Thirty to 50% of patients with juvenile- or mature-onset diabetes mellitus develop clinically evident diabetic nephropathy 10 to 20 years from the onset of diabetes mellitus1. Thus, it is important to elucidate the mechanisms involved in the progression of renal complications of this disease. Diabetic nephropathy is characterized by glomerular hypertrophy, extracellular matrix (ECM) accumulation in the mesangium, and thickening of glomerular capillary walls, leading to glomerulosclerosis and loss of renal function2,3.
Rats administered streptozotocin or alloxan and genetically selected animals fed a high-sucrose diet have been employed as the models for type 2 diabetes mellitus4. In this study, we used the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, which was developed from a strain of Long-Evans rat by selective breeding and is now recognized as an adequate model for human type 2 diabetes mellitus5,6. The characteristic features of OLETF rats are5,6 (1) a chronic course of diabetes; (2) hyperlipidemia; (3) mild obesity; (4) male inheritance; (5) hyperplastic foci of pancreatic islets; and (6) renal complications. In OLETF rats, plasma glucose levels are high at 8 weeks of age and then significantly exceed that of the genetic controls, the Long-Evans Tokushima Otsuka (LETO) rats, after 18 weeks of age. Hypercholesterolemia and hypertriglycemia appear at 8 weeks of age, but deteriorate with age. OLETF rats past the age of 30 weeks show significant proteinuria compared with LETO rats, and the proteinuria progressively increases with time5,6. Pathological changes of the kidney in OLETF rats past the age of 40 weeks include ECM accumulation in the mesangium, thickening of the glomerular basement membrane, and glomerulosclerosis, changes resembling human diabetic glomerulopathy5,6. Thus, this model is appropriate for observing the long-term disease course.
Several growth factors or metabolic products, including transforming growth factor-
(TGF-), insulin-like growth factor-I, platelet-derived growth factor, prostaglandins, angiotensin II (Ang II), low-density lipoprotein (LDL) and advanced glycation end products, have been shown to be contributing factors involved in the progression of diabetic glomerulopathy7,8,9. These factors can promote mesangial cell (MC) proliferation, glomerular hypertrophy, and ECM overproduction, or alter the glomerular hemodynamics.
The present study was performed to elucidate the early events in the evolution of diabetic glomerulopathy. MCs from OLETF rats at two different stages of the disease, that is, MCs from young rats prior to development of nephropathy at 12 to 14 weeks old (y-OLETF MC) and MC from old rats showing overt nephropathy at 48 to 50 weeks old (o-OLETF MC), were compared with respects to their response to native (n)-LDL/oxidized (ox)-LDL and Ang II.
METHODS
Animals and reagents
All procedures were in accordance with institutional guidelines for animal research. Male OLETF rats and control male LETO rats were supplied by Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan). They were fed standard laboratory chow and given tap water ad libitum. Human n-LDL and ox-LDL were purchased from Biogenesis (Sandown, NH, USA). n-LDL labeled with 1,1'-dioactadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (Dil-n-LDL) and Dil-acetylated-LDL were from Biomedical Technologies Inc. (Stoughton, MA, USA) and Harbor Bio-Products (Norwood, MA, USA), respectively. [125I]-sodium salt, [3H]-thymidine and [32P]-dCTP were obtained from Amersham Japan (Tokyo, Japan). Rabbit antibody to LDL (reactive with n-LDL, but not with ox-LDL) was purchased from Alpha Biomedical Laboratories (Bellevue, Washington, USA). Mouse antibody to proliferating cell nuclear antigen (PCNA) was from Chemicon (Temecula, CA, USA). Rabbit antibodies to c-Fos and c-Myc were from Oncogene Science (Cambridge, MA, USA). Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG were purchased from Cappel (Durham, NC, USA). Rabbit antibodies to rat collagen types I and III were from Chemicon. Fetal calf serum (FCS) was purchased from HyClone (Logan, UT, USA). The following reagents were purchased from Sigma (St Louis, MO, USA): Ang II, trypsin, collagenase, RPMI 1640, Dulbecco's modified Eagle's medium (DMEM), ampicillin-streptomycin solution, insulin, Nu-serum and fatty acid-free bovine serum albumin (BSA). Salmon sperm DNA was obtained from Stratagene (Heidelberg, Germany). 50 
Denhardt's solution was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Glomerular isolation and MC culture
