Application of iron sucrose in prevention of ischemia-reperfusion injury of transplanted kidney

Abstract

The invention relates to application of iron sucrose in prevention of transplanted kidney ischemia-reperfusion (I / R) injury. The safety and effectiveness of the iron sucrose in prevention of mouse kidney I / R injury are mainly investigated, the application of the iron sucrose in preparation of the medicine for prophylactically relieving the kidney I / R injury is provided, and mouse in-vivo tests show that the inflammatory response of the I / R kidney can be remarkably reduced by intravenous injection of 3.0 mg (150 mg / kg body weight) of the iron sucrose one day before an operation; according to the present invention, the serum creatinine and urea nitrogen levels of mice are reduced, the kidney injury caused by I / R is effectively and safely reduced, the important basis is provided for the clinical application of the iron sucrose before the operation of the kidney transplantation recipient so as to reduce the ischemia-reperfusion injury during the transplantation process, and the new strategy is provided for reducing the delayed recovery of the transplanted kidney function and improving the long-term survival of the transplanted kidney.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of iron sucrose in preventing ischemia-reperfusion injury of transplanted kidney. Background Art

[0002] Patients with end-stage renal disease who are waiting for kidney transplantation surgery often have iron metabolism disorders and renal anemia. Iron deficiency will further aggravate the acute kidney injury caused by ischemia / reperfusion (I / R) during the transplantation process, thereby causing delayed recovery of transplanted kidney function and affecting the long-term survival of the transplanted kidney.

[0003] At present, there is no specific drug for preventing and treating renal I / R injury in clinical practice. In order to improve the long-term survival rate of kidney transplantation, it is of great significance to develop a specific drug for preventing and treating renal I / R injury. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of sucrose iron that can reduce renal injury caused by I / R in preventing ischemia-reperfusion injury of transplanted kidney.

[0005] The technical solution of the present invention is as follows:

[0006] Application of iron sucrose in preventing ischemia-reperfusion injury in transplanted kidneys.

[0007] Furthermore, the intravenous injection dose of iron sucrose is 150 mg / kg body weight.

[0008] Furthermore, the intravenous injection time of iron sucrose is one day before the operation.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] 1. The present invention mainly investigates the safety and effectiveness of iron sucrose in preventing renal I / R injury, and provides the use of iron sucrose in the preparation of a drug for preventively reducing renal I / R injury. In vivo experiments in mice show that intravenous injection of 3.0 mg (150 mg / kg body weight) of iron sucrose one day before surgery can significantly reduce the inflammatory response of I / R kidneys, reduce the levels of blood creatinine and urea nitrogen in mice, and effectively and safely reduce renal injury caused by I / R. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the experimental result of iron sucrose protecting the renal function of I / R mice;

[0012] Figure 2 This is the experimental result showing that iron sucrose alleviates renal inflammatory damage in I / R mice. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] like Figures 1 to 2 As shown, the application of iron sucrose in preventing ischemia-reperfusion injury in transplanted kidneys;

[0015] 1. Experimental Materials

[0016] Experimental materials: Sucrose iron (Aisu), brown colloidal solution, 5 mL per tube, containing 100 mg iron and 1.6 g sucrose, stored away from light.

[0017] Experimental animals: 25 male C57BL / 6J mice, 8 weeks old, weighing 20-25 g, were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd. and raised in the Experimental Animal Center of Zhengzhou University, SPF grade.

[0018] 2. Experimental groups:

[0019] Male C57BL / 6J mice were randomly divided into 5 groups: sham group, ischemia-reperfusion (I / R) group, 50 mg / kg iron sucrose injection and I / R group, 100 mg / kg iron sucrose injection and I / R group, and 150 mg / kg iron sucrose injection and I / R group, with 5 mice in each group. The mice were weighed one day before surgery, and iron sucrose was injected through the tail vein. The activity status of the mice was observed and recorded.

[0020] 3. Construction of mouse renal I / R model:

[0021] Male C57BL / 6J mice were anesthetized by injection of 1% sodium pentobarbital and fixed on the operating table in a supine position. The mouse's abdominal hair was shaved and disinfected. An incision was made in the middle of the abdomen. The kidneys were visible after cutting the skin and muscles. The renal arteries of both kidneys were carefully separated and quickly clamped with non-traumatic artery clamps. After 25 minutes, the clamps were removed to open bilateral blood flow, and the abdominal incision was sutured and closed layer by layer. The sham group of the control group only underwent an abdominal incision without vascular clamping. After the mice woke up, they were put back into clean cages and returned to the breeding room for continued breeding. The activity status of the mice was closely observed and recorded. Kidney tissue and blood samples were collected from the mice 24 hours later.

