Application of < 18 > F-lactitol in preparation of diabetic nephropathy detection product

Abstract

The invention discloses application of < 18 > F-lactitol in preparation of a diabetic nephropathy detection product, and belongs to the technical field of biomedical detection. In order to solve the problem that the action and mechanism of STRs in the kidney through SGLT2-dependent glucose reabsorption and transport are not clear yet, 18F-labeled lactitol is used, the in-vivo glucose level, kidney pathology and SGLT2 expression are detected through PET / CT, and the glucose uptake rate is labeled through in-vitro fluorescence, so that the effect and mechanism of STRs in the kidney through SGLT2-dependent glucose reabsorption and transport are determined. The method is used for evaluating the glucose reabsorption effect and mechanism of the STRs in STZ (streptozotocin)-induced diabetic mice and HK-2 cells. The result shows that the 18F-lactitol can be sensitively and specifically combined with the STRs, the biological distribution of the 18F-lactitol and the expression of the STRs in the kidney of a diabetic mouse are obviously reduced, the expression of the SGLT2 is up-regulated, and the lactitol or siRNA-STRs can obviously reduce the influence of high glucose on the expression of the STRs and the SGLT2 and glucose absorption. Therefore, the STRs can be used as a new therapeutic target for diabetes, and the < 18 > F-lactitol labeled PET / CT can become a novel probe for detecting diabetic kidney injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to the application of 18F-lactitol in the preparation of diagnostic products for diabetic nephropathy. Background Technology

[0002] Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes and is associated with increased cardiovascular mortality. Currently, according to the American Diabetes Association (ADA) guidelines, screening and diagnosis of DN, in addition to diagnosing diabetes and retinopathy, primarily rely on measurements of urinary albumin excretion rate (UAE), glomerular filtration rate (GFR), and renal biopsy. However, many factors such as urinary tract infections, hematuria, and acute febrile illnesses can affect the accuracy of UAE measurements. Invasive examinations such as renal biopsy are not the first choice. Therefore, it is necessary to find a non-invasive, highly specific imaging method to aid in the monitoring and auxiliary diagnosis of DN.

[0003] Positron emission tomography (PET) combined with X-ray computed tomography (CT) is widely used in the clinical auxiliary diagnosis of metabolic diseases. For example, 18F-fluorodeoxyglucose (FDG)-PET / CT has been used clinically for about 40 years, helping to stage, restate, follow up and monitor the treatment of various glucose-high affinity tumors, and reflecting the functional status of adipose tissue.

[0004] Sweet taste receptors (STRs) are heterodimers composed of taste receptor 1 member 2 (T1R2) and taste receptor 1 member 3 (T1R3), widely distributed throughout the body, including the kidneys. Recent reviews indicate that STRs are responsible for recognizing sweet substances such as carbohydrates. Strong evidence shows that STRs mediate basal insulin secretion and are involved in the pathogenesis of obesity and type 2 diabetes mellitus (T2DM). Previous studies have confirmed that in diabetic states, STRs and related signaling components (Gα-gutrin, PLCβ2, and TRPM5) are significantly downregulated in the kidneys. Furthermore, STRs may be involved in the activation of ROS-NLRP3 inflammasome signaling in the pathogenesis of diabetic nephropathy (DN).

[0005] Sodium-glucose cotransporter 2 (SGLT2) is primarily expressed in proximal tubular epithelial cells of the kidney and is responsible for the reabsorption of 90% of filtered glucose. Knocking out SGLT2 in mice inhibits renal glucose reabsorption, reducing hyperglycemia associated with a high-fat diet (HFD) and obesity, while simultaneously increasing glucose-stimulated insulin secretion. Recently, substantial evidence suggests that SGLT2 inhibitors, as novel antidiabetic drugs, have direct protective effects against diabetic nephropathy (DN), including reducing proteinuria, preventing disease progression, and modulating NLRP3 inflammasome activity.