Mesangial cells were separated from glomeruli of male OLETF rats according to the procedure described by Harper et al10 as described in our earlier publications11,12. Animals at two different stages of the disease were selected: young rats (y-OLETF) prior to development of nephropathy at 12 to 14 weeks of age and weighing 400 to 500 g, and old rats (o-OLETF) showing developed nephropathy at 48 to 50 weeks of age that weighed 600 to 700 g. Glomeruli were harvested by sieving the renal cortices, which were then digested with 0.25% trypsin, followed by incubation with 0.05% collagenase for 10 to 15 minutes each at 37°C in culture chambers. As the primary culture, the digested glomeruli were incubated at 37°C in RPMI 1640 supplemented with 20% FCS, 100 U/mL ampicillin, 100 
g/mL streptomycin and 0.6 U/mL insulin. After two to three weeks, MCs appeared and obliterated the epithelial cells. Cultured cells were detached with trypsin/ethylenediaminetetraacetic acid (EDTA), and glomeruli were removed by filtration. Mesangial cells were characterized by their typical stellate morphology on light microscopy, positive staining for smooth muscle-specific actin, and negative staining for factor VIII antigen and for cytokeratin, as described previously11,12. Mesangial cells from control LETO rats, 12 to 14 weeks old and weighing 400 to 450 g (y-LETO), or 48 to 50 weeks old and weighing 500 to 550 g (o-LETO), also were cultured. LETO rats never became diabetic. MCs were expanded and passaged every seven days, and the cells between the fifth and eighth passages were utilized in this study. The cells maintained the characteristics of MCs described above. Confluent cells were made quiescent by incubation in serum-free medium for 24 to 48 hours before the addition of test agents.
Assay of DNA synthesis
Rat MCs, cultured in 24-well plastic plates until confluence and starved in FCS-free RPMI 1640 for 48 hours, were incubated with n-LDL/ox-LDL, Ang II or FCS (for baseline line culture) for the indicated times. [3H]-thymidine was added to the cultures at a final concentration of 1 Ci/mL and the cultures were incubated for an additional four hours. The cells were rinsed three times with ice-cold phosphate-buffered saline (PBS) followed by incubation with 10% trichloroacetic acid for 30 minutes on ice. After rinsing with 100% ethanol, acid-precipitable materials were dissolved in 0.25 N NaOH/0.1% sodium dodecyl sulfate (SDS) and counted by scintillation spectrometry to determine the incorporation of [3H]-thymidine into the acid-insoluble cellular DNA fraction13.
RNA isolation and Northern blot analysis
Total RNA was extracted from cultured MC using Isogen solution (Nippon Gene, Tokyo, Japan). Aliquots of 15 g of RNA were electrophoresed in each lane, transferred onto S & S Nytran nylon membranes (Schleicher & Schuell, Keene, NH, USA), and fixed by exposure to UV light (Funakoshi Ltd, Tokyo, Japan). The membranes were prehybridized in a solution consisting of 50% formamide, 5 standard sodium citrate (SSC), 0.5% (wt/vol) SDS, 5 Denhardt's solution and 100 mg/mL sonicated salmon sperm DNA at 42°C for at least four hours. Blots were then hybridized with 0.5 to 1.0 107 cpm/mL of 32P-labeled cDNA probes at 42°C for 12 to 18 hours. The cDNA probes used were the 1.3-kb PstI-BamHI fragment of rat type I collagen, 2.23-kb EcoRI-BamHI fragment of rat type III collagen, rat transforming growth factor- (a gift from Dr. Enyu Imai, The First Department of Internal Medicine, Osaka University Graduate School of Medicine, Osaka, Japan)14, and 2.2-kb EcoRI fragment of matrix metalloproteinase (MMP)-2 (a gift from Dr. R.C. Harris, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA). Probes were labeled with 32P-dCTP (>108 cpm/g) by the random priming method using the Megaprime DNA Labeling System (Amersham, Buckinghamshire, UK). After washing twice with 2 SSC for 15 minutes at room temperature and with 1 SSC containing 0.1% SDS for 15 minutes at 65°C, and finally with 0.2% SSC containing 0.1% SDS for an additional 15 minutes, the membranes were exposed to Amersham Hyper-ECl film at -70°C with intensifying screens. The blots were stripped and reprobed with a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Clontech, Heidelberg, Germany) under the same conditions described above. Expression levels of mRNA were quantified by densitometry and normalized relative to GAPDH expression. Data were presented as means 
SE of the ratios of optical density (OD) areas for mRNAs, divided by OD areas for GAPDH mRNA.
