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NDT Advance Access originally published online on April 23, 2007
Nephrology Dialysis Transplantation 2007 22(9):2485-2496; doi:10.1093/ndt/gfm229
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Combined effects of moderately elevated blood glucose and locally produced TGF-ß1 on glomerular morphology and renal collagen production

Søren Krag1,2, Jens R. Nyengaard2 and Lise Wogensen1

1Research Laboratory for Biochemical Pathology and 2Stereology and Electron Microscopy Research Laboratory, Aarhus University Hospital, The Institute of Clinical Medicine, The University of Aarhus, Denmark

Correspondence and offprint requests to: Søren Krag, PhD, Research Laboratory for Biochemical Pathology, Aarhus Sygehus, Noerrebrogade, 44, 8000 Aarhus C, Denmark. Email: soeren.krag{at}ki.au.dk



   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Background. There is a correlation between renal graft rejection and blood glucose (BG) levels. Furthermore, diabetic patients may develop non-diabetic renal diseases, which in some circumstances progress rapidly. Since transforming growth factor-ß1 (TGF-ß) levels are elevated in many renal diseases, the accelerated progression may be due to interactions between glucose and locally produced TGF-ß1. Therefore, we investigated the effect of mild hyperglycaemia on glomerular morphology and collagen production in TGF-ß1 transgenic mice.

Methods. To achieve BG concentrations of ~15 mmol/l in TGF-ß1 transgenic and non-transgenic mice, we used multiple streptozotocin (STZ) injections, and after 8 weeks, we measured the changes in glomerular morphology and total collagen content. We also analysed extracellular matrix (ECM) and protease mRNA levels using real-time polymerase chain reaction (PCR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK) expression by immunohistochemistry.

Results. Mild hyperglycaemia alone had no effect on glomerular structure or ECM deposition. Over-expression of TGF-ß1 increased basement membrane thickness (BMT) and the mesangial volume fraction. Furthermore, it augmented ECM, Matrix metalloproteinase-2 (MMP), MMP-9, plasminogen activator inhibitor-1 (PAI) and tissue inhibitor of metalloproteinase-1 (TIMP) gene expression and pERK1/2 immunostaining. Elevated BG in combination with TGF-ß1 resulted in enlargement of glomerular volume, total mesangial volume and renal collagen content. Moreover, high BG exaggerated TGF-ß1-induced changes in the BMT, MMP-2 and TIMP-1 expression and pERK1/2 staining.

Conclusion. Even moderate elevations in BG accelerate the progression of those kidney diseases in which TGF-ß1 is involved. This emphasizes the importance of strict BG control in renal transplant patients and diabetic patients with renal malfunctions unrelated to diabetes.

Keywords: diabetes; fibrosis; kidney disease; non-diabetic glomerulopathy; post-transplant diabetes; TGF-ß1



   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Patients who have diabetes, including the 5–10% of renal transplant recipients that develop post-transplant diabetes mellitus (PTDM) may encounter renal problems after kidney transplantation. Some clinical studies have unveiled a correlation between increased blood glucose (BG) and acute graft rejection or renal graft survival in recipients with or without preceding diabetes [1–3]. This finding is controversial, however, since other investigators have found similar graft survivals in normoglycaemic and hyperglycaemic patients [4,5]. Furthermore, some diabetic patients may develop non-diabetic renal diseases. Renal lesions not typical of diabetic nephropathy are encountered in biopsy specimens from patients with type 2 diabetes. Retrospective studies of biopsies have shown a prevalence of 12–81% for non-diabetic glomerulopathies alone, or superimposed on diabetic nephropathy—although prospective studies have found a lower prevalence, between 6–39% [6]. It is known that the rate of increase in serum creatinine is higher in diabetic patients with end-stage renal disease (ESRD) than in non-diabetic ESRD patients [7]—an effect that could be due to pathophysiological differences between diabetic nephropathy or ESRD of other causes, or it could be the result of a more rapid progression of any renal insult in situations with even minor elevations in BG levels.