[0022] 4. Detection of the protective effect of sucrose iron on renal function in mice

[0023] 4.1. Methods for detecting renal function in mice

[0024] (1) The main function of the kidney is to produce urine through the filtration of the glomeruli and the reabsorption and secretion of the renal tubules, thereby excreting metabolic waste from the body and maintaining water, electrolyte and acid-base balance. Serum creatinine (Scr) is the end product of muscle metabolism and is mainly excreted from the body through glomerular filtration. Blood urea nitrogen (BUN) is the end product of protein metabolism in the body. The ammonia produced by protein decomposition is converted into urea through the urea cycle in the liver, enters the blood circulation, and is then mainly excreted from the body through glomerular filtration. Under normal renal function, Scr and BUN can be effectively cleared and the blood concentration is maintained at a low level; when renal function is impaired, the glomerular filtration rate decreases, resulting in an increase in the concentration of Scr and BUN in the blood. Therefore, Scr and BUN are widely used in clinical renal function testing and are important indicators for diagnosing renal damage.

[0025] (2) After 24 h of reperfusion, the eyeballs of mice in each group were removed and blood was collected. The whole blood was placed in a 1.5 mL sterile EP tube and allowed to stand at 4°C for 1 h. The tube was then centrifuged at 3000 g for 15 min at 4°C. The supernatant was placed in a new EP tube to obtain the serum sample to be tested, which was then stored in a -80°C refrigerator for subsequent testing.

[0026] (3) Detection of Scr content in mouse serum using sarcosine oxidase method (Nanjing Jiancheng, creatinine determination kit, C01121):

[0027] 1) First, 6 μL of the serum sample to be tested, 442 μmol / L creatinine standard (standard group) and double distilled water (blank group) were added to a sterile 96-well plate, and then 180 μL of enzyme mixed solution A (creatinine amide hydrolase, creatine amino hydrolase, sarcosine oxidase, and peroxidase were mixed in a ratio of 1:1:1:1) was added to each well;

[0028] 2) After incubation in a 37°C incubator for 5 min, use a full-wavelength microplate reader to detect the absorbance value of each well at 546 nm, which is recorded as A1;

[0029] 3) Then, 60 μL of 4-aminoantipyrine was added to each detection well, and after incubation at 37°C for 5 min, the absorbance value of each detection well was detected at 546 nm using a multifunctional microplate reader, which was recorded as A2;

[0030] 4) Calculate the Scr value of the serum sample to be tested using the formula: ΔA = A2-K*A1, K = 0.756, Scr (μmol / L) = (ΔA 测定 -ΔA 空白 ) / (ΔA 标准 -ΔA 空白 )×C 标准 , C 标准(Standard concentration) = 442 μmol / L.

[0031] (4) Detection of BUN content in mouse serum using urease method (Nanjing Jiancheng, BUN test kit, C01321):

[0032] 1) First, prepare urease buffer and 10mmol / L BUN standard application solution;

[0033] ①. Urease buffer solution: Prepare urease buffer solution according to the ratio of urease stock solution to urease diluent = 3:1000, and prepare it before use;

[0034] ②, Preparation of 10mmol / L BUN standard solution: Weigh 6.006mg urea and dissolve it in 1mL double distilled water to prepare 100mmol / L BUN standard storage solution. Take 100μL 100mmol / L BUN standard storage solution, add 900μL double distilled water to prepare 10mmol / L BUN standard application solution;

[0035] 2) Add 20 μL of the serum sample to be tested, 10 mmol / L BUN standard application solution (standard group) and double distilled water (blank group) to a 10 mL EP tube, and then add 250 μL of freshly prepared urease buffer, mix well and place in a 37°C water bath for 10 min;

[0036] 3) Add 1 mL of phenol colorimetric agent and 1 mL of alkaline sodium hypochlorite to each 10 mL EP tube, mix thoroughly and place in a 37°C water bath for 10 min, and pipette 200 μL of sample into a 96-well plate. Set three replicate wells for each sample, and use a multifunctional microplate reader to detect the absorbance of each well at 640 nm, and adjust to zero with double distilled water.

[0037] 4) Calculate the BUN value in serum using the formula: Urea nitrogen (BUN) concentration (mmmmol / L) = (ΔA 测定 -ΔA 空白 ) / (ΔA 标准 -ΔA 空白 )*C 标准 , C 标准 (Standard concentration) = 10 mmol / L.