[125I]-LDL binding assay
[125I]-Low-density lipoprotein was prepared using a commercial kit (Iodo-Gen; Pierce, Rockford, IL, USA). Free [125I] was removed by gel filtration. The final radioactivities ranged from 0.5 106 to 1 107 CPM/g LDL. The LDL binding assay was carried out according to the method described by Goldstein, Basu and Brown15 with slight modifications16. After reaching confluence, MCs were incubated in medium containing lipoprotein-deficient serum consisting of DMEM supplemented with 1% FCS, 8% Nu-serum and 1% ampicillin-streptomycin solution for 36 hours before the experiment. The medium was then removed and replaced with 1 mL of medium consisting of Dulbecco's PBS with 100 mg% of glucose, 2 mg/mL fatty acid-free BSA and [125I]-labeled n-LDL or ox-LDL (1 106 CPM/mL). After incubation at 4°C for the times indicated, the medium was removed, and the MCs were washed three times with ice-cold PBS containing 1% BSA. Additional solution was added to the culture plates, the plates were incubated for five minutes, and the solution was removed. Finally, cells were washed with PBS without BSA. Then, the cells were osmolyzed in 1.0 N NaCl and radioactivity was counted with a gamma counter.
Incorporation of LDL into MCs
Incorporation of LDL into MCs was examined using a Dil-labeled fluorescent probe as described previously16. Subconfluent MCs were incubated with Dulbecco's PBS with 100 mg% glucose and either 10 g/mL of Dil-n-LDL or Dil-acetylated-LDL at 37°C for two hours. The cells were washed vigorously with PBS and examined by immunofluorescence microscopy.
Morphological and immunohistological studies
OLETF and LETO rats were sacrificed and studied at 15, 25, 35, 45, 55, 65, and 75 weeks. Each group consisted of five rats. The number of intraglomerular cells per glomerular cross-section was assessed by counting the total cell nuclei under a microscope using PAS-stained sections containing at least 20 equatorially cut glomeruli from each animal, as described previously17,18,19. Indirect immunofluorescent staining was performed as described previously20,21. In brief, sections were reacted with the primary antibody followed by incubation with the appropriate secondary antibodies labeled with FITC. As a control, kidney sections were incubated with non-immune rabbit serum or unrelated mouse IgG antibody, followed by appropriate FITC-labeled secondary antibodies or secondary antibody alone. These controls gave entirely negative results. The sections containing at least 20 glomeruli (N = 5) were viewed under an immunofluorescent or a phase-contrast immunofluorescent microscope (Nikon Eclipse E600, Tokyo, Japan) and the glomerular cells immunoreactive for PCNA, c-Fos, or c-Myc were counted, as described previously18,22. The data were expressed as the number of positive cells per glomerular cross-section.
Statistics
Data are presented as mean SE. Statistical comparisons were performed using the Mann-Whitney U test with the software program StatView 4.5. P < 0.05 was considered statistically significant.
RESULTS
Morphology of glomeruli of OLETF rats
Representative light micrographs of glomeruli of OLETF and control LETO rats of various ages are shown in Figure 1 a, b, c, d and e. In 15-week-old OLETF rats, there were no obvious alterations in glomeruli. At 25 weeks of age, MC proliferation was observed. After 45 weeks, mesangial expansion accompanied by the accumulation of ECM and thickening of glomerular capillary walls occurred. At 65 weeks, fully developed diabetic glomerulopathy accompanied by nodular sclerosis was seen. In LETO rats, there were no obvious findings except minor alterations due to aging in glomeruli even at 65 weeks.
Figure 1.
Microscopic findings (A–E) and low-density lipoprotein (LDL) deposition (F–J) in glomeruli of Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats. Glomerular changes were minimal in 15-week-old OLETF rats (A). An increase of mesangial cells was seen in 25-week-old OLETF rats (B). ECM accumulation was evident in 45-week-old OLETF rats (C) and "nodular lesions" were present in 65-week-old OLETF rats (D), while only minor glomerular changes were seen in 65-week-old LETO rats (E). Glomerular LDL deposition was seen in 15- (F) and 25-week-old (G) OLETF rats. LDL deposition was intense in glomeruli of 45- (H) and 65-week-old (I) OLETF rats, whereas it was weak in glomeruli of 65-week-old LETO rats (J). Original magnifications were 400.
