Tinospora cordifolia consumption ameliorates changes in kidney chondroitin sulphate/dermatan sulphate in diabetic rats

Abstract

Diabetes is known to alter kidney extracellular matrix (ECM) components. Chondroitin sulphate (CS)/dermatan sulphate (DS), an ECM component, which plays an essential role in kidney is altered during diabetes. The focus of this study has been to examine the effect of Tinospora cordifolia (TC) consumption, a potent plant widely used to treat diabetes, on kidney CS/DS. Experimentally induced diabetic rats were fed with diet containing TC at 2·5 and 5 % levels and the effect of it on kidney CS/DS was examined. The CS/DS content and CS:heparan sulphate ratio which was decreased during diabetic condition were ameliorated in TC-fed groups. Disaccharide composition analysis of CS/DS by HPLC showed that decreases in 'E' units and degree of sulphation were modulated in 5 % TC-fed groups. Apparent molecular weight of purified CS/DS from the control rat kidney was found to be 38 kDa which was decreased to 29 kDa in diabetic rat kidney. Rats in 5 % TC-fed groups showed chain length of 38 kDa akin to control rats. Expression of chondroitin 4-O-sulfotransferase-1, dermatan 4-O-sulfotransferase-1 and N-acetylgalactosamine 4 sulphate 6-O-sulfotransferase, enzymes involved in the synthesis of 'E' units which was reduced during diabetic condition, was significantly contained in the 5 % TC-fed group. Purified CS/DS from 5 % TC-fed group was able to bind higher amounts of ECM components, namely type IV collagen and laminin, when compared with untreated diabetic rats. The present results demonstrate that consumption of a diet containing TC at the 5 % level modulates changes in kidney CS/DS which were due to diabetes.

Keywords: 2AB 2-aminobenzamide; AIN, American Institute of Nutrition; CS, chondroitin sulphate; Chondroitin sulphate/dermatan sulphate; DMMB, 1,9-dimethylmethylene blue; DS, dermatan sulphate; Diabetes; ECM, extracellular matrix; GAG, glycosaminoglycan; GFR, glomerular filtration rate; GalNAc, N-acetyl-d-galactosamine; Glycosaminoglycans; HS, heparan sulphate; HexUA, hexuronic acid; IdoA-l-iduronic acid; Kidneys; SFC, starch-fed control; SFD, starch-fed diabetic; STZ, streptozotocin; TC, Tinospora cordifolia; TFD, TC-fed diabetic.; Tinospora cordifolia.

Fig. 1

Effect of Tinospora cordifolia (TC) on kidney index (A), glomerular filtration rate (GFR) (B) and microalbuminuria (C) in control (n 6) and diabetic rats (n 11 per group). SFC, starch-fed control; SFD, starch-fed diabetic; TFD, TC-fed diabetic (2·5 and 5 %). Values are means, with standard deviations represented by vertical bars. * Mean value was significantly different from that of the SFC rats (P < 0·05). † Mean value was significantly different from that of the SFD rats (P < 0·05).

Fig. 2

Glomerular area of kidney from control, diabetic and Tinospora cordifolia (TC)-fed rats. (A) Kidneys were paraffin blocked and 5 µm sections were made. These paraffin sections were hydrated and stained with haematoxylin and eosin as detailed in the Experimental methods section. The areas of glomerulus were calculated using Image J software (B). Analysis was carried out for at least twenty glomeruli per rat. SFC, starch-fed control; SFD, starch-fed diabetic; TFD, TC-fed diabetic (2·5 and 5 %). Values are means, with standard deviations represented by vertical bars, for control rats (n 6) and diabetic rats (n 11 per group). * Mean value was significantly different from that of the SFC rats (P < 0·05). † Mean value was significantly different from that of the SFD rats (P < 0·05).

Fig. 3

Sulfated glycosaminoglycans (GAG) in the kidney of control, diabetic and Tinospora cordifolia (TC)-fed diabetic rats. Sulfated GAG were isolated from the kidneys of control, diabetic and TC-fed diabetic rat kidney (A). The amount of chondroitin sulphate/dermatan sulphate (CS/DS) was determined by the 1,9-dimethylmethylene blue (DMMB) assay after differential digestion with either chondroitinase A, B or C (see Experimental methods) (B). Ratio of CS/heparan sulphate (HS) was calculated from the amounts of CS/DS and HS obtained (C). SFC, starch-fed control; SFD, starch-fed diabetic; TFD, TC-fed diabetic (2·5 and 5 %). Values are means of two independent experiments carried out in triplicates, with standard deviations represented by vertical bars, for pooled control (n 6), diabetic (n 11) and TC-fed diabetic rat kidney (n 11). * Mean value was significantly different from that of the SFC rats (P < 0·05). † Mean value was significantly different from that of the SFD rats (P < 0·05).

