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.
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Date
2014
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Abstract
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.
Description
Ph. D. University of KwaZulu-Natal, Durban 2014.
Keywords
Streptozotocin--Physiological effect., Insulin--Physiological transport., Diabetes--Treatment., Renal pharmacology., Theses--Human physiology.