ECM and LDL deposition in glomeruli of OLETF rats
Glomerular deposition of LDL was weak in 15-week-old OLETF rats, and it progressively increased from 25 to 65 weeks of age Figure 1 f, g, h and i. In LETO rat glomeruli, LDL was negative from 15 to 45 weeks of age and was weakly deposited after 65 weeks of age Figure 1j. Type I and type III collagens were accumulated in the glomeruli in 45- to 65-week-old OLETF rats, but were not as extensively accumulated in the age-matched controls Figure 2.
Figure 2.
Immunofluorescent staining for type III collagen in OLETF and LETO rat glomeruli. The accumulation of type III collagen was evident in the mesangial areas in 45-week-old OLETF rats (B), but much less evident in 15-week-old OLETF (A) and 45-week-old LETO (C) rats. A negative control section of a 45-week-old OLETF rat, stained with non-immune rabbit serum, followed by FITC-labeled secondary antibodies, is shown in (D). Original magnifications were 400.
Glomerular cell number and expression of PCNA, c-Fos and c-Myc in OLETF rats
Total glomerular cellularity and the glomerular cells expressing PCNA, c-Fos and c-Myc were assessed using kidney sections from OLETF and LETO rats Figure 3. Glomerular cell numbers were significantly higher in OLETF rats from 25 to 65 weeks of age than in age-matched LETO rats. Glomerular cells expressing PCNA were significantly increased in OLETF compared to LETO rats at 35 week of age. Immunostaining revealed that the number of c-Fos- or c-Myc-positive glomerular cells was significantly higher in OLETF rats than in LETO rats at 15 weeks of age.
Figure 3.
Total glomerular cellularity and the glomerular cells expressing PCNA, c-Fos and c-Myc in OLETF rat kidneys. Immunofluorescent staining for PCNA (a) and c-Myc (b) in glomeruli of 35-week-old and 15-week-old OLETF rats, respectively. Note the diffuse nuclear staining (arrows) within the glomeruli. A negative control section, stained with non-immune rabbit serum, followed by FITC-labeled secondary antibodies, of 35-week-old OLETF rat is shown in (c). Arrowheads in panels a to c denote the Bowman's capsule; G is glomerulus. Original magnifications were 400. Cell number (d) and expression of PCNA (e), c-Fos (f), and c-Myc (g) in glomeruli of OLETF (
) and LETO (
) rats. Glomerular cell numbers were significantly higher in OLETF than in LETO rats between 25 and 65 weeks of age (d). PCNA expression (e) in glomeruli was significantly increased in 35-week-old OLETF rats compared with that in LETO rats. The number of glomerular cells expressing c-Fos (f) and c-Myc (g) was significantly higher in 15-week-old of OLETF rats than in LETO rats. Total glomerular cells and glomerular cells positive for PCNA, c-Fos, and c-Myc were counted in at least 20 glomeruli in each section (N = 5 per group). Data are presented as mean SE. *P < 0.05, **P < 0.01.
Effects of LDL on DNA synthesis in cultured MCs
In cultures of MCs in 10% FCS, the growth rate of MCs from y-OLETF rats was similar to that of MCs from o-OLETF, y-OLETF and o-LETO rats for up to 48 hours. From 72 to 120 hours, the growth of MCs from y-OLETF rats was faster, as shown by an approximately 1.6-fold increase in cell numbers compared with y- and o-LETO MCs, and an approximately 1.3-fold increase compared with o-OLETF MCs.
Mesangial cells, cultured until confluent and starved in FCS-free medium for 48 hours, were incubated with 10 g/mL, 50 g/mL, or 100 g/mL of n-LDL or ox-LDL Figure 4. DNA synthesis evaluated by [3H]-thymidine incorporation was compared between LDL-stimulated cells and unstimulated controls (=100%). The results are expressed as the percent of the control DNA synthesis. After exposure of MCs to 10 g/mL of n-LDL, DNA synthesis in y-OLETF MCs increased to approximately 300% of control level. DNA synthesis in o-OLETF, y-LETO and o-LETO MCs increased to 180 to 200% of the control level; the difference between y-OLETF MCs and o-OLETF or LETO MCs was significant (P < 0.05). DNA synthesis in MCs from OLETF and LETO rats was weakly stimulated by 50 g/mL of n-LDL. The addition of 100 g/mL of n-LDL suppressed DNA synthesis in OLETF and LETO MCs, while the addition of 10 g/mL, 50 g/mL, or 100 g/mL of ox-LDL minimally affected DNA synthesis in OLETF and LETO MCs.
