessed by manual tag counting using Image-Pro Plus 3.0.
Statistical Analysis
Data are presented as mean ± SE. Groups were compared by unpaired Student’s t-test. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical characteristics of the experimental mice are shown in
Table 1
. The diabetic mice were frankly
hyperglycemic by the end of the experimental period. At age 16 weeks,
the body weights of the db/db mice were significantly
greater than those of the db/m mice. Kidney weights were
also significantly greater in the db/db mice. However,
average kidney-to-body weight ratios were predictably lower in the
db/db group because the diabetic animals were much heavier.
The db/db mice developed renal insufficiency as evidenced by
the elevated plasma creatinine concentration. To assess
glomerulosclerosis, we measured the glomerular fraction occupied by
mesangial matrix and divided the analysis by location in the inner or
outer cortex, because glomeruli are normally larger in the inner
cortex. The mesangial matrix fraction was significantly increased in
both inner and outer cortices in db/db compared with
db/m mice (Figure 1, A and B
;
Table 1
). Ultrastructural examination of the kidney of db/db
mice revealed increased mesangial extracellular matrix (Figure 1, C and D)
. The size of the mesangial cell was slightly increased
in db/db mice compared with db/m mice. However,
the number of mesangial cells in the glomerulus was not different
between the two groups (Table 1)
. In db/db mice, the
glomerular basement membrane was thickened with irregular distortions
and multifocal foot process effacement of the podocytes (Figure 1, E and F
; Table 1
). No electron dense deposits were seen.
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To evaluate the renal TGF-ß system in the db/db
mouse, we first examined the expression of TGF-ß1 mRNA by in
situ hybridization. As shown in Figure 2, A and B
, the expression of TGF-ß1
mRNA was significantly increased in the glomerular tuft, in a
mesangio-capillary pattern, in db/db mice compared with
controls (Figure 2C)
. Although the staining of TGF-ß1 mRNA was
weaker in the cortical tubules of both nondiabetic and diabetic mice,
formal quantitative analysis revealed a significant increase in the
tubular TGF-ß1 mRNA in db/db versus
db/m mice as well (Figure 2D)
.
|
. In diabetic
mice, the intensity of staining in the tubules was slightly but not
significantly increased (Figure 3, A and B)
. In addition, the
glomerular staining was particularly prominent in the juxtaglomerular
areas of diabetic mice (Figure 3, B
versus
A). Although the db/db group displayed increased
TGF-ß1 protein in both glomerular and tubular compartments, these
differences did not reach statistical significance when compared with
the db/m group (Figure 3, C and D)
. Thus, the relative
changes in the quantity of TGF-ß1 protein in the diabetic mouse were
less pronounced than the changes in the TGF-ß1 mRNA discussed above.
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Whereas TGF-ß1 mRNA staining was stronger in the glomeruli than
in the tubules, TßRII mRNA staining was stronger in the tubules than
in the glomeruli of both groups of mice (Figure 4, A and B)
. Quantitatively, the
expression of TßRII mRNA within the tubules was clearly and
significantly increased in db/db mice (Figure 4D)
, but
the expression of TßRII mRNA within the glomeruli was also
significantly increased in db/db compared with control
db/m mice (Figure 4C)
.
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,
and overall expression was markedly enhanced in diabetic
versus nondiabetic mice (Figure 5, A–D)
. Like its mRNA
counterpart, the TßRII protein stained more heavily in the tubular
than in the glomerular compartments (Figure 5, D
versus B).
Compared with the nondiabetic controls, the diabetic mice displayed
significantly greater staining densities for TßRII protein in the
tubules as expected (Figure 5F)
but also in the glomeruli (Figure 5E)
.
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Recent pieces of evidence have demonstrated that Smad proteins are
important intracellular transducers of TGF-ß
signaling.26
Therefore, we hypothesized that the Smad
pathway is activated in the diabetic kidney, and Smad proteins would
accumulate in renal cell nuclei. Immunohistochemical staining for Smad3
protein revealed diffusely increased cytoplasmic and nuclear staining
in db/db mice compared with controls. By visual inspection,
nuclear staining of Smad3 protein was noticeably more prominent in the
db/db mice (Figure 6, B and E)
compared with db/m mice (Figure 6, A and D)
. By manual tag
counting, Smad3 nuclear staining was confirmed to be significantly
increased in both glomeruli and tubules in db/db mice
(Figure 6, G and H)
.
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After Smad3 is activated in response to TGF-ß, it translocates
to the nucleus where it binds to certain DNA sites such as the
SBE.29
We next examined whether nuclear protein binding to
SBE is enhanced in the diabetic kidney. To evaluate the extent of SBE
binding, we performed Southwestern histochemistry.28,30
As
shown in Figure 7
, labeled SBE localized
more often to the nuclei of both tubular (Figure 7, A and B)
and
glomerular (Figure 7, C and D)
cells in db/db mice than in
db/m controls. Figure 7, E and F
, which quantitate the
percentage of glomerular or tubular cells that display nuclear SBE
binding, showed significant increases in the nuclear localization of
SBE in diabetic compared with nondiabetic mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been well established that the TGF-ß cytokine is up-regulated in the kidney of animal models of type 1 diabetes. The intrarenal expression of TGF-ß mRNA, assessed by Northern analysis or reverse transcriptase-polymerase chain reaction of whole kidney RNA, is increased in streptozotocin-induced diabetic animals.4,5,7 Using immunohistochemistry and in situ hybridization, Park and colleagues6 reported that the TGF-ß protein and mRNA were increased in the glomerular and interstitial compartments during the early stages of streptozotocin diabetes in rats. Our group has shown with Northern analysis that the expression of renal TGF-ß1 mRNA is increased in the BioBreeding rat and the nonobese diabetic mouse, other models of type 1 diabetes.8 Together, these results strongly support a pathogenic role for this cytokine in type 1 diabetic nephropathy.