Glucose is an upstream mediator in mechanisms that underlie renal pathologies. Those are followed by signal pathways more downstream, involving, for example, autocrine- or paracrine-produced growth factors, of which transforming growth factor-ß1 (TGF-ß1), vascular endothelial growth factor, platelet-derived growth factor, connective tissue growth factor (CTGF), fibroblast growth factor, epidermal growth factor, and insulin-like growth factor-1 have drawn the most attention [8].

TGF-ß1 is important in the regulation of extracellular matrix (ECM) metabolism, favouring ECM deposition. Elevated TGF-ß1 is evident in numerous human kidney diseases and animal models thereof, including diabetic nephropathy, chronic cyclosporine nephrotoxicity and renal transplant grafts [9,10]. Accordingly, chronic kidney disease, including graft rejection, is characterized by increased ECM, which results from the disruption of the delicate balance between ECM synthesis and degradation that exists under normal conditions. The regulation of ECM degradation is especially complex since many proteases and protease inhibitors are involved. The most important participants in matrix degradation are the matrix metalloproteinases (MMPs) and plasminogen activators (PA). These are inhibited by tissue inhibitors of metalloproteinases (TIMPs) and plasminogen activator inhibitor (PAI) [11]. We speculated that even small elevations in BG combined with an increase in renal TGF-ß1 content may worsen different kidney diseases or accelerate graft rejection. Therefore, we set out to investigate the separate and combined effects of moderate elevations of BG and locally produced TGF-ß1 on glomerular morphology and interstitial collagen production, using a transgenic model with juxtaglomerular expression of active TGF-ß1.



   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Animals
Active TGF-ß1 was targeted to the kidney by the Ren1c- promoter in the C57 BL/6J mouse strain [12]. One group of 8-week-old TGF-ß1 transgenic (Tg) male mice (n = 12) and one of the non-transgenic (WT) (n = 12) male controls were divided into two groups. One TG and one WT group received five consecutive daily intraperitoneal injections of streptozotocin (STZ) (40 mg/kg). The remaining two groups received only vehicle (VEH) (n = 6 in each of 4 groups). BG was measured in a drop of tail venous blood just before the first injection (day 0) and weekly thereafter. The mice in the STZ groups were included in the experiment when the BG concentrations were higher than 8.5 mmol/l. If at any point BG was <8.5 mmol/l in the STZ-treated animals, they received an additional injection of STZ. Body weight (BW) was recorded weekly during the experiment.

The mice were housed at the animal facility at the University of Aarhus, and they were handled and sacrificed according to the guidelines and principles of laboratory animal care recommended by the Animal Experiments Inspectorate, Denmark. They were kept at 21°C with a 12-h day/night cycle and were given free access to standard chow and water. The project was approved by the Animal Experiments Inspectorate, Denmark (#1999/561–218) and the Danish Working Environment Service (2001-0011479/12).

Collection of material
After having elevated BG for 8 weeks, the mice were placed in individual metabolic cages, for collecting 24-h urines and recording water intake. The mice were next sacrificed by cervical dislocation. One kidney of each animal, chosen randomly between the right and the left, was weighed, and in preparation for electron microscopy (EM) was fixed in Thyrode's buffer containing 1% glutaraldehyde and 3% paraformaldehyde. The other kidney was halved. One half was placed in optimal cutting temperature compound (OCT) (Sakura, The Netherlands) and snap frozen in liquid nitrogen while the other half was placed in 4% phosphate-buffered paraformaldehyde and was later embedded in paraffin.

Albumin excretion
The collected urine was spun for 2 min at 10 000 r.p.m; afterwards it was diluted 1 : 250 and the albumin concentration was measured using a commercial ELISA according to instructions from the manufacturer (Bethyl Laboratories Inc., TX, USA), and the 24-h albumin excretion was calculated. Two mice, one in each of the VEH groups, produced inadequate volumes of urine to allow determination of albumin concentration, therefore, the number of samples was 5/6 in each group.

Glomerular volume
The kidney was cut into 1-mm slices, and every third slice was chosen by systematic, uniformly random sampling and the slices from each kidney were embedded together in glycolmethacrylate plastic. A 15 µm-thick disector pair and one 3 µm section were cut and stained with periodic acid-Schiff [13]. The counting was done using a microscope and the CAST software (Visiopharm, Denmark).