Figure 4.
DNA synthesis in mesangial cells (MCs) exposed to native (n)-low-density lipoprotein (LDL)/oxidized (ox)-LDL. MCs were incubated with 10 g/mL, 50 g/mL, or 100 g/mL of n-LDL or ox-LDL for 4 hours. The unstimulated basal level in young (y)-OLETF MCs (), old (o)-OLETF (
), y-LETO (
), and o-LETO (
) was 2.3 104 cpm, 0.8 104 cpm, 1.0 104 cpm, and 0.9 104 cpm, respectively. After stimulation with 10 g/mL of LDL, [3H]-thymidine incorporation in y-OLETF, o-OLETF, y-LETO, and o-LETO was 7.0 104 (mean) cpm, 1.5 104 cpm, 2.1 104 cpm, and 1.9 104 cpm, respectively. y-OLETF MCs showed an approximately 300% increase from the unstimulated basal level, compared with o-OLETF MCs (
180% increase) or control LETO MC (200% increase). DNA synthesis in MCs from OLETF and LETO rats was weakly stimulated by 50 g/mL of n-LDL, while 100 g/mL of n-LDL suppressed DNA synthesis in MCs from OLETF and LETO rats. Exposure to 10 g/mL, 50 g/mL or 100 g/mL of ox-LDL did not markedly affect DNA synthesis in OLETF and LETO MCs. Symbols and bars represent means SE of 3 experiments, each carried out in quadruplicate; *P < 0.05.
Analysis of LDL receptors/scavenger receptors in MC
Incorporation of fluorescence-labeled n-LDL/acetylated LDL and binding of 125I-labeled n-LDL/ox-LDL to MCs were studied. After a two-hour incubation, fluorescence of Dil-n-LDL was strong in y-OLETF MCs, but weak in o-OLETF or LETO MCs Figure 5a. Binding of 125I-labeled n-LDL to MCs reached equilibrium after one to two hours of incubation. The maximum binding to y-OLETF MCs was approximately twofold greater than that to LETO MCs and fourfold greater than that to o-OLETF MCs Figure 5b. Addition of excess amounts of unlabeled n-LDL (100 g/mL) displaced approximately 40 to 50% of the 125I-labeled LDL in a time-dependent manner.
Figure 5.
Uptake of fluorescence-labeled n-LDL into cultured MCs and binding of 125I-labeled n-LDL to cultured MCs. Dil-n-LDL-uptake (a) and binding to 125I-labeled n-LDL (b) were higher in y-OLETF MCs (c) than in o-OLETF or LETO MCs. Symbols and bars and bars represent means SE of 3 experiments, each carried out in quadruplicate. Symbols in panel d are: () y-OLETF; (
) y-LETO; (
) o-LETO; (
) o-OLETF.
Similarly, incorporation of fluorescence-labeled acetylated-LDL and binding of 125I-labeled ox-LDL to MCs were studied. Uptake of Dil-acetylated-LDL was high in o-OLETF MCs Figure 6a. The level of 125I-ox-LDL binding was elevated in o-OLETF MCs (1.5-fold and 3 to 4-fold higher in o-OLETF MCs than in y-OLETF and LETO MCs, respectively).
Figure 6.
Uptake of fluorescence-labeled modified LDL into cultured MCs and binding of 125I-labeled ox-LDL to cultured MCs. Dil-acetylated-LDL uptake (a) and 125I-labeled ox-LDL binding (b) were elevated in o-OLETF MCs compared with y-OLETF or LETO MCs. Symbols and bars and bars represent means SE of 3 experiments, each carried out in quadruplicate. Symbols in panel d are: () o-OLETF; () y-OLETF; () o-LETO; () y-LETO.
