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PLoS One
2014 Jan 01;99:e106929. doi: 10.1371/journal.pone.0106929.
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The protective role of fucosylated chondroitin sulfate, a distinct glycosaminoglycan, in a murine model of streptozotocin-induced diabetic nephropathy.
Gomes CL
,
Leão CL
,
Venturotti C
,
Barreira AL
,
Guimarães G
,
Fonseca RJ
,
Fortunato RS
,
Mourão PA
,
Delgado AG
,
Takiya CM
,
Leite M
.
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BACKGROUND: Heparanase-1 activation, albuminuria, and a decrease in glomerular heparan sulfate (HS) have been described in diabetic nephropathy (DN). Glycosaminoglycan (GAG)-based drugs have been shown to have renoprotective effects in this setting, although recent trials have questioned their clinical effectiveness. Here, we describe the effects of fucosylated chondroitin sulfate (FCS), a novel GAG extracted from a marine echinoderm, in experimentally induced DN compared to a widely used GAG, enoxaparin (ENX).
METHODS: Diabetes mellitus (DM) was induced by streptozotocin in male Wistar rats divided into three groups: DM (without treatment), FCS (8 mg/kg), and ENX (4 mg/kg), administered subcutaneously. After 12 weeks, we measured blood glucose, blood pressure, albuminuria, and renal function. The kidneys were evaluated for mesangial expansion and collagen content. Immunohistochemical quantifications of macrophages, TGF-β, nestin and immunofluorescence analysis of heparanase-1 and glomerular basement membrane (GBM) HS content was also performed. Gene expression of proteoglycan core proteins and enzymes involved in GAG assembly/degradation were analyzed by TaqMan real-time PCR.
RESULTS: Treatment with GAGs prevented albuminuria and did not affect the glucose level or other functional aspects. The DM group exhibited increased mesangial matrix deposition and tubulointerstitial expansion, and prevention was observed in both GAG groups. TGF-β expression and macrophage infiltration were prevented by the GAG treatments, and podocyte damage was halted. The diabetic milieu resulted in the down-regulation of agrin, perlecan and collagen XVIII mRNAs, along with the expression of enzymes involved in GAG biosynthesis. Treatment with FCS and ENX positively modulated such changes. Heparanase-1 expression was significantly reduced after GAG treatment without affecting the GBM HS content, which was uniformly reduced in all of the diabetic animals.
CONCLUSIONS: Our results demonstrate that the administration of FCS prevented several pathological features of ND in rats. This finding should stimulate further research on GAG treatment for this complication of diabetes.
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25192337
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Figure 1. The time course for the urinary albumin excretion rate after 20 weeks of the study.A significantly increased level of albuminuria was evident in the DM group compared to the control group, with prevention in the DM rats treated with FCS and ENX. In A, Total albumin excretion in 24 hours (miligrams), at baseline and at the end of the twentieth week and in B, albumin (micrograms) to creatinine (miligrams) ratio in a random urinary sample at the end of the experiment. The data represent the means ± SEMs obtained from 5 rats per group. *p<0.01 DM vs. the baseline groups and final control group, #p<0.05 DM vs. the final GAG groups.
Figure 2. Morphological aspects of the mesangial axis and tubulointerstitial area in the experimental animals at the end of the study.A–D show PAS-stained glomerular photomicrographs with a significant increase in the mesangial area in the DM group (B) compared to the control group (A) and no increase in the DM groups treated with FCS (C) or ENX (D). In E, a semi-quantitative analysis demonstrated a 1.4-fold increase in the mesangial area in the DM group compared to the other groups. In F–I, PAS-stained renal sections show expansion of the interstitial area with tubular dilation in the DM group (G), compared to the control animals (F) and GAG-treated groups (H and I). In J, the semiquantitative analysis shows a discrete but significant expansion in the DM group. In L–N, the Sirius Red staining area shows a trend toward increased deposition of collagen fibers in the interstitial area in the DM animals (M) compared to the other groups but without statistical significance, as shown in (O). In N–P, Masson’s trichrome staining depicts the expansion of the interstitial area in the DM rats due to edema and tubular dilation compared to the treated groups. The data represent the means ± SEMs. *p<0.001, **p<0.0001. A–N, bar = 25 µm; N–P, bar = 50 µm.
Figure 3. Immunohistochemical analysis at the end of the experiment.From A–C, representative photomicrographs of ED-1–stained glomerular sections showing macrophage infiltration (arrows) in the DM glomeruli (A) and indicating an increased micro-inflammatory response in the diabetic milieu compared to the normal glomeruli in the control (not shown) and FCS- (B) and enoxaparin- (C) treated animals. D shows a semi-quantitative analysis of the ED-1 surface density. E–G show photomicrographs of the TGF-β–stained glomerular sections, and I–L show the tubulointerstitial area with increased localization of TGF-β in the DM group compared to controls (not shown) and both GAG groups. H and M demonstrate a semi-quantitative analysis of the TGF-β density, confirming the histopathological findings. N–P show reduced expression of nestin in podocytes from the DM group (N) compared to a significant preservation of nestin expression in the DM groups treated with FCS (O) or ENX (P). Q shows a semi-quantitative analysis of glomerular nestin density. The data represent the means ± SEMs. *p<0.0001 DM vs. all of the groups, #p<0.001 ENX vs. Control, **p<0.01 ENX vs. Control, ##p<0.05, ENX vs. FCS. Bar = 25 µm.
Figure 4. The GAGs effectively reduced heparanase-1 expression in the glomeruli of treated DM rats.From A–D, immunofluorescence photomicrographs of heparanase-1–stained renal glomerular sections with an evident increase in expression in the DM (B) animals that was prevented by FCS (C) and ENX (D) administration, as confirmed by a semiquantitative scoring system (E). The data represent the means ± SEMs. *p<0.0001 vs. groups; #p<0.01 vs. ENX. Bar = 25 µm.
Figure 5. A diabetic state promoted a uniform loss of linear heparan sulfate staining in the GBM independent of the treatment.From A–D, the representative immunofluorescence photomicrographs of the heparan sulfate epitope JM-403 in the renal glomerular sections. In the control group (A), a normal linear distribution of heparan sulfate was identified in the GBM, whereas in all of the DM groups (B–D), independent of the treatment, a decreased presence of heparan sulfate in the GBM (as recognized by the JM403 epitope) was evident. This result was confirmed using a semiquantitative scoring system (E). The data represent the means ± SEMs. *p<0.0001 vs. groups. Bar = 25 µm.
Figure 6. Gene expression of proteoglycan core proteins in DM and GAGs treated groups related to control.Gene expression of: agrin, the major GBM proteoglycan; perlecan and collagen XVIII, predominantly mesangial proteoglycans; decorin, a small rich-leucine proteoglycan and glypican-1, a cell suface associated proteoglycan. Gray columns, DM; dark gray, FCS and black, ENX. *p<0.01 vs. control; #p<0.05 vs. DM.
Figure 7. Gene expression analysis for glycosaminoglycan modifying/degrading enzymes in DM and GAGs treated groups related to control.Gray columns, DM; dark gray, FCS and black, ENX. *p<0.05 vs. control. HPSE, heparanase-1; CS, chondroitin sulfate; NDST-1, N-deacetylase/N-sulfotransferase; HS-3-ST-1, heparan sulfate-3-O-sulfotransferase 1.
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