The glomerular density Nv(glom/kidney) in a disector was calculated as:


Formula

where Q– is the number of glomeruli counted, t is the thickness of the disector sections, (a/p) is the area associated with each test point and P (kidney) is the number of points overlaying the kidney. The total on-screen magnification was 94x and the a/p = 307*103 µm2.

The volume fraction of glomeruli was estimated on the 3-µm sections using point-counting with 6 points for counting the total kidney and 150 points for the glomeruli. The mean glomerular volume was then calculated as:


Formula

Mesangial volume and basement membrane thickness
From the remaining slices, small tissue biopsies were punched randomly from the cortex using a plastic grid with equidistantly spaced holes. These were embedded in Epon, contrasted using an automated stainer (EMstain, Leica, Germany), and 2–3-µm sections were cut until at least one glomerulus appeared. A 50-nm section was then cut with an ultramicrotome (Leica, Germany). The glomeruli were photographed using a Phillips CM10 electron microscope with a Kodak 1.6 camera (Kodak, NY, USA) and Analysis software version 3.1 (Soft imaging systems, Germany). The mesangial volume fraction was point-counted on a 3400x original magnification using a point grid (1 : 9). The glomerular area was defined as the polygon obtained when the outer capillary profiles were connected by straight lines. At 7900x original magnification, the basement membrane thickness (BMT) was measured where it intersected with the test lines, and the harmonical mean was calculated [14].

Total collagen content
Kidneys slices were defatted in 2 : 1 chloroform: methanol that included protease inhibitors, for 21 h at 4°C, dried at room temperature for 16 h and pulverized. Then 200 µl 6N HCl was added to 10 mg of the pulverized tissues and hydrolysed at 118°C for 18 h. This was neutralized using 120 µL 10 N sodiumhydroxide. Hydroxyproline was measured in the supernatant diluted 1 : 80 after centrifugation. Collagen content was calculated from the total hydroxyproline content using 7.46 as a correction factor (n = 5/6).

Immunohistochemistry
Paraffin embedded sections were deparaffinized, rehydrated and blocked with 20 mmol/l glycine. The epitopes were demasked by microwave treatment for 12 min in a 10 mmol/l citrate buffer, pH 6.0. After blockage with 2% normal serum, the primary antibody was added: anti-collagen type I (1 : 100, Biodesign, ME, USA), anti-collagen type III (1 : 15, Chemicon, CA, USA), anti-FSP1 (1 : 3000, a kind gift from Dr E. Neilson, Vanderbilt University, TN, USA) and anti-phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2) (1 : 50, Cell Signalling Technology, MA, USA). Sections were incubated with biotin-labelled secondary antibody (1:200, Vector Laboratories, CA, USA), which then was detected by the ABC kit (Vetor Laboratories, CA, USA) and 3,3 diaminobenzidine (Sigma-Aldrich, MI, USA). A blinded observer did the semiquantitative scoring of the p-ERK 1/2 sections (n = 3 in each group).

Quantitative RT-PCR
Renal tissue stored in OCT was removed from the medium while still frozen and immersed immediately in Trizol (Invitrogen, CA, USA). After the material was treated with DNase, 2 µg of RNA was reverse-transcribed by Superscript III using random hexamer primers according to the instructions provided by the manufacturer (Invitrogen, CA, USA). Polymerase chain reaction (PCR) was performed and analysed using an iCyclerTM (Bio-rad, CA, USA). For the sequence of the primers used for real-time PCR see Table 1.


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  		Table 1. The nucleotide sequence of primers used for real-time PCR

 
Pilot experiments revealed that it was possible to perform all reactions except uPA under the following conditions: 95°C for 5 min followed by 35–45 cycles of 95°C/30 s, 60°C/1 min and 72°C/45 s, using SYBR-green (SYBR-green, Bio-rad, CA, USA). Reaction conditions for uPA were 95°C/5 min followed by 40 cycles of 95°C/30 s, 63°C/1 min and 72°C/45 s. Samples of cDNA from all mice were mixed and a standard curve (1 : 5, 1 : 50, 1 : 500) was included in each PCR reaction. The results were located within the standards in all reactions and the PCR efficiency was 95–105%—if not, we repeated the PCR reaction. The ratio of the target gene to the quantity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), analysed in parallel, was computed. The results were standardized with reference to the control group. Due to the low RNA yield only five samples were available in the Tg–STZ group.