[0006] As natural ligands of STRs, glucose binding to STRs can activate sweet taste signals. However, the role and mechanism of STRs in the renal glucose reabsorption and transport via SGLT2-dependent processes under hyperglycemic stress remain unclear. Summary of the Invention

[0007] To address the aforementioned problems, this invention aims to provide the application of 18F-lactitol in the preparation of diabetic nephropathy detection products. By using a micro-PET / CT system, the dynamic distribution of STRs in the kidneys of non-diabetic / diabetic mice is monitored non-invasively and longitudinally, confirming the interaction between STR signaling and SGLT2, and demonstrating its functional relevance to renal glucose transport and blood glucose homeostasis.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides the application of 18F-lactitol in the preparation of diagnostic products for diabetic nephropathy.

[0010] Specifically, lactitol in 18F-lactitol downregulates the expression of sweet taste receptors, upregulates SGLT2 expression, and mitigates the effects of glucose on the expression of STRs and SGLT2 and glucose reabsorption.

[0011] Specifically, the products include reagents, kits, probes, and detection systems.

[0012] Specifically, the detection system is 18F-lactitol-PET / CT.

[0013] On the other hand, the present invention also provides a probe that can specifically bind to sweet taste receptors, the probe being 18F-lactitol.

[0014] The beneficial effects of this invention are:

[0015] This invention utilizes PET / CT to detect the distribution of streptozotocin-dependent stromal tract (STR) structures in vivo. Using 18F-labeled lactitol, and applying either the STR inhibitor lactitol or siRNA-STRs, in vivo glucose levels, renal pathology, and SGLT2 expression were measured, along with in vitro fluorescently labeled glucose uptake. This study aimed to evaluate the role and mechanism of STRs in glucose reabsorption in streptozotocin (STZ)-induced diabetic mice and human proximal tubular epithelial cells (HK-2). Results showed that 18F-lactitol sensitively and specifically binds to STRs. The biodistribution of 18F-lactitol and STR expression were significantly downregulated in the kidneys of diabetic mice, while SGLT2 expression was upregulated. Lactitol or siRNA-STRs significantly mitigated the effects of high glucose on STR and SGLT2 expression and glucose uptake. In summary, this invention is the first to explore the relationship between STRs and SGLT2. The results support the possibility that the kidney may regulate SGLT2-dependent glucose reabsorption through STRs, suggesting that STRs may serve as novel therapeutic targets for diabetes, and that 18F-lactitol-labeled PET / CT may become a novel probe for detecting diabetic kidney injury. Attached Figure Description

[0016] Figure 1 This invention relates to the synthesis of 18F-lactitol and its binding with STRs on HK2.

[0017] Figure 2 This is the distribution result of 18F-lactitol in mice in this invention.

[0018] Figure 3 This shows the distribution of 18F-lactitol in mice in this invention.

[0019] Figure 4 The results show the expression of SGLT2, T1R2, and T1R3 under different concentrations of glucose and glucose plus lactitol treatment in this invention.

[0020] Figure 5 The images show immunofluorescence staining of HK2 under high glucose and high lactitol treatment in this invention, as well as the quantitative results of relative glucose uptake and average intensity of SGLT2.

[0021] Figure 6 The results show the expression of SGLT2, T1R2, and T1R3 under high glucose plus T1R2 or T1R3 siRNA treatment in this invention.

[0022] Figure 7 The images show immunofluorescence staining of HK2 under high glucose plus T1R2 or T1R3 siRNA treatment in this invention, as well as the quantitative results of relative glucose uptake and average intensity of SGLT2. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0024] I. Experimental Materials and Methods

[0025] 1.1 Cell lines

[0026] HK2 (human proximal tubular epithelial cells) were provided by the Sichuan Provincial Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Diseases and cultured in a mixture of HAM'S F12 and Dulbecco modified Eagle medium (DMEM / HAM'S-F12; EuroClone, CelBio), supplemented with 10% heat-inactivated FBS (HI-FBS), 2 mM L-glutamine and 100 U / ml penicillin.