Fig. 4

Disaccharide composition and hybrid structure analysis of kidney chondroitin sulphate/dermatan sulphate (CS/DS) from control diabetic and Tinospora cordifolia (TC)-fed diabetic rats. (A) Isolated glycosaminoglycans (GAG) were subjected to HNO₂ digestion followed by gel filtration and hyaluronidase digestion to obtain pure CS/DS. Disaccharide composition analysis was carried out by digesting 4 µg (as sulfated GAG) by chondroitinase ABC followed by 2AB labelling and analysed by anion-exchange HPLC on a PA-03 column using a 16-530 mm-NaH₂PO₄ gradient over a 1 h period. Fractions were monitored by fluorescence detection, as detailed in Experimental methods. Peak areas of disaccharides were integrated and expressed as percentages. ΔDi-0S, (Δ4,5HexUAα1-3GalNAc); ΔDi-6S, (Δ4,5HexUAα1-3GalNAc(6S)); ΔDi-4S, (Δ4,5HexUAα1-3GalNAc(4S)); ΔDi-diS_D, (Δ4,5HexUA(2S)α1-3GalNAc(6S)); Di-diS_E, (Δ4,5HexUAα1-3GalNAc(4S,6S)). (B) Purified kidney CS/DS (8 µg as sulfated GAG) from all groups was digested with either chondroitinase ABC, AC, B or none. Undigested GAG were estimated by 1,9-dimethylmethylene blue (DMMB) as detailed in Experimental methods. ■, Starch-fed control; □, starch-fed diabetic; ░, TC-fed diabetic (2·5 %); ▓, TC-fed diabetic (5 %). Values are means of analyses carried out on purified CS/DS preparations of two independent experiments, with standard deviations represented by vertical bars. * Mean value was significantly different from that of the SFC rats (P < 0·05). † Mean value was significantly different from that of the SFD rats (P < 0·05).

Fig. 5

Molecular weight determination of the purified chondroitin sulphate/dermatan sulphate (CS/DS). Molecular weight of the purified CS/DS from kidney was determined by gel filtration chromatography on a column of Superdex 200, calibrated with known molecular mass markers as detailed in Experimental methods. Purified CS/DS from control, diabetic and Tinospora cordifolia (TC)-fed groups, 20 µg each, was loaded individually onto the Superdex 200 column and the fractions collected and analysed for glycosaminoglycans by complexation with the metachromatic dye, 1,9-dimethylmethylene blue (DMMB), with absorbance at 525 nm. –▴–, Starch-fed control; –*–, starch-fed diabetic; –◆–, TC-fed diabetic (2·5 %); –■–, TC-fed diabetic (5 %).

Fig. 6

mRNA expression of chondroitin 4-O-sulfotransferase (C4ST-1), dermatan 4-O-sulfotransferase (D4ST-1) and N-acetylgalactosamine 4 sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in kidney. Total RNA was isolated, reverse transcribed and amplified by adding requisite primers. Amplicon size was observed by agarose gel electrophoresis (A). Bands were quantified by densitometry using Win-32 software and normalised against actin which was used as an internal control. The expression of mRNA was evaluated for C4ST-1 (B), D4ST-1 (C) and GalNAc4S-6ST (D). Expression was carried out in triplicates for at least five animals per group. SFC, starch-fed control; SFD, starch-fed diabetic; TFD, Tinospora cordifolia-fed diabetic (2·5 and 5 %). Values are means, with standard deviations represented by vertical bars. * Mean value was significantly different from that of the SFC rats (P < 0·05). † Mean value was significantly different from that of the SFD rats (P < 0·05).

Fig. 7

Binding of purified chondroitin sulphate/dermatan sulphate (CS/DS) to extracellular matrix components. Purified CS/DS from the kidney of control, diabetic and Tinospora cordifolia (TC)-treated rats was immobilised in a ninety-six-well plate with prior coating of poly-l-lysine as detailed in Experimental methods. Extracellular matrix components such as type IV collagen (A) and laminin (B) in varying amounts were evaluated for binding to CS/DS by immunoassay. ■, Starch-fed control; □, starch-fed diabetic; ░, TC-fed diabetic (2·5 %); ▓, TC-fed diabetic (5 %). Values are means of assays done in quadruplicates, with standard deviations represented by vertical bars. * Mean value was significantly different from that of the SFC rats (P < 0·05). † Mean value was significantly different from that of the SFD rats (P < 0·05).