Statistical methods
The separate effects of TGF-ß1 and STZ were compared by general linear model analysis (GLMTGF-ß1 and GLMSTZ). When comparing all four groups we applied ANOVA followed by the Student–Newmans–Keuls test for multiple comparisons. In case of unequal variance, the Kruskal–Wallis test followed by Mann–Whitney's non-parametric test for multiple comparisons was used. Data are shown as mean and coefficient of variation (CV = SD/mean). A P-value of ≤0.05 is considered significant.



   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 Conclusion
 Acknowledgements
 References
 
Functional data
Mean BG concentrations in the two STZ groups were comparable [Tg: 14.8 mmol/l (19%) and WT: 14.3 mmol/l (22%)] and higher than those in the respective VEH-treated groups [Tg: 7.4 mmol/l (4%) and WT: 7.3 mmol/l (2%)] (both P = 0.004) (Figure 1A).


Figure 1
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  		Fig. 1. (A) Blood glucose concentration and (B) body weight in the experimental period. (C) Urine albumin excretion per 24 h at sacrifice. TG (filled triangle, filled circle), WT (open triangle, open circle), VEH (filled triangle, open triangle) or STZ (filled circle, open circle). *P < 0.05 vs WT VEH and vs Tg-VEH mice. Mean ± SD.

 
BW was similar in all the four groups at day 0. After 8 weeks, the two STZ groups had significantly lower BW [Tg: 26.4 g (3%) and WT: 24.7 g (13%)] vs VEH [Tg: 28.6 g [5%] and WT: 29.1 g [10%]) (GLMstz P = 0.002) (Figure 1B). Water intake, urine output and kidney weight did not differ between the four groups of mice.

The 24-h urinary albumin excretion at sacrifice was increased in the two groups receiving STZ vs the VEH treated groups (both P < 0.05 and GLMstz P < 0.001). No difference was observed between the Tg and WT groups (Figure 1C).

Glomerular volume
Most importantly, glomerular volume [Vn(glom)] was increased in the Tg-STZ group vs the three other groups of mice (ANOVA P < 0.05) (Figure 2A), which exhibited similar Vn(glom). We did not observe any effect on glomerular volume of moderate hyperglycaemia alone. This signifies that both TGF-ß1 and elevated BG are essential for the enlargement of glomerular volume in this C57Bl/6J model.


Figure 2
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  		Fig. 2. (A) Glomerular volume [Vn(glom)], (B) basement membrane thickness (BMT), (C) volume fraction of the mesangium [Vv(mes/glom)], and (D) total mesangial volume per glomerulus [V(mes/glom)] in Tg and WT mice with normal blood glucose levels (VEH) or elevated blood glucose levels induced by streptozotocin (STZ). Tg (filled triangle, filled circle), WT (open triangle, open circle), VEH (filled triangle, open triangle) or STZ (filled circle, open circle). *P < 0.05 vs Tg-VEH. #P < 0.05 vs WT-VEH and vs WT-STZ mice.

 
BMT
Glomerular basement membrane thickened during exposure in vivo to TGF-ß1 (GLMTGF-ß1 P < 0.001) and this effect was further augmented by moderate hyperglycaemia. As seen in Figures 2B and 3, the BMT rose from 169 nm (9.3%) in the VEH-treated Tg mice to 187 nm (9.5%) in the Tg-STZ animals (P < 0.05 by ANOVA). Elevated BG alone did not significantly increase the BMT in the WT animals [136 nm (6.6%) and 145 nm (8.5%) in WT VEH and STZ mice, respectively]. These data indicate that moderate increases of BG levels and TGF-ß1 act together to increase the BMT.