The role of TGF-ß has not been as extensively studied in type 2 diabetic nephropathy. One study found by immunohistochemistry that the TGF-ß1 protein was increased in the renal cortex of the Otsuka-Long-Evans-Tokushima-Fatty rat, another model of type 2 diabetes.31 Another group showed that TGF-ß was overexpressed in the glomeruli of db/db mice in association with increased expression of fibronectin and type IV collagen.22 Similarly, the present study demonstrated that the expression of TGF-ß1 mRNA, assessed by in situ hybridization, was markedly enhanced in the glomeruli of db/db mice. Although this finding seems to conflict with our previous study in which Northern analysis of whole kidney RNA failed to detect an increase of TGF-ß1 mRNA in db/db mice versus control,32 these disparities may be reconciled if one considers that TGF-ß1 overexpression occurred predominantly in the glomerular compartment only. Hybridization signals for TGF-ß1 mRNA were also increased in the cortical tubular compartment between diabetic and nondiabetic groups, but the staining was patchy and irregular. Furthermore, the renal medulla did not show any increase in TGF-ß1 expression by in situ hybridization (data not shown). Because glomeruli comprise <10% of the renal parenchyma, the local increase in glomerular TGF-ß1 was masked by the lack of increase in tubular and medullary TGF-ß1 when whole kidney RNA was examined. In addition, Northern analysis of the whole kidney may not be sensitive enough to resolve small differences in message expression in the various renal compartments.
The marked increase in glomerular TGF-ß1 mRNA, however, did not result in a proportional increase in TGF-ß1 protein. This contrasts with the situation in type 1 diabetic animals that show significant elevations of both TGF-ß1 message and protein in the glomerulus.7 The answer may lie with insulin, which has metabolic and growth-promoting effects on the kidney in diabetes.33,34 We speculate that the hyperinsulinemia of type 2 diabetes, absent in type 1, may inhibit the translation of TGF-ß1 mRNA to protein. In fact, preliminary work on cultured renal tubular cells has revealed that exogenous insulin may alter the production of TGF-ß1 protein through a posttranscriptional mechanism.35,36
On the other hand, TßRII protein was up-regulated in accord with the mRNA expression in both glomeruli and tubules of the diabetic mice. These findings are consistent with our previous study using Western and Northern analyses of whole kidney tissue.23,32 In experimental diabetic conditions, the up-regulation of TßRII by high glucose may sensitize the mesangial cell to the effects of exogenous TGF-ß1, which will considerably enhance its type IV collagen production for example.17 Extrapolated to the in vivo situation, these findings suggest that increased TßRII expression may enhance the responsiveness to TGF-ß, thus accentuating the extracellular matrix accumulation in the kidneys of db/db mice.
Interestingly, nuclear accumulation of Smad3 and nuclear protein binding to SBE were increased in the kidney of diabetic db/db mice. To our knowledge, these in vivo observations are the first to prove that the specific downstream signaling pathway of TGF-ß is activated in the kidney in diabetes. After the binding of the TGF-ß ligand, Smad3 is phosphorylated and translocates into the nucleus where it participates in the transcriptional regulation of TGF-ß target genes.26 Several TGF-ß target genes relevant to renal fibrosis, such as plasminogen activator inhibitor PAI-1 and type I collagen, have been reported to be regulated by Smad3 through Smad binding sites in their promoters.37-39 Furthermore, Smad proteins have been shown to interact with other transcription factors, such as activator protein-1 and the family of cAMP-responsive element-binding proteins, which are required for TGF-ß-induced transcription of other fibrosis-related genes.26,40,41 Although the precise involvement of the Smad pathway in the regulation of extracellular matrix proteins needs to be clarified, it is possible that the activation of the Smad pathway leads to up-regulation of not only type I collagen but also other extracellular matrix proteins.
Our histological observations and their quantitation prove that the critical components of the TGF-ß system are up-regulated throughout the renal cortex of the db/db mouse model of type 2 diabetes. Although the level of TGF-ß1 protein was variably increased, the expression of TßRII was particularly pronounced in both glomerular and tubular compartments of the renal cortex, and this translated into the downstream activation of Smad3, an important element of the TGF-ß signaling system. Given the well-known prosclerotic effects of TGF-ß, these findings strongly support a central role for the TGF-ß system in the pathogenesis of glomerulosclerosis and tubulointerstitial fibrosis in the nephropathy of type 2 diabetes.
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Acknowledgements |
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Footnotes |
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Supported in part by the Juvenile Diabetes Foundation International (to F. N. Z., M. I., and S. C.), and the National Institutes of Health (grants DK-44513, DK-45191, and DK-54608 to F. N. Z.; training grant DK-07006, and National Research Service Award to S. C.). S. W. H. and D. C. H are visiting scholars at the University of Pennsylvania and are supported by Yonsei University, and Hyonam Kidney Laboratory/Soon Chun Hyang University, respectively, Seoul, Korea. M. C. Iglesias-de la Cruz is a postdoctoral fellow at the University of Pennsylvania and is supported by the Ministerio de Educación y Cultura of Spain.
Accepted for publication January 23, 2001.
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 J. P. Grande, G. M. Warner, H. J. Walker, A. N. K. Yusufi, J. Cheng, C. E. Gray, J. B. Kopp, and K. A. Nath TGF-{beta}1 Is an Autocrine Mediator of Renal Tubular Epithelial Cell Growth and Collagen IV Production Experimental Biology and Medicine, March 1, 2002; 227(3): 171 - 181. [Abstract] [Full Text] [PDF]
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