The effects of insulin and Syzygium aromaticum-derived oleanolic acid containing dermal patches on kidney function and renal expression of glucose transporters in streptozotocin-induced diabetic rats.
Ngubane, Phikelelani Siphosethu.
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Introduction The tight glycaemic control required to attenuate chronic complications in type 1 diabetes mellitus requires multiple daily injections of bolus insulin which have been reported to be associated with Na+ retention resulting in hyperinsulinaemic oedema and hypertension. Current research on insulin delivery methods include buccal, oral, nasal, and transdermal delivery systems. Transdermal delivery system is of great interest as this offers sustained controlled release of insulin into the systemic circulation. We have previously reported that transdermal application of pectin hydrogel insulin (PI) matrix patches sustain controlled insulin delivery into the bloodstream of STZ-induced diabetic rats to perhaps ameliorate diabetic complications. Since we have previously reported that STZ-induced diabetic rats retain Na+ following hypotonic saline challenge, this study investigated whether insulin-containing dermal patches can avert and improve the impaired renal fluid and electrolyte handling of STZ-induced diabetic rats. We have also shown that oral administration of OA in addition to possessing hypoglycaemic effects, improves kidney function STZ-induced diabetic rats. The study therefore also investigated whether OA-containing dermal patches can improve kidney function STZ-induced diabetic rats. Materials and methods Pectin insulin (PI)-containing dermal patches of various doses (3.99, 9.57, 16.80 μg/kg) and pectin oleanolic acid (P-OA) containing dermal patches of various doses (21, 42, 84 mg/kg) were prepared by dissolving pectin/insulin or pectin/OA in deionized water and solidified with CaCl2. Short-term (5 weeks) effects on renal function of thrice daily treatments with PI and P-OA patches 8 hours apart were assessed in diabetic animals. Rats sham treated with the pectin drug free patch and insulin (175 μg/kg sc) acted as negative and positive controls, respectively. Daily urine volume, urinary glucose, Na+, K+ and creatinine excretion rates were monitored over 5-weeks. Blood was collected 6 h following treatments for insulin determination. Blood and kidney samples were also collected after 5 weeks for hormonal analysis and measurement of selected biochemical parameters. Results Untreated STZ-induced diabetic rats exhibited elevated weekly urinary glucose, K+ outputs and depressed urinary Na+ outputs throughout the 5-week compared to non-diabetic control animals. Application of PI-containing dermal patches significantly increased urinary Na+ output and reduced urine volume and urinary outputs of glucose and K+ in weeks 4 and 5. Plasma AVP concentrations of untreated STZ-induced diabetic rats were significantly low at end of the 5-week experimental period by comparison with control non-diabetic animals while plasma aldosterone levels were significantly elevated. The highest dose of the insulin-containing dermal patch (16.80 μg/kg) significantly (p < 0.05) elevated plasma AVP concentrations while decreasing plasma aldosterone concentrations of STZ-induced rats by comparison to untreated STZ-diabetic rats. GFR of untreated STZ-induced diabetic rats was significantly decreased while plasma creatinine concentrations were significantly elevated by comparison to non-diabetic control animals. PI containing dermal patches increased GFR of STZ-induced diabetic rats with a concomitant reduction of plasma creatinine concentrations by comparison to untreated STZ-induced diabetic rats. Interestingly, P-OA dermal patches also increased GFR of STZ-induced diabetic rats while reducing plasma creatinine concentrations. The effects of both PI and P-OA containing dermal patch compared with subcutaneous insulin. Significant increase of MDA and decreases of SOD and GPx were found in the skin, kidney and heart tissues of STZ-diabetic animals as compared to non-diabetic control animals. PI (16.80 μg/kg) -treated STZ-induced diabetic animals however showed low concentrations of MDA and increased the activities of SOD and GPx in the skin, kidney and heart tissues compared to untreated STZ-induced diabetic animals. P-OA-treated STZ-induced diabetic animals similarly and significantly showed decreased MDA, and increased activity of antioxidant enzymes; SOD and GPx in skin, kidney and heart tissues. H and E kidney stained sections of untreated non-diabetic control, untreated STZ-induced diabetic rats and diabetic animals topically applied with insulin and OA-containing dermal patches were observed under light microscope. However, STZ-induced diabetic rats showed thickened basement membrane of the Bowmans capsule, thickened glomerular basement membrane and hypercellularity of the proximal tubules by comparison to the non-diabetic animals after 5 weeks of the study. Treatment with insulin containing dermal patches and subcutaneous insulin for 5 weeks however attenuated these features when compared with the untreated STZ-diabetic rats. Like PI dermal patches, OA containing dermal patches also ameliorated structural changes of kidney of STZ-induced diabetic rats. The increased urinary glucose concentrations of the untreated STZ-induced diabetic rats were associated with increased expression of GLUT 1 and SGLT 1 to normalcy by comparison to nondiabetic rats. The highest dose of PI containing dermal patch however, like subcutaneous insulin, significantly decreased the expressions of GLUT 1 and SGLT 1 by comparison to STZ-induced diabetic controls. Plasma insulin concentrations of untreated STZ-induced diabetic rats were significantly low in comparison with control non-diabetic animals. Acute (6 h) and short-term (5 weeks) daily application of PI containing dermal patches to STZ induced diabetic rats significantly elevated plasma insulin concentrations by comparison with untreated diabetic animals. However, the plasma insulin concentrations in animals treated with the high insulin doses (9.57, 16.80 μg/kg) were significantly higher than those found in diabetic groups treated with the low insulin dose (3.99 μg/kg). There were no differences in the plasma insulin concentrations in STZ-induced diabetic animals treated with P-OA containing dermal patches by comparison to STZ- diabetic untreated controls both acutely and chronically. To determine whether insulin was transported across skin of STZ-induced diabetic rats following topical application of PI and P-OA containing dermal patches, we also monitored the density of phosphorylated insulin receptor substrates (IRS) in the skin by immunohistochemical staining with specific insulin receptor antibodies. Non-diabetic treated skin sections showed slight immunostaining of insulin receptors in comparison STZ-induced diabetic rats which stained negative. Immunohistochemical staining for phosphorylated IRS in the skin of animals following application of insulin and sc insulin treatment for 5 weeks clearly demonstrated widespread localization of IRS in cell bodies of the dermis, collagen and subcutaneous layer. Interestingly, OA-containing dermal patches also showed widespread localization of IRS in cell bodies of the dermis, collagen and subcutaneous layer. H and E skin sections of untreated non-diabetic control, untreated STZ-induced diabetic rats and diabetic animals topically applied insulin and OA-containing dermal patches showed no significant histological differences in dermis compared to the untreated non diabetic control skin sections. Discussion Previous studies indicate compromised renal function in experimental diabetes and diabetic patients. The results herein however indicate that insulin containing dermal patches increase Na+ excretion probably by decreasing plasma aldosterone and increasing plasma AVP concentrations of STZ-induced diabetic rats. PI containing dermal patches also improve kidney function by increasing GFR with concomitant reduction of plasma creatinine concentrations. Like PI containing dermal patches, P-OA containing dermal patches increased Na+ excretion by decreasing plasma aldosterone and increasing plasma AVP concentrations of STZ-induced diabetic rats. P-OA containing dermal patches also increased GFR and reduced plasma creatinine concentrations of STZ-induced diabetic rats. Conclusion From these results, we conclude that PI and P-OA dermal patches deliver physiological amounts that can improve kidney function in diabetes.