ECM production by MCs after stimulation with n-LDL/ox-LDL
Type I and type III collagen synthesis in MCs after stimulation with 50 g/mL of n-LDL or ox-LDL was analyzed by Northern blotting Figure 7. In o-OLETF MCs, type I collagen mRNA expression was up-regulated after addition of n-LDL or ox-LDL. y-OLETF MCs and LETO MCs showed only a slight increase in type I collagen mRNA levels. Similar results were observed for type III collagen mRNA expression in these cells (data not shown).
Figure 7.
Type I collagen mRNA expression in MCs after exposure to n-LDL or ox-LDL. (A) Northern blots of representative experiments and (B) the ratio of optical density (OD) areas for type I collagen mRNA divided by OD areas for DAPDH mRNA. Symbols and bars represent means SE of 3 experiments, each carried out in quadruplicate. After exposure to 50 g/mL of n-LDL or ox-LDL, type I collagen mRNA expression in o-OLETF MCs was up-regulated and peaked within 2 to 8 hours of incubation (right panel). Type I collagen mRNA expression was only slightly increased in y-OLETF MCs (left panel) and OLETO MCs (data not shown) after stimulation with n-LDL or ox-LDL.
Induction of TGF-
mRNA and MMP-2 mRNA expression by n-LDL/ox-LDL
Northern blot analyses revealed that TGF- mRNA expression in MCs from OLETF and LETO rats was minimal in untreated cultures in FCS, but was markedly elevated in o-OLETF MCs compared with y-OLETF or LETO MCs after stimulation with 50 g/mL n-LDL or ox-LDL Figure 8. Induction of MMP-2 mRNA by n-LDL/ox-LDL was similar in y-OLEFT, o-OLETF, and LETO MCs (data not shown).
Figure 8.
Transforming growth factor- (TGF-) mRNA expression in MCs treated with n-LDL/ox-LDL. (A) Northern blot analysis showed that, after 18 hours of incubation with 50 g/mL of ox-LDL, TGF- mRNA expression was higher in o-OLETF MCs than in y-OLETF or LETO MCs. (B) The ratio of TGF- mRNA signal to GAPDH mRNA signal in OLETF and LETO MCs with stimulation by 50 g/mL of n-LDL (
) or ox-LDL () or or without stimulation of LDL (). n-LDL and ox-LDL significantly increased TGF- mRNA expression in o-OLETF, compared with y-OLETF (P < 0.01) and o-LETO (P < 0.01). Symbols and bars represent means SE of 3 experiments, each carried out in quadruplicate; **P < 0.01.
Effects of Ang II on DNA synthesis and ECM production in MC
Exposure to Ang II (10-7 mol/L) caused a significantly greater elevation of DNA synthesis in y-OLETF MCs (300% of control level in y-OLETF vs. 120 to 160% of control level in o-OLETF, y-LETO and o-LETO, P < 0.05; Figure 9). Northern blot analyses for type I and III collagen mRNA revealed that ECM production was high in unstimulated o-OLETF MCs and was further increased after incubation with Ang II Figure 10a, while there was no marked increase in y-OLETO or LETO. After stimulation of MCs with Ang II, TGF- mRNA was also more markedly up-regulated in o-OLETF MCs than in MCs from y-OLETF or LETO rats Figure 10b.
Figure 9.
DNA synthesis in OLETF and LETO MCs exposed to angiotensin II (Ang II 10-7 mol/L) for 18 hours. [3H]-thymidine incorporation was high in unstimulated y-OLETF MCs, and was further increased by exposure to Ang II, while it was low in o-LETF and LETO MCs with or without Ang II exposure. Symbols and bars represent means SE of 5 separate experiments (N = 3 rats per group); **P < 0.01.
Figure 10.
Northern blot analysis for type I and III collagen mRNA (A) and TGF- mRNA (B) in Ang II-stimulated MCs from OLETF and LETO rats. (A) Incubation with Ang II (Ang II+) resulted in up-regulation of type I and III collagen mRNAs in o-OLETF MCs, but not in y-OLETF and LETO MCs. (B) After incubation of MCs with Ang II, TGF- mRNA expression was stronger in o-OLETF MCs than in y-OLETF or o-LETO MCs. In untreated MC cultures in FCS, TGF- mRNA expression was minimal in OLETF and LETO rats (as shown in Figure 8b).