Figure 3
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  		Fig. 3. EM photographs of basement membranes (arrows) from the following groups: WT VEH (A), WT-STZ (B), Tg VEH (C), Tg STZ (D) (original magnification 7900x).

 
Mesangial expansion
The volume fraction of the mesangium in the glomerulus [V v(mes/glom)] was increased by TGF-ß1 (GLMTGF-ß1 P < 0.05). However, hyperglycaemia was without influence on the Vv(mes/glom) in both WT and Tg mice (Figure 2C). The total mesangial volume per glomerulus [V(mes/glom)] in Tg-STZ mice was higher than in the other three groups (ANOVA P < 0.05) (Figure 3D).

Collagen content
In concert TGF-ß1 and elevated BG augmented the deposition of collagen in Tg-STZ animals [80.4 µg/mg tissue (25%)] compared with the other three groups (as follows)(ANOVA P <0.05)(Figure 4A). A mean BG concentration of 14.3 mmol/l for 8 weeks was without impact on the collagen content in WT mice [VEH: 44.1 µg/mg tissue (22%) and STZ: 45.1 µg/mg tissue (13%)] while TGF-ß1 alone resulted in higher collagen content [57.2 µg/mg tissue (24%)], but without reaching statistical significance when compared with the WT group (P = 0.23).


Figure 4
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  		Fig. 4. (A) Total collagen content in WT and Tg mice with normal blood glucose levels (VEH) or elevated blood glucose levels induced by streptozotocin (STZ). Tg (filled triangle, filled circle), WT (open triangle, open circle), VEH (filled triangle, open triangle) or STZ (filled circle, open circle). *P < 0.05 vs the other three groups by ANOVA. (B) Kidneys from mice immunostained with antibodies against collagen type I (a–d) and III (e–h). WT (a,b,e,f) and Tg (c,d,g,h) receiving VEH (a,c,e,g) or STZ (b,d,f,h). Original magnification 40x.

 
Collagen immunohistochemistry
Collagen I and III showed a very subtly increased immunostaining in the tubulointerstitium in the Tg compared with the WT animals. The Tg-STZ animals exhibited slightly more collagen type I and III than Tg-VEH mice (Figure 4B). There was no discernible difference between the STZ and VEH WT animals.

ECM and TGF-ß1 gene expression and ßig-h3/TGF-ß1 ratio as estimated by real-time PCR
Moderate hyperglycaemia alone did not stimulate the RNA expression of the selected ECM components, TGF-ß1 or ßig-h3/TGF-ß1 ratio (Figure 5). Compared with the WT animals, TGF-ß1 Tg mice exhibited increased mRNA expression of collagens type I ({alpha}1) and III ({alpha}1), fibronectin and endogenous TGF-ß1 (all ANOVA or non-parametric test P < 0.05) (Figure 5). The collagen type IV ({alpha}1) expression was marginally increased in the two Tg groups compared with WT mice (ANOVA = 0.058). For all ECM components analysed, mRNA levels were similar in Tg-VEH and Tg-STZ animals. The ßig-h3/TGF-ß1 ratio is recognized as a marker for TGF-ß1 bioavailability [15]. The ratio was increased in the Tg-VEH mice compared with the three other groups (Figure 5F) (ANOVA P < 0.05). Thus, the ratio of active TGF-ß1 to total TGF-ß1 is highest in that group.


Figure 5
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  		Fig. 5. Real-time RT-PCR analysis of collagen type I ({alpha}1), III ({alpha}1) and IV ({alpha}1), fibronectin, TGFß1 mRNA and ßig-h3/TGF-ß1 ratio in Tg (filled triangle, filled circle) and WT (open triangle, open circle) mice with normal blood glucose (VEH) (filled triangle, open triangle) or elevated blood glucose (STZ) (filled circle, open circle). Results are expressed as the ratio between ECM molecules and GAPDH mRNA expression, and standardized to WT VEH animals. *P < 0.05 vs Tg-STZ, #P < 0.05 vs WT VEH and WT STZ mice.