[0038] 4.2 Experimental results on the protective effect of iron sucrose on renal function in mice

[0039] The results of the experimental study on the protective effect of sucrose iron on the renal function of mice are as follows Figure 1 As shown in B;

[0040] in, Figure 1 A is a schematic diagram of constructing a model of acute kidney injury induced by bilateral renal I / R in mice; Figure 1Sham in the figure indicates sham operation group, which is the negative control group. The statistical data are presented in a bar graph of mean ± standard deviation. The results were analyzed by ONE-ANOVA statistical analysis. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0041] Figure 1 B is the concentration of renal function markers creatinine (Scr) and urea nitrogen (BUN) in the serum of mice after renal I / R injury after mice were pretreated with 50 mg / kg, 100 mg / kg and 150 mg / kg sucrose iron in the present invention. Figure 1 As shown in Figure B, renal I / R causes a significant increase in the concentrations of Scr and BUN in mouse serum, while pretreatment with sucrose iron can significantly reduce the levels of Scr and BUN in mouse serum, and the protective effect of sucrose iron on the kidney is concentration-dependent.

[0042] 5. Detection of the protective effect of sucrose iron on renal inflammatory damage in mice

[0043] 5.1. Methods for detecting kidney inflammation in mice

[0044] (1) The concentrations of proinflammatory factors IL-6 and TNF-α in mouse serum were detected by enzyme-linked immunosorbent assay (ELISA). Antibodies to IL-6 and TNF-α were coated on a 96-well polystyrene plate to form an antigen-antibody solid phase immune reaction system. Six standard wells, 41 sample wells to be tested, and one blank well were set up. 50 μL of 20, 40, 80, 160, 320, and 480 pg / mL standard samples were added to the six standard wells in sequence. 50 μL of the mouse serum sample to be tested was added to the detection well. Then, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each of the standard wells and sample wells. The reaction wells were sealed with a sealing film and placed in a 37°C incubator in the dark for 60 min. Discard the liquid and pat dry on absorbent paper. Add 350 μL of washing solution (PBST, i.e., PBS with 0.05% Tween20, pH 7.4) to each well, let stand for 1 min, then discard the washing solution, and repeat the washing 5 times. Then add 50 μL of substrate buffer A (0.2 mol / L sodium phosphate citrate, pH 5.0) and 50 μL of substrate buffer B (20 μg / mL tetramethylbenzidine) to each well, and incubate at 37°C in the dark for 15 min. Finally, add 50 μL of 2 mol / L sulfuric acid to each well to terminate the reaction, and use a multifunctional microplate reader to detect the OD value of each well at a wavelength of 450 nm within 15 min.

[0045] (2) After 24 hours of reperfusion, blood was collected from the orbits of the mice and the mice were immediately killed by cervical dislocation. The renal tissues of the mice were immediately dissected out and the renal capsule was torn off. The renal tissues were evenly divided into two halves along the sagittal plane. One half was placed in 4% paraformaldehyde and placed in a 4°C refrigerator for tissue fixation; the other half was placed in tissue preservation solution and stored in an 80°C refrigerator.

[0046] (3) After fixation in 4% paraformaldehyde for 24 hours, the tissue was dehydrated with gradient ethanol and then transparentized with xylene, followed by wax immersion and embedding. The embedded wax block was sliced ​​using a Leica pathology slicer, and the sliced ​​tissue slices were placed in a 40°C water bath for spreading. The anti-slip glass slide was inserted into the water surface at an angle to pick up the slices, so that the slices were attached to the appropriate position of the slide, and the slices were baked in an oven at 60°C for 3 hours. The slices were then dewaxed in turn, and then placed in hematoxylin staining solution and 1% eosin staining solution for 5 minutes. The cell nucleus contains acidic nucleic acid, which has a strong affinity with the alkaline dye hematoxylin, while the cytoplasm contains alkaline substances with a strong affinity with the acidic dye eosin. Therefore, after the tissue slices were stained with HE, the cell nucleus was stained blue-purple by hematoxylin, and the cytoplasm, muscle fibers, collagen fibers and red blood cells showed different degrees of red. Finally, an advanced upright microscope was used to observe the tissue morphology and collect images.