[0027] 1.2 Mice

[0028] All mice used in this study had a C57BL / 6 genetic background. Mice were randomly assigned to three groups: a control group (CTL group, n=6), a diabetes group (DM group, n=6), and a DM plus lactitol group (DM+Lac group, n=6). Mice in the DM and DM+Lac groups were induced to have diabetes by intraperitoneal injection of streptozotocin (STZ, 0.1M, 40 mg / kg) four times daily. Mice in the DM+Lac group were additionally injected with citrate-buffered saline (CtLac) solution. Simultaneously, mice in the CTL and DM groups received the same volume of CtLac solution intraperitoneally. To test the effect of lactitol on DM mice, DM+Lac mice received lactitol (10 mM) dissolved in their drinking water for 12 weeks, starting from the first day of STZ injection. Fasting blood glucose (FBG) was measured by tail vein sampling four days after STZ injection; a FBG level ≥16.7 mmol / L for three consecutive days was considered a diagnosis of diabetes. Mice were housed under a 12-hour light-dark cycle, with free access to food and specific pathogens.

[0029] Mice were weighed every two weeks, and 24-hour urinary microalbumin (mAlb) was collected in metabolic cages (Hattera, USA). Urinary protein and creatinine concentrations were measured according to the kit instructions (Afifinion TMACR, Axis-Shield PoCAS, Norway), and the protein-to-creatinine ratio (ACR) was calculated. After 12 weeks, all mice underwent further micro-PET / CT analysis. All procedures complied with Chinese animal welfare regulations.

[0030] 1.3 Antibodies

[0031] Primary antibodies: Rabbit anti-SGLT2 (for Western blot, 1:1000; for immunostaining, 1:50; CellSignaling, catalog number #14210S), rabbit polyclonal anti-T1R2 (for Western blot, 1:1000; Santa Cruz, catalog number #sc-50305), rabbit polyclonal anti-T1R3 (for Western blot, 1:1000; Santa Cruz, catalog number #sc-50352), mouse monoclonal anti-GAPDH (1:1000; abcam, catalog number #ab8245).

[0032] Secondary antibodies: AlexaFluor488-labeled anti-rabbit (1:200; Invitrogen, catalog number R37118), AlexaFluor555-labeled anti-rabbit (1:200; Invitrogen, catalog number A21429), and HRP-labeled anti-mouse (1:5000; Invitrogen, catalog number 31430).

[0033] 1.4 Other reagents

[0034] Streptozotocin (STZ, Sigma, catalog number 572201), glucose (D-(+)-glucose, Sigma, catalog number G7021), lactitol (Lac, (±)-2-(p-methoxyphenoxy)propionic acid, Sigma, catalog number M6546).

[0035] 1.518F-Lactitol Binding and Competitive Inhibition Study in HK2

[0036] 18F-lactitol was used to synthesize a radionuclide-labeled precursor by modifying a specific nanobody Lac, which was then labeled with 18F to achieve sweet receptor immunoPET imaging.

[0037] Cell binding studies were conducted to test whether the newly synthesized 18F-lactitol could stably bind to cells over a period of time. HK2 cells were pre-cultured for 24 hours and then treated with different concentrations (0, 1, 3, 5, 10, 20 and 40 μCi) of 18F-lactitol. After binding for 1 hour, the cells were washed three times with cold PBS and then radioactivity was measured using a gamma counter (model PE2470).

[0038] The binding specificity of 18F-lactitol to sweet taste receptors (STRs) of HK2 cells was tested by a cell competitive inhibition study. HK2 cells were pre-cultured for 24 hours and then incubated with lactitol for one hour. Subsequently, they were treated with different concentrations (0, 1, 3, 5, 10, 20 and 40 μCi) of 18F-lactitol. After binding for one hour, the cells were washed three times with cold PBS and then radioactivity was measured using a gamma counter (model PE2470).

[0039] 1.618F-Lactitol Binding and Competitive Inhibition Study in Mice

[0040] This in vivo binding study tested whether 18F-lactitol could stably bind to STRs in different organs over a period of time. Three groups of mice were simultaneously injected with 18F-lactitol (80±0.4 μCi; tail vein injection). Mice were sacrificed at 30, 60, and 180 minutes post-injection (CTL group sacrificed at 30 minutes, DM group at 60 minutes, and DM+Lac group at 180 minutes). Kidneys, livers, tongues, hearts, and spleens were removed, washed three times with cold PBS, and radioactivity was measured using a gamma counter (PE2470). To promote the excretion of free 18F-lactitol, furosemide (5 mg / kg) was injected intraperitoneally 30 minutes after injection.