 
Because statistical testing identified their data as outliers, one mouse in the Tg-STZ group was discarded in the collagen type IV ({alpha}1) analysis and one Tg-VEH mouse was omitted from the fibronectin expression analysis.

Protease and protease inhibitor gene expression
The expressions of MMP-2 and MMP-9 were increased by factors of 5.8 and 2.4, respectively, in the Tg-VEH animals compared with WT VEH (P < 0.01). MMP-2 expression was further elevated by a factor of 1.5 in Tg-STZ mice, while the expression of MMP-9 mRNA was unaffected by elevated BG (Figure 6A and B). There was no difference in gene expression of the two PA, tPA and uPA (Figure 6C and D). The amounts of PAI-1 and TIMP-1 mRNA were increased by TGF-ß1 (P < 0.01 vs WT VEH), but it had no effect on the level of TIMP-2 mRNA (Figure 6E–G). Interestingly, hyperglycaemia further elevated the expression of the TIMP-1 gene [Tg-STZ 7.46 (3.8%) vs Tg-VEH 3.44 (28.7%)] (P < 0.05), but PAI-1 was unaffected [Tg-STZ 3.94 (57%) vs Tg-VEH 4.29 (89%)]. TIMP-1 data from one mouse in the two Tg groups were excluded from statistical analysis as outliers.


Figure 6
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  		Fig. 6. Real-time RT-PCR analysis of MMP-2, MMP-9, tPA, uPA, PAI-1, TIMP-1 and TIMP-2 mRNA in Tg (filled triangle, filled circle) and WT (open triangle, open circle) mice with normal blood glucose levels (VEH) (filled triangle, open triangle) or elevated blood glucose levels (STZ) (filled circle, open circle). Results are expressed as the ratio between ECM molecules and GAPDH mRNA expression, and standardized to WT VEH animals. *P < 0.05 vs Tg-VEH, #P < 0.05 vs WT VEH and WT STZ mice.

 
Phospho-ERK 1/2 immunohistochemistry
In control mice, phospho-ERK 1/2 (p-ERK 1/2) was observed mainly in distal tubuli and collecting ducts with minute amounts of positive staining in interstitial and glomerular cells. Elevated BG alone did not alter this staining pattern. TGF-ß1 increased staining intensity in the interstitium, and sporadic signals were found in glomeruli and Bowman's capsules. In the Tg-STZ mice, glomerular and interstitial staining were increased compared with Tg-VEH and WT mice, while tubular staining was unchanged. In glomeruli, mesangial and endothelial cells both seem to harbour p-ERK 1/2 (Figure 7A). Immunohistochemistry on serial sections revealed that some of the p-ERK 1/2-positive interstitial cells also expressed the fibroblast marker FSP-1 (Figure 7B). This staining pattern is comparable with that reported by other groups [16,17]


Figure 7
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  		Fig. 7. (A) Immunohistochemical staining using p-ERK 1/2 antibody on kidney sections. Photos show tubular profiles (a–d original magnification 200x), glomeruli (e–h, original magnification 1000x) and interstitial cells (i–l, original magnification 1000x) from WT VEH (a,e,i), WT STZ (b,f,j), Tg VEH (c,g,k) and Tg STZ (d,h,l) mice. Arrows show positive p-ERK 1/2 staining, arrowheads point at tubular cells. (B) Immunohistochemistry using p-ERK 1/2 (a) and FSP-1 (b) antibodies on serial renal sections illustrating co-localization (arrow) between p-ERK 1/2 (a) and FSP-1 (b) but also some FSP-1-positive cells that do not stain for p-ERK 1/2 (arrowhead) (original magnification 400x).

 


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
Conclusion
 Acknowledgements
 References
 
In a transgenic mouse model, on the C57BL/6J background with renal over-expression of TGF-ß1 and moderately increased BG concentration, we made the following observations: local expression of TGF-ß1 in the kidney results in increased BMT and Vv(mes/glom), augmented TGF-ß1 activation and increased expressions of endogenous (murine) TGF-ß1, collagen type I ({alpha}1) and III ({alpha}1), fibronectin, MMP-2, MMP-9 and the protease inhibitors PAI-1 and TIMP-1. Moderate hyperglycaemia in combination with local overproduction of TGF-ß1 enlarges Vn(glom), V(mes/glom) and total collagen deposition. It further augments the TGF-ß1-induced increases in the BMT, MMP-2 and TIMP-1 expression. A mean BG concentration of 14.3 mmol/l for 8 weeks has no impact on glomerular morphology, TGF-ß1 expression, expression of ECM components, or on any of the tested proteases or inhibitors of proteases in WT mice.

