Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical appli-cations. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di-or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di-and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.

Objective To observe the effects of intermedin (IMD) on nitric oxide synthetase (NOS) in renal ischemia-reperfusion (IR) rat models and the action mechanism.Methods A total of 24 rats were divided into four groups (n =6 each).Group Ⅰ underwent right nephrectomy one week prior to the exposure of left renal pedicles,but did uot receive any I/R.Group Ⅱ underwent right nephrectomy one week prior to left renal I/R surgery.Group Ⅲ underwent right nephrectomy and left renal IMD-pCDNA3.1 ( + ) transfection by ultrasound-mircobubbles and renal I/R surgeries were performed one week after gene transfection.Group Ⅳ was treated with the same way as group Ⅲ except that empty control vector was transfected.All the animals were killed at the end of 24 h of reperfusion.The expression and site of IMD were determined by using immunohistochemistry.Serum levels of BUN and creatinine were determined.The kidney formaldehyde-fixed and paraffin-embedded sections were stained with HE and PAS by standard methods and then histological changes were analyzed semiquantitatively.The mRNA expression levels of endothelial NOS (eNOS),inducible NOS (iNOS) and neuronal NOS (nNOS) in the kidneys of the four groups were detected by using RT-PCR.The protein expression levels of the three NOS mentioned above in the kidneys were semiquantitatively analyzed by Western blotting.Results IMD was weakly expressed in the plasma of tubulointerstitial cells in sham-operated group; whereas IMD expression in the kidneys subject to I/R was increased.Moreover,as compared with I/R group,IMD expression levels were obviously increased (P＜0.01 ).The degree of morphological changes as well as renal dysfunction in group Ⅲ was obviously lessened as compared with group Ⅱ.The mRNA and protein expression levels of eNOS in group Ⅲ were notably increased as compared with group Ⅱ,while the mRNA and protein expression levels of iNOSin group Ⅲ were obviously reduced as compared with I/R group not transfeeted with IMD (P＜0.05).Meanwhile,there were no significant differences in the mRNA and protein expression levels of nNOS among groups Ⅱ,Ⅲ and Ⅳ.Conclusion IMD gene in the kidneys of rats can promote the expression of eNOS and attenuate over-expression of iNOS in the kidneys following I/R,thus protecting against tubulointerstitial damages and renal dysfunction in rat I/R models.