[0041] The binding specificity of 18F-lactitol to STRs in mice was tested using an in vivo competitive inhibition study. Mice were pretreated with lactitol (5 mM; tail vein injection), and one hour later, they were injected with 18F-lactitol (80 ± 0.4 μCi; tail vein injection). Mice in the three groups were sacrificed at 30, 60, and 180 minutes after injection (CTL group was sacrificed at 30 minutes after injection, DM group at 60 minutes after injection, and DM+Lac group at 180 minutes after injection). Kidneys, livers, tongues, hearts, and spleens were removed, washed three times with cold PBS, and radioactivity was measured using a gamma counter (model PE2470).

[0042] 1.7 Micro-PET / CT imaging of 18F-lactitol in mice

[0043] Mice were injected with 18F-lactitol (80±0.4 μCi, via tail vein injection), and the 18F-lactitol signal was detected by micro-PET / CT 30 minutes later. Then, furosemide (5 mg / kg) was injected intraperitoneally, and the 18F-lactitol signal in the CTL, DM, and DM+Lac groups was detected again by micro-PET / CT at 60 and 180 minutes.

[0044] 3D images reconstructed from PET / CT data were analyzed using VivoQuant ver 1.23 (InviCRO, Boston, Massachusetts) software. For microPET / CT, files were first cropped to reduce file size and remove experimental artifacts such as the scan panel and the tube containing the carotid artery. After obtaining images containing only kidney signals, the color intensity of both modalities was manually adjusted to match the histological / autoradiographic signals as closely as possible. Then, the distance / annotation function was selected to perform linear cross-sections of 5–8 mm on the kidney transverse plane. After downloading the raw data, a graph showing microCT density and microPET units along the cross-section was reconstructed using Matlab software. The 3D ROI tool function was selected to quantify PET+ / CT-, PET+ / CT+, and PET- / CT+ regions at the 3D level. First, the microPET data was thresholded using the Otsu method to delineate the range of PET+ signals. On the microCT plane, densities exceeding 1000 Hounsfield units (HU) were defined as calcification. To detect PET+ / CT+ signals, we selected the 3D region defined by PET+ and applied a global threshold of ≥1,000 HU to the micro-CT data within that region. To detect the PET- / CT+ region, we selected all remaining micro-CT data and applied a global threshold of ≥1,000 HU. The raw data containing information from different modal regions was downloaded and analyzed using Image-Pro Plus 6.0 software.

[0045] 1.8 Instantaneous transfection

[0046] HK2 cells were transiently transfected with human T1R2 siRNA or T1R3 siRNA SMARTpool siGENOME siRNA duplex (300 nM), or a non-specific control duplex, using Dharmafect reagent according to the manufacturer's instructions. 42 to 48 hours after transfection, HK2 cells were exposed to glucose (30 mM) for 72 hours. The knockdown of mRNA and protein expression was verified by RT-PCR and Western blotting, respectively.

[0047] The forward sequence of T1R2 siRNA is as follows:

[0048] 5'-GUGAAGGUGAUAGGCUACA-3'

[0049] The reverse sequence of T1R2 siRNA is:

[0050] 5'-UGUAGCCUAUCACCUUCAC-3'

[0051] The forward sequence of T1R3 siRNA is:

[0052] 5'-CUGUCUACGCAGCUGUGUA-3'

[0053] The reverse sequence of T1R3 siRNA is:

[0054] 5'-UACACAGCUGCGUAGACAG-3'

[0055] 1.9 Real-time quantitative PCR

[0056] Total RNA was extracted from HK2 cells using an RNA extraction kit (ComWin Biotech, Beijing, China) and reverse transcribed using a PrimeScript RT kit (TaKaRa). The synthesized cDNA was used as a template for quantitative PCR analysis, which was performed using a SYBR Green Supermix (BioRad) in a real-time quantitative thermal cycler (BioRad). For relative quantification, changes in gene expression were measured using gaph expression as an internal control.