It is known that glomerular changes attributable to high BG are characterized by early glomerular hypertrophy followed by widening of the BMT and expansion of the mesangium [18]. The same is observed in experimental models of diabetes [19]. Most of the experiments reporting morphological changes in the kidney are performed with BG levels maintained around 25 mmol/l or higher for many weeks. However, very few patients with diabetes have such high BG concentrations over extended periods. In the present investigation, we report that a BG concentration around 15 mmol/l for 8 weeks is not sufficient to induce measurable glomerulopathy or changes in total renal collagen content. It is slightly surprising that Vn(glom) remained normal in the WT STZ animals. This conflicts with data that show increased glomerular volume in mice with BG levels in the range of 14–22 mmol/l for 9 days or 18 mmol/l over 60 days [19,20]. There are several possible explanations for this discrepancy. The first of the former studies used C57 mice, as did our experiments. However, data suggests that the initial hypertrophy is reversible by, e.g. insulin administration; and since our mice must have had some persistent insulin secretory capacity, that may have been enough to prevent hypertrophy. The second study used db/db mice; and here, strain differences could play a role. Interestingly, elevated BG in combination with locally produced TGF-ß1 is able to induce glomerular hypertrophy. This is in line with data that demonstrate that STZ-induced glomerular enlargement is inhibited by TGF-ß1 antibodies, indicating that TGF-ß1 is involved in this hypertrophy [19].

Moderate hyperglycaemia is without any effect on BMT in WT mice. Importantly, TGF-ß1 local in the kidney increases the BMT, and this effect is exaggerated by elevated BG. Likewise, elevated BG alone has no impact on total collagen content; but, in combination with TGF-ß1, it stimulates collagen deposition. Vv(mes/glom) is also enlarged by TGF-ß1, but apparently hyperglycaemia has no additional effect. This might suggest that the mesangium expanded under the influence of TGF-ß1 is less responsive to the concomitant elevation of BG. It should be remembered, however, that the combination of high BG and TGF-ß1 enlarges the V(mes/glom). Overall, our data suggest that even moderate increases of BG, such as seen in most patients with diabetes, including PTDM, may accelerate pathological processes in which TGF-ß1 is involved, leading to an accelerated development of glomerulopathy and tubulo-interstitial fibrosis.

Renal collagen content reflects the balance between deposition and degradation. Our data show that TGF-ß1 increases the RNA expression of interstitial collagen types in the kidney, even though the superimposition of elevated BG increases the total collagen content—the mRNA level is not further increased. A likely explanation is found in the analysis of the protease system. TGF-ß1 stimulates the expression of both MMP-2 and TIMP-1, and this increase is augmented by elevated BG. Apparently, the increase of MMP-2 should neutralize the inhibitory effect of TIMP-1 on ECM degradation. However, this is not always the case, for low degradative capacity in the presence of increased MMP-2 is seen in response to diabetes and CTGF [21,22]. Furthermore, a transgenic mouse with MMP-2 over-expression targeted to the proximal tubules develops progressive renal injury, most probably from increased epithelial mesenchymal transition [23]. Thus, it is most likely that the balance in ECM turnover when local TGF-ß1 levels are high and BG is elevated is dominated by decreased matrix degradation. On the other hand, with high local TGF-ß1 production alone the balance is dominated by increased synthesis of ECM components and only to a minor extent is influenced by reduced degradation.