DISCUSSION
Using OLETF rats, which exhibit chronic-course diabetes resembling human type 2 diabetes mellitus, we found a significant increase in glomerular cell number in OLETF compared to LETO rats between 25 and 75 weeks of age. Glomerular cells in OLETF rats in the early stage showed transient up-regulation of PCNA as well as of the immediate early gene products c-Fos and c-Myc. Previous studies investigating glomerular cell proliferation in the early phase of diabetes reported conflicting results. Earlier morphological observations performed using kidneys from patients with type I diabetes mellitus23,24 or streptozotocin-induced diabetic rats25 revealed glomerular hypertrophy and basement membrane thickening as the initial structural alterations. Subsequent detailed studies using autoradiography indicated that glomerular hypercellularity occurs concomitantly with glomerular enlargement in the early phase of streptozotocin-induced diabetes in rats26. Mahadevan et al reported a significant increase in the number of MC in rats 21 days after induction of streptozotocin-induced diabetes27. PCNA, a nuclear protein that is maximally activated during the S-phase of the cell cycle28, and immediate early genes (c-fos, c-myc, and Egr-1), transcriptional factors that are expressed in the G1 phase of the cell cycle29, have been studied in diabetic models27,30,31 and MCs cultured in high-glucose media32. Increased expression of PCNA has been reported in glomerular cells from rats with streptozotocin-induced diabetes19,30. Young et al observed early and self-limited MC proliferation in this model, and showed that staining for PCNA was evident by day 1, and peaked at day 319. PCNA as well as immediate early genes (c-myc and Egr-1) were induced in MCs cultured in high glucose conditions32. Transient increases in c-fos and c-jun were reported after the onset of hyperglycemia in diabetic rats31. Thus, our data, together with those of others19,27,30,31,32, indicate that proliferation of glomerular cells occurs in the diabetic milieu preceding the development of nephropathy.
Abnormal lipid metabolism is a frequent complication in various types of immune-mediated or nonimmune-mediated glomerular diseases33,34. Previous studies in humans and animals have suggested that altered lipid metabolism causes glomerular injury and promotes deterioration of glomerular function35,36. Hypercholesterolemia and hypertriglycemia, commonly observed during the course of human diabetes mellitus37, are also found in OLETF rats5,6. Quantitative abnormalities of the major cholesterol carrier LDL as well as qualitative alterations of LDL such as enhanced glycation and oxidation have been implicated in diabetic microvascular complications38,39. Glomerular cells, including MCs and epithelial cells, possess LDL receptors, and thus are affected by abnormal lipid metabolism16,40. In addition, MCs can express scavenger receptors21,41. As shown for the development of atherosclerosis42, glomerular cells can be damaged by the uptake of n-LDL or ox-LDL via their specific receptors.
We studied the glomerular LDL deposition and the response of OLETF MCs to n-LDL or ox-LDL. In this model, plasma levels of cholesterol and triglyceride increased from 8 weeks of age5,6. We found that glomerular LDL deposition progressively increased with age, whereas the LDL receptor in o-OLETF rats appeared to be down-regulated. These observations suggest that, in addition to LDL binding to its specific receptor, other mechanisms, such as affinity of LDL to glycosaminoglycans in glomerular matrix components43 or phagocytosis of LDL by mesangial cells44, are involved in glomerular LDL deposition.
Young-OLETF MCs, expressing high levels of LDL receptors, markedly proliferated after exposure to low concentrations (10 g/mL) of n-LDL. In contrast, proliferative response to this dose of n-LDL in o-OLETF MCs was similar to that in LETO MCs. It has been shown that low concentrations of LDL induce proliferation of rat MCs but high concentrations (100 g/mL) of it are instead cytotoxic45, as we found in OLETF and LETO MCs. It is generally considered that LDL concentrations of 100 to 500 g/mL are well within the physiological range in the plasma of non-diabetic rats45. In OLETF rats that develop hyperlipidemia after 8 weeks of age6, the plasma lipoprotein levels are expected to be increased with age. It is unknown, however, what the prevailing concentration of LDL is for mesangial cells in vivo, because the passage of LDL particles into the mesangial area may be influenced by the accessibility to fenestrated endothelium and by charged endothelium45.