[0057] Primer sequences used for relative quantification of gene expression include:

[0058] Forward primer sequence for human SGLT2:

[0059] 5'-ACACGGTACAGACCTTCGTCA-3'

[0060] Reverse primer sequence for human SGLT2:

[0061] 5'-GCTGCTCCCAGGTATTTGTC-3'

[0062] Forward primer sequence for human T1R2:

[0063] 5'-GTTCTTCCTCCTATGGGTCCT-3'

[0064] Reverse primer sequence for human T1R2:

[0065] 5'-AGGTTGTAGCCTATCACCTTCA-3'

[0066] Forward primer sequence for human T1R3:

[0067] 5'-CCGCCTACTGCAACTACACG-3'

[0068] Reverse primer sequence for human T1R3:

[0069] 5'-CTAGCACCGTAGCTGACCTG-3'

[0070] Forward primer sequence for human gapdh:

[0071] 5'-GGAGCGAGATCCCTCCAAAAT-3'

[0072] Reverse primer sequence for human gapdh:

[0073] 5'-GGCTGTTGTCATACTTCTCATGG-3'

[0074] 1.10 Western Blotting

[0075] Total protein was extracted from HK2 cells using a protein extraction kit (Kaiji, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes for Western blotting using various antibodies.

[0076] 1.11 Immunofluorescence staining

[0077] HK2 cells were cultured on coverslips in 6-well plates. After overnight adhesion, cells were incubated for 72 hours in glucose (30 mM) with or without lactitol (5 mM). For transiently transfected HK2 cells, cells were treated with glucose (30 mM) for 72 hours after transfection, 42 to 48 hours later. Cells were then fixed with 4% paraformaldehyde and blocked with 5% goat serum. Cells were incubated overnight at 4°C with anti-SGLT2 primary antibody and then at room temperature for 1 hour with anti-rabbit secondary antibody and DAPI. Images were captured using a fluorescence microscope (Leica, Germany), and mean intensity was semi-quantitatively analyzed using Image-Pro Plus 6.0 software.

[0078] 1.12 Statistics

[0079] *p≤0.05, compared with the CTL group; #p≤0.05, compared with the HG group; used for unpaired t-tests.

[0080] II. Experimental Results

[0081] 2.1 Generation and Evaluation of 118F-Lactitol Probe

[0082] This study synthesized an 18F-lactitol probe via a nucleophilic substitution chemical reaction, as shown in the attached diagram. Figure 1 As shown in Figure A. The results of 18F-lactitol binding and competitive inhibition in HK2 are attached. Figure 1 As shown in B, in the appendix Figure 1In Figure B, the blue line represents the radiometric measurement results of HK2 cells after treatment with 0, 1, 3, 5, 10, 20, and 40 μCi of 18F-lactitol for one hour, which is also the binding result of 18F-lactitol in HK2 cells; the red line represents the radiometric measurement results of HK2 cells after incubation with lactitol for one hour, followed by treatment with different concentrations (0, 1, 3, 5, 10, 20, and 40 μCi) of 18F-lactitol for one hour, which is also the competitive inhibition result of 18F-lactitol in HK2 cells.

[0083] From the appendix Figure 1 As can be seen, the 18F-lactitol probe can bind to STRs on HK2 sensitively and specifically.

[0084] 2.2 Distribution of 1,8F-lactitol in mice

[0085] Mice were euthanized at 30, 60, and 180 minutes after injection of 18F-lactitol. The liver, tongue, right kidney, left kidney, spleen, and heart were removed, washed three times with cold PBS, and subjected to radioactivity testing. The results corresponded to the attached... Figure 2 The blue of AF.

[0086] Mice were first pretreated with lactitol (5 mM; tail vein injection), then injected with 18F-lactitol. Mice were sacrificed at 30, 60, and 180 minutes. Liver, tongue, right kidney, left kidney, spleen, and heart were removed, washed three times with cold PBS, and subjected to radioactivity testing. Results corresponded to the attached... Figure 2 The red in AF.