The mechanisms of the interactions between elevated BG levels and locally expressed TGF-ß1 are not fully understood. One possibility is suggested by in vitro studies that show that increased glucose enhances the sensitivity of mesangial cells to TGF-ß1 via activation of ERK 1/2 and protein kinase C{delta} (PKC{delta}) [24]. The authors found that collagen type I promoter activity in response to TGF-ß1 stimulation is doubled in mesangial cells grown in 20 mM/l glucose compared with those grown in 6.5 mM/l glucose, without any concomitant difference in TGF-ß1 activity. Furthermore, TGF-ß1 itself activates ERK 1/2 [25]. In line with this, we found that staining of activated ERK 1/2 increased in glomeruli and interstitium of Tg-STZ compared with the other 3 groups. Thus, it seems that ERK 1/2 is a likely intermediary for the additive interaction of BG and TGF-ß1. Furthermore, cell culture studies show that a high concentration of glucose is able to potentiate TGF-ß1 synthesis in response to stimuli like PDGF or mechanical stress [26,27].

TGF-ß1 expression was similar in our Tg-VEH and Tg-STZ animals, and the ßig-h3/TGF-ß1 ratio was highest in the TG-VEH group. Other investigators have found that glucose increases ßig-h3 in vitro through a TGF-ß1-dependant pathway [28]. It is, therefore, unlikely that the lower ßig-h3 expression in the Tg-STZ group is caused by glucose-mediated ßig-h3 suppression. It seems more probable that the lower ßig-h3/TGF-ß1 ratio in the Tg-STZ group is associated with a TIMP-1-mediated reduction in protease activity, since proteases are known to play an important role in the activation of TGF-ß [29]. It should be remembered that we studied late events and it is possible that TGF-ß1 levels are different in earlier stages in our model of renal disease.

The only impact of moderate hyperglycaemia alone was the development of albuminuria, which was of equal severity in STZ-treated Tg and WT mice. One explanation could be that hyperglycaemic mice have polyuria due to osmotic diuresis. However, the 24-h urinary volume was similar in all four groups of mice. Another explanation could be alterations in the tubular protein reabsorption machinery induced by either diabetes or STZ. Megalin is important for the reabsorption of albumin in the proximal tubules; and it is known that STZ-induced diabetes down-regulates megalin and albumin reabsorption [30]. Furthermore, STZ is able to induce proteinuria independent of increased BG concentrations—though it seems that albumin is exempted from this toxic effect at least in rats [31].

Previous studies have shown that non-diabetic glomerulopathies do not significantly worsen the prognosis for diabetic patients with diabetic nephropathy [32]. However, to the best of our knowledge there is no study that compares the prognosis of non-diabetic kidney disease in diabetic and non-diabetic patients. Based on studies of PTDM, it is known that diabetes is associated with an increased risk of graft failure. The present investigation shows, in a model of progressive kidney disease, that diabetes speeds up the progression of the TGF-ß1-mediated disease.



   Conclusion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Conclusion
Acknowledgements
 References
 
Moderate elevation of BG exacerbates TGF-ß1-induced BMT, MMP-2 and TIMP-1 expression and acts together with TGF-ß1 to enlarge glomerular volume, mesangial area and total renal collagen content. This suggests that diabetic patients may have a worse prognosis than non-diabetic patients when they have a kidney disease not originally related to diabetes or have chronic allograft nephropathy. It would be interesting to investigate this possibility further in other experimental models of kidney disease and in diabetic patients.



   Acknowledgements
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
References
 
This project was supported by grants from the Danish Diabetes Association, the Fougner-Hartmann foundation, Michaelsen Foundation and the Novo Nordisk Research Foundation. The authors greatly appreciate the ever faithful assistance of Lotte Arentoft, Karin Vestergaard, Ulla Hovgaard, Lone Lysgaard, Mai-Britt Lundorf, Anette Berg and Herdis Krunderup. Finally, they would also like to thank The Institute of Clinical Medicine, Aarhus University. S.K. was the recipient of a PhD scholarship from the Faculty of Health Sciences, Aarhus University.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 

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Received for publication: 11. 1.06
Accepted in revised form: 23. 3.07


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K. Rossing, H. Mischak, M. Dakna, P. Zurbig, J. Novak, B. A. Julian, D. M. Good, J. J. Coon, L. Tarnow, P. Rossing, et al.
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