Oxidized-LDL is produced by conversion from n-LDL by free radicals excreted by macrophages or intrinsic glomerular cells33,46,47,48. Cultured MCs from non-diabetic rats have been demonstrated to bind and incorporate ox-LDL to a greater extent than n-LDL41, which results in the alterations of eicosanoid synthesis and cytokine production, influencing local glomerular hemodynamics, vascular permeability and ECM production41,49. LDL binding studies indicated that y-OLETF MCs possess a relatively low concentration of scavenger receptors compared with LDL receptors, which may be related to the poor response of DNA synthesis to ox-LDL. In contrast, o-OLETF MCs expressed high amounts of scavenger receptors and showed the down-regulation of LDL receptors. Following stimulation with n-LDL or ox-LDL, o-OLETF MCs produced ECM proteins, while y-OLETF synthesized rather small amounts of these proteins. It appears that functional changes of MCs with respect to the response to n-LDL/ox-LDL occur with the development of diabetes in OLETF rats. Although the precise mechanisms of the functional switch in OLETF MCs are unclear, the switch may be explained by a transition from cellular expression of LDL receptors to expression of scavenger receptors and also by co-expression of TGF- in o-OLETF MCs, but not in y-OLETF MCs. Furthermore, it is speculated that the intracellular signaling processes are different in MCs from y-OLETF and o-OLETF rats. Stimulation by n-LDL or modified LDL has been reported to induce activation of extracellular signal-regulated kinases (ERK), one of the mitogen-activated protein kinase signaling pathways, in non-diabetic rat MCs50, which affects cell proliferation and also matrix production. The activation of this pathway may contribute to MC proliferation or ECM synthesis in diabetic conditions as well. Gene induction downstream of the ERK cascade might be different in y-OLETF and o-OLETF rats, thus inducing different responses to the same stimulus.
Various lines of evidence indicate that the renin-angiotensin system is locally activated in diabetic nephropathy, thus influencing parenchymal cell functions and hemodynamics in the kidney51,52. In several human studies, angiotensin-converting-enzyme inhibitors or Ang II receptor blockades have been demonstrated to be renoprotective, independent of their systemic antihypertensive actions53,54. Ang II contributes to control of hypertrophy, DNA synthesis and ECM production in glomerular cells9,52,55. These actions are exerted in part by synergy with other growth factors such as TGF- or by co-inhibition of proteolytic enzymes to retard the degradation of ECM9,56. In this study, y-OLETF MCs stimulated with Ang II showed a significant increase in DNA synthesis compared with o-OLETF and LETO MCs. In contrast, o-OLETF MCs stimulated with Ang II produced type I and type III collagen and showed the enhancement of TGF- expression, whereas ECM synthesis was not markedly affected by Ang II in y-OLETO and LETO MCs. The precise mechanisms of the shift from cell proliferation toward ECM production in Ang II-stimulated MCs are unclear. In the late stage of diabetes in OLETF rats, prolonged hyperglycemia or other metabolic abnormalities might facilitate expression of other relevant growth factors such as TGF-. Similar to our findings, Yagi et al reported that, in OLETF rats, glomerular type I collagen mRNA and TGF- mRNA were up-regulated at diabetic stage (30- and 54-weeks-old), but not at the prediabetic stage (14-weeks-old)57. Previous reports19,27,30,31,32,57 and our present findings collectively indicate that stage-specific gene regulation is induced in multipotential MCs of diabetic rats. The precise mechanisms of this process has not been fully elucidated, but expression of specific receptors, as observed in n-LDL/ox-LDL, coexpression of TGF-, as we found in o-OLETF MCs stimulated with ox-LDL and Ang II, and other mechanisms such as different activation or control of genes downstream of intracellular signals in y-OLETF and o-OLETF rats may be involved.
In summary, our observations showed that, upon stimulation with n-LDL/ox-LDL and Ang II, functional transformation from proliferation to ECM biosynthesis occurred in cultured OLETF MCs during the evolution of diabetes. The status of these elements in diabetic conditions in vivo remain speculative, but in vitro experiments using cultured MCs may reflect certain processes of abnormal regulation of proliferation and ECM production in vivo. Further studies are needed to demonstrate that intervention with agents modifying MC responses in the early phase can retard or prevent the progression of diabetic nephropathy.
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Acknowledgments
This work was supported by a grant from Morinaga Hohshikai, Japan (to M.O., AA03979) and a grant from Osaka Kidney Foundation, Japan (to M.O., OKF00-001).