[0087] From the appendix Figure 2 It can be seen that 18F-lactisole can specifically bind to the STRs of the tongue, heart, liver, spleen and kidneys 30 minutes after intravenous injection in the tail vein, while free 18F-lactisole is mainly excreted through the kidneys.

[0088] 2.3 Micro-PET / CT imaging of 18F-lactitol in mice

[0089] The micro-PET / CT imaging results of 18F-lactitol in mice are attached. Figure 3As shown, A represents the micro-PET / CT imaging results 30 minutes after mice received a tail vein injection of 80±0.4μCi (approximately 4μg, 100μL) of 18F-lactitol, from left to right: DM+Lac group, DM group, and CTL group. B represents the micro-PET / CT imaging results 180 minutes after mice received a tail vein injection of 80±0.4μCi (approximately 4μg, 100μL) of 18F-lactitol. Thirty minutes after micro-PET / CT imaging, furosemide (5mg / kg) was injected intraperitoneally to excrete excess 18F-lactitol that had not bound STRs, thus enabling the visualization of specific STRs; from left to right: DM+Lac group, DM group, and CTL group. C represents the quantitative results of signal area and intensity in the right and left kidneys of mice in the DM+Lac, DM, and CTL groups during 180-minute micro-PET / CT imaging. *p<0.05, compared with the NC group; #p<0.05, compared with the DM group.

[0090] 30-minute micro-PET / CT images showed high uptake of 18F-lactitol in the liver and kidneys, with a significant increase in the renal region of DM mice compared to CTL mice, while lactitol treatment partially reduced the renal region of DM mice, as shown in the attached image. Figure 3 As shown in Figure A. Furosemide (5 mg / kg) was then injected intraperitoneally, and the biodistribution of 18F-lactitol in CTL, DM, and DM+Lac mice was detected again at 180 minutes using micro-PET / CT. The results are shown in the attached figure. Figure 3 As shown in Figures B and C, 180-minute microPET / CT images revealed a significant downregulation of 18F-lactitol biodistribution in the kidneys of diabetic nephropathy (DM) mice, and lactitol treatment partially reversed the high glucose-induced downregulation. These results suggest that 18F-lactitol microPET / CT can be used to monitor the occurrence and progression of diabetic nephropathy.

[0091] 2.4 Lactitol regulates SGLT2 expression and glucose transport in HK2

[0092] The results of real-time PCR studies on lactitol-regulated SGLT2 expression and glucose transport in HK2 are attached. Figure 4 and Figure 5 As shown, in the appendix Figure 4In the table, A shows the Western blot results of SGLT2, T1R2, and T1R3 under different glucose concentrations (10, 20, 30 mM). B shows the relative protein and mRNA expression quantification of SGLT2 under different glucose concentrations (10, 20, 30 mM) (mean ± standard deviation; n = 3). C shows the relative protein and mRNA expression quantification of T1R3 under different glucose concentrations. D shows the relative protein and mRNA expression quantification of T1R2 under different glucose concentrations. E shows the Western blot results of SGLT2, T1R2, and T1R3 under high glucose (30 mM) plus different concentrations of lactitol (3, 5, 10 mM). F shows the relative protein and mRNA expression quantification of SGLT2 under high glucose plus different concentrations of lactitol. G shows the relative protein and mRNA expression quantification of T1R3 under high glucose plus different concentrations of lactitol. H represents the quantitative analysis of relative protein and mRNA expression of T1R2 under high glucose and different concentrations of lactitol treatment. (See attached...) Figure 5 In the table, A shows the immunofluorescence staining image of HK2 under high glucose (30 mM) and lactitol (5 mM) treatment (scale bar = 100 μm). B shows the mean intensity quantification of SGLT2 (mean ± standard deviation; n = 3). C shows the relative glucose uptake of HK2 under high glucose (30 mM) and different concentrations of lactitol (3, 5, 10 mM) treatment (mean ± standard deviation; n = 3). *p<0.05, compared with the CTL group; #p<0.05, compared with the HG group.

