Dual-color Immunofluorescent Labeling Of AR And TLR4 With Quantum Dots In Cell Model And Kidneys Of Diabetic Rats

Background:Diabetes is one of the major chronic diseases diagnosed worldwide with a common complication of diabetic nephropathy (DN). There are multiple possible mechanisms associated with DN, among which aldose reductase (AR) and toll-like receptor 4 (TLR4) may be involved in the occurrence and development of DN. AR is a key enzyme in the polyol pathway. It not only plays an important role in diabetic complications, but also may be involved in a variety of signaling pathways, especially inflammation. A number of studies show that DN is an immune and inflammation-related disease, inflammation contributes to renal tubular injury in DN. TLR4 is critical transmembrane protein mediated inflammation in mammals, which triggers the signal transduction pathway and involves in inflammation through recognition and binding pathogen-associated molecules. Effective detection of specific protein markers has great value for the diagnosis and treatment of diseases. The common pathology detection methods include immunohistochemistry (IHC) and immunofluorescence staining. However, they are not easy to detect multiple proteins simultaneously in the same tissue section. As a new type of nanosized fluorophore, quantum dots (QDs) have been recognized in imaging applications and have broad prospects in the biomedical research, especially in multiplexed staining.Objective:To explore the expressions and roles of AR and TLR4 in cell model and diabetic rats, and provide a new insight in the diagnosis and treatment of DN. Meanwhile, to explore the application of QD-based double-color labeling method in the detection of AR and TLR4 in diabetic rat renal tissues simultaneously, and study the relationship between two proteins.Methods:1、COS-7 cells was transient transfected with plasmid contained human AR gene to construct a cell model overexpressed AR;2、QD-anti-AR、QD-anti-TLR4 conjugated probes were prepared and detected;3、Detecting and localization the expression of AR in COS-7 cell model by immunocytochemistry and quantum dots immunofluorescence;4、Hematein and Eosin stained the renal tissues of diabetic rats;5、 Using conventional IHC and QDs immunofluorescence to detect the expressions of AR and TLR4 in the renal tissues of diabetic rats;6、A dual-color immunofluorescent labeling technique based on QDs was applied to detect the expressions of AR and TLR4 in the renal tissues of diabetic rats simultaneously.Results:1、A cell model with overexpression of AR was successfully constructed by transfecting plasmid pHAR into COS-7 cells. The QDs immunofluorescence revealed the expression of AR was located in cells cytoplasm and membrane;2、HE staining revealed the diabetic rats exhibited glomerular enlargement and mesangial matrix expansion, notably the most obvious change was the tubules which emerged tubule swelling and tubular extensive vacuolar degeneration, and indicates that the diabetic rats have occurred the early renal pathological changes;3、The expression level of AR was up-regulated in diabetic rats, and mainly located in the glomerular capillary and the interstitial capillary;4、The expression level of TLR4 protein in diabetic nephropathy was up-regulated, and mainly located in renal cortex tubules, predominantly in vacuoles degenerated tubules;5、The positive signals of AR and TLR4 in diabetic kidney tissues were first observed simultaneously by multiplexed QDs imaging method. The two protein expression sites were completely consistent with their individual detection;6、The QDs-620 were successfully conjugated with AR and TLR4 antibodies respectively by EDC. And the optimal conjugate ratio between QDs-620 and antibody was found by agarose gel electrophoresis;7、The optimized QD-based immunofluorescence technique has not only shown a satisfying sensitivity and specificity for the detection of biomarkers in cells and tissues, but also is more simple and quickly.Conclusion:1、The cell model was constructed and the expression site of AR in cells was correctly located, which laid a foundation for screening ARIs;2、The expression and distribution of AR in kidney tissues of diabetic rats were first observed intuitively by IHC,which suggested AR involved in the occurrence and development of DN;3、TLR4 overexpressed in the vacuoles degenerated tubules of diabetic rats, which indicated TLR4 involved in the occurrence and development of DN through inflammation;4、The expressions of AR and TLR4 in renal tissues of diabetic rats were first simultaneously observed by multiplexed QDs imaging method. It reveals AR and TLR4 may be jointly participated in the occurrence and development of DN through inflammation;5、The optimized QD-based immunofluorescence technique has not only shown a satisfying sensitivity and specificity, but also is a valuable supplement of IHC in multiplexed imaging. The technology provides a new insight into the mechanistic study on the correlation among biological factors as well as the potential applications in diagnosis and treatment of diseases.