Antidiabetic and renoprotective effects of Cladophora glomerata Kützing extract in experimental type 2 diabetic rats: a potential nutraceutical product for diabetic nephropathy

Abstract

Cladophora glomerata extract (CGE) has been shown to exhibit antigastric ulcer, anti-inflammatory, analgesic, hypotensive, and antioxidant activities. The present study investigated antidiabetic and renoprotective effects of CGE in type 2 diabetes mellitus (T2DM) rats. The rats were induced by high-fat diet and streptozotocin and supplemented daily with 1 g/kg BW of CGE for 12 weeks. The renal transport function was assessed by the uptake of para-aminohippurate mediated organic anion transporters 1 (Oat1) and 3 (Oat3), using renal cortical slices. These two transporters were known to be upregulated by insulin and PKCζ while they were downregulated by PKCα activation. Compared to T2DM, CGE supplemented rats had significantly improved hyperglycaemia, hypertriglyceridemia, insulin resistance, and renal morphology. The baseline uptake of para-aminohippurate was not different among experimental groups and was correlated with Oat1 and 3 mRNA expressions. Nevertheless, while insulin-stimulated Oat1 and 3 functions in renal slices were blunted in T2DM rats, they were improved by CGE supplementation. The mechanism of CGE-restored insulin-stimulated Oat1 and 3 functions was clearly shown to be associated with upregulated PKCζ and downregulated PKCα expressions and activations. These findings indicate that CGE has antidiabetic effect and suggest it may prevent diabetic nephropathy through PKCs in a T2DM rat model.

Figure 1

Acute antihyperglycemic effect of Cladophora glomerata in normal rats. Oral administration of 0.25, 0.5, and 1 g/kg BW of CGE and 20 mg/kg BW of metformin was given to normal rats for 30 min. Subsequently, 50% glucose solution at a concentration of 2 g/kg BW was administered into each rat. Blood samples were individually collected and blood glucose levels were then determined (n = 6–8). ^∗ P < 0.05 indicates significant differences from blood glucose level before treatment.

Figure 2

Micrographs of conventional Hematoxylin and Eosin (H&E) staining of rat kidneys. A sagittal half of kidney from each experimental group was removed, fixed, embedded, cut, and stained by H&E (original magnification 200x for all panels). The data were repeated at least 3 times from separate sets of animals. The results were analyzed using bright-field microscopy. (a) Normal (ND), (b) normal diet supplemented with CGE (ND + CGE), (c) T2DM (DM), (d) T2DM treated with metformin (DM + metformin), and ((e) and (f)) T2DM supplemented with CGE (DM + CGE). Arrow “B” in all panels indicates Bowman's capsule space. Arrow labeled “P” indicates the proximal tubular epithelial cells and the tubular lumen space. Asterisk (∗) indicates infiltration of blood cells and lymphocytes. Glomerulus (G), proximal tubular epithelial cells and the tubular lumen space (P), and glomerular basement membrane (GBM).

Figure 3

Micrographs of Periodic Acid-Schiff (PAS) staining of rat kidneys. Light microscopies of sagittal half of kidney sections stained with PAS and counterstained with hematoxylin are shown (original magnification 200x for all panels). (a) Normal (ND), (b) normal diet supplemented with CGE (ND + CGE), (c) type 2 diabetes mellitus (DM), and (d) T2DM supplemented with CGE (DM + CGE). Bowman's capsular spaces are indicated by arrow “B”; the proximal tubular epithelium and the lumen are indicated by arrow “P.” Glomerulus (G).

Figure 4

Effects of Cladophora glomerata on PAH transport by rOat1 and rOat3 and their mRNA expressions. (a) Rat renal cortical slices were incubated for 30 min in the buffer containing 5 μM of [³H]-PAH at room temperature. Data are expressed as tissue to medium ratios (T/M), that is, tissue content (DPM/mg) ÷ medium (DPM/μL), and represented as mean ± SD. Each experiment was performed from separate animals and at least 5 renal slices were used in each condition (n = 6–8). (b) Rat renal cortical slices were preincubated for 30 min in the presence or absence of 30 μg/mL of insulin and followed by incubation with classical substrate of organic anion transporters, 5 μM of [³H]-PAH for 30 min. Data are expressed as fold over control (without insulin). Each experiment was performed from separate sets of animals and 5 renal slices were used in each condition (n = 6). ^∗ P < 0.05 indicates significant differences from the slices incubated with buffer alone. (c) Rat Oat1 and (d) Rat Oat3 mRNA expressions in renal cortical tissues. Total RNAs were extracted from rat cortical tissues from each animal and rOat1 and 3 mRNA levels were measured using quantitative real-time PCR (qPCR). The data are expressed as mean ± SD and repeated from separate animals (n = 5–7).

Figure 5

Effects of Cladophora glomerata extract on PKCα expression, activation, and translocation in renal cortical tissues. Whole cell lysate, cytosolic, and nuclei fractions were extracted from rat renal cortical tissues. The samples were then separated using electrophoresis and western blotting. (a) Anti-PKCα and (b) p-PKCα antibodies were subsequently detected while anti-Na⁺-K⁺-ATPase and anti-β-actin antibodies were used as a membrane marker and loading control, respectively. The data are expressed as mean ± SD and repeated from separate sets of animals (n = 5). A representative blot of PKCα and p-PKCα protein expressions is shown in top panel ((a) and (b)) and quantification of relative protein expression in each fraction is presented in bottom panel ((a) and (b)).

Figure 6

Effects of Cladophora glomerata extract on PKCζ expression, activation, and translocation in renal cortical tissues. Whole cell lysate, cytosolic, and nuclei fractions were extracted from rat renal cortical tissues. The samples were then separated using electrophoresis and western blotting. (a) Anti-PKCζ and (b) p-PKCζ antibodies were subsequently detected while anti-Na⁺-K⁺-ATPase and anti-β-actin antibodies were used as a membrane marker and loading control, respectively. The data are expressed as mean ± SD and repeated from separate sets of animals (n = 5). A representative blot of PKCζ and p-PKCζ protein expressions is shown in top panel ((a) and (b)) and quantification of relative protein expression in each fraction is presented in bottom panel ((a) and (b)).

Figure 7

Effects of Cladophora glomerata extract on regulatory function of rOat1 and rOat3 functions. (a) Downregulation of rOat1 and 3 by PMA-inhibited PKCα activity. Rat renal cortical slices were preincubated for 30 min in the presence or absence of 100 nM of PMA or PMA with GÖ6976 and followed by incubation with 5 μM of [³H]-PAH for 30 min. Data are expressed as tissue to medium ratios (T/M) (n = 5). ^∗ P < 0.05 indicates significant differences from control. ^# P < 0.05 indicates significant differences from PMA preincubation in renal slices from ND. (b) Upregulation of rOat1 and 3 by insulin-stimulated PKCζ activity. Rat renal cortical slices were preincubated for 30 min in the presence or absence of 30 μg/mL of insulin or insulin with PKCζ-PS and followed by incubation with 5 μM of [³H]-PAH for 30 min. Data are expressed as tissue to medium ratios (T/M) (n = 5–7). ^∗ P < 0.05 indicates significant differences from respective control. ^# P < 0.05 indicates significant differences from insulin preincubation in renal slices from ND. ^≠ P < 0.05 indicates significant differences from insulin preincubation in renal slices from ND + CGE.

Figure 8

Cladophora glomerata extract qualification using HPLC-DAD/MSD analysis. CGE was analyzed against their phenolic standards. Semiquantitative data was analyzed by peak area under the curve relative to the content of each component in the extract.