[0093] From the appendix Figure 4 and attached Figure 5 As can be seen, after treating HK2 cells with different concentrations of glucose (10, 20, 30 mM) for 72 hours, the mRNA and protein expression levels of SGLT2 transporter protein increased (see attached figure). Figure 4 (As shown in Figures A and B). The mRNA and protein expression levels of STR components T1R2 and T1R3 were downregulated (as shown in the attached figure). Figure 4 (As shown in A, C, and D). Lactitol treatment (3, 5, and 10 mM) partially reversed high glucose-induced SGLT2 upregulation (as shown in the attached diagram). Figure 4 Middle E, F and Appendix Figure 5 (as shown in Figure B) and the downregulation of T1R2 and T1R3 (as shown in the appendix) Figure 4 (As shown in E, G, and H). It is noteworthy that lactitol can also regulate glucose transport at HK2, partially reversing the hyperglycemic-induced increase in glucose transport (as shown in Appendix). Figure 5 (As shown in C).

[0094] 2.5 Knockdown of STRs regulates SGLT2 expression and glucose transport in HK2

[0095] To assess the interaction between STRs and SGLT2, this study further investigated the regulation of SGLT2 expression and glucose transport in HK2 by knocking down STRs. The results are shown in the attached figure. Figure 6 and attached Figure 7 As shown, in the appendix Figure 6 In the table, A shows the Western blot results of SGLT2, T1R2, and T1R3 after treatment with high glucose (30 mM) and T1R2 or T1R3 siRNA. B shows the relative protein and mRNA expression quantification of SGLT2 (mean ± standard deviation; n = 3). C shows the relative protein and mRNA expression quantification of T1R3. D shows the relative protein and mRNA expression quantification of T1R2. (See appendix...) Figure 7 In the table, A shows immunofluorescence staining images of HK2 under high glucose (30 mM) treatment with T1R2 or T1R3 siRNA (scale bar = 100 μm). B shows the mean intensity quantification of SGLT2 (mean ± standard deviation; n = 3). C shows the relative glucose uptake of HK2 under high glucose (30 mM) treatment with T1R2 or T1R3 siRNA (mean ± standard deviation; n = 3). *p<0.05, compared with the CTL group; #p<0.05, compared with the HG group.

[0096] From the appendix Figure 6 and attached Figure 7 As can be seen, siRNA can knock down the expression of T1R2 and T1R3 in HK2 cells (see attached image). Figure 6 (As shown in A, C, and D). Notably, knockdown of T1R2 / T1R3 led to downregulation of SGLT2 protein and mRNA expression under high glucose treatment (as shown in the attached figure). Figure 6 (As shown in A and B). Consistent with this, T1R2 / T1R3 siRNA significantly inhibited glucose transport in HK2 cells, as shown in the attached figure. Figure 7 As shown in the figure. In summary, these data indicate that STRs can partially regulate SGLT2 expression and function, suggesting that STRs may be another therapeutic target for diabetes.

[0097] In summary, this study established an STZ-induced diabetic (DM) mouse model. 18F-lactitol PET / CT results showed downregulation of renal STRs in the DN mouse model, and lactitol treatment partially reversed the hyperglycemic-induced downregulation of STRs. Notably, in HK2 cell experiments, this study is the first to discover that lactitol treatment and transient STR knockdown can partially reverse the effects of prolonged hyperglycemic stimulation (72 hours) on SGLT2. Hyperglycemia significantly upregulated SGLT2 mRNA and protein expression and promoted glucose transport. The interaction between STRs and SGLT2 may indicate that STRs are another pharmacological target for DN.

[0098] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. 18F-Lactitol in the preparation of diagnostic products for diabetic nephropathy.

2. The application according to claim 1, characterized in that: Lactitol in 18F-lactitol downregulates the expression of sweet taste receptors, upregulates SGLT2 expression, and reduces glucose-induced STRs and SGLT2 expression and glucose uptake.

3. The application according to claim 1, characterized in that: The products include reagents, kits, probes, and detection systems.

4. The application according to claim 3, characterized in that: The detection system is 18F-lactitol-PET / CT.

5. A probe capable of specifically binding to sweet taste receptors, characterized in that: The probe is 18F-lactitol.

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