[0047] (4) When tissue cells are oxidatively damaged, lipid peroxidation will occur. Malondialdehyde (MDA) is one of the end products of lipid peroxidation. It can react with biological macromolecules such as DNA and protein to form adducts. It is an important indicator for evaluating oxidative damage to tissue cells. The paraffin-embedded mouse kidney tissue was sliced ​​and dewaxed, and then antigen repair was performed using an electric ceramic furnace. The dewaxed and hydrated tissue sections were placed on a high-temperature plastic slice rack in a beaker, and an appropriate volume of 0.01M citrate buffer (pH6.0) was added to the beaker so that the sliced ​​tissue was completely immersed in the repair solution and heated for repair for 15 minutes. Then the beaker was removed and placed in cold water to cool down. After the repair solution cooled to room temperature, the slide was removed and gently rinsed with PBS (pH7.4) three times, each time for 3 minutes. 3% hydrogen peroxide was added to the sliced ​​tissue to block endogenous peroxidase, incubated at room temperature for 15 minutes, and rinsed with PBS three times, each time for 3 minutes. Wipe the slides dry with absorbent paper, add diluted normal goat serum, and block at room temperature for 30 minutes to reduce nonspecific staining. Shake off excess liquid, then add 1:200 diluted MDA antibody (i.e. primary antibody) and incubate overnight in a 4°C wet box. Rinse the sections with PBS 3 times, 3 minutes each time, wipe the sections dry with absorbent paper, add HRP-labeled goat anti-mouse secondary antibody, and incubate at 37°C for 30 minutes. Rinse the sections with PBS 4 times, 3 minutes each time, shake off the PBS solution, wipe the sections dry with absorbent paper, and add freshly prepared DAB color development solution to each section. When the positive signal is observed to be brown or brown under the microscope, rinse the sections with tap water to stop color development. Then counterstain the nuclei with hematoxylin for 2 minutes, dehydrate and seal the sections with neutral resin, and observe and collect images under a high-level upright microscope after drying.

[0048] (5) After the kidney is injured by I / R, apoptosis of tissue cells is induced. The DNA of apoptotic cells is broken, resulting in free hydroxyl groups at the 3' end. TUNEL staining uses terminal deoxynucleotidyl transferase to add fluorescein-labeled dUTP (deoxyuridine triphosphate) to these 3' hydroxyl groups, thereby dyeing apoptotic cells green. Paraffin-embedded mouse kidney tissue was sliced ​​and dewaxed. After the slices were slightly dried, a circle was drawn around the tissue with a tissue pen to prevent the liquid from flowing away. Proteinase K working solution was added to the circle and incubated in a 37°C incubator for 25 minutes. The slides were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, each time for 5 minutes. After the slices were slightly dried, membrane breaking working solution was added to the circle to cover the tissue and incubated at room temperature for 20 minutes. The slides were placed in PBS and washed on a decolorizing shaker for 3 times, each time for 5 minutes. TdT and dUTP were mixed at a ratio of 1:9, added to the sliced ​​tissue, placed in a humidified box, and incubated at 37°C for 2 hours. Wash with PBS 3 times, 5 min each time. After removing PBS, add DAPI dye solution and incubate at room temperature for 10 min away from light. After washing, seal the slides with anti-fluorescence quenching sealing agent, observe and collect images under a fluorescence microscope. The cell nuclei stained with DAPI are blue under ultraviolet excitation (excitation wavelength 330-380nm, emission wavelength 420nm), and the positive apoptotic cell nuclei are green under FITC excitation (excitation wavelength 465-495nm, emission wavelength 515-555nm).

[0049] 5.2 Experimental results of detecting renal inflammatory damage in mice

[0050] The results of the experimental study on the protection of iron sucrose against renal inflammatory damage in mice are as follows Figure 2 As shown in middle AF;

[0051] Among them, A is the level of proinflammatory factors IL-6 and TNF-α in mouse serum; B is the concentration of alanine aminotransferase (ALT) in serum reflecting liver function; C is the pathological section of mouse kidney tissue, stained with hematoxylin & eosin (H&E), malondialdehyde (MDA) and TUNEL respectively; D is the quantitative analysis result of MDA staining of mouse kidney tissue section in Figure C; E is the level of MDA in mouse kidney tissue; F is the quantitative analysis result of TUNEL staining of mouse kidney tissue section in Figure C. Statistical data are presented in a bar graph of mean ± standard deviation, and the results were analyzed by ONE-ANOVA statistical analysis of the differences between groups, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0052] Figure 2A in the middle is the concentration of proinflammatory factors IL-6 and TNF-α in the serum of mice after renal I / R injury after mice were pretreated with 50 mg / kg, 100 mg / kg and 150 mg / kg of sucrose iron in the present invention. The results show that renal I / R can cause a significant increase in the concentration of IL-6 and TNF-α in the serum of mice, while pretreatment with sucrose iron can significantly reduce the levels of IL-6 and TNF-α in the serum of mice, and the protective effect of sucrose iron on the kidney is concentration-dependent.

[0053] Figure 2 B is the concentration of alanine aminotransferase (ALT), an indicator of liver function, in the serum of mice after they underwent renal I / R injury after being pretreated with iron sucrose. The results showed that renal I / R caused a significant increase in the ALT concentration in the serum of mice, while pretreatment with iron sucrose significantly reduced the serum ALT level of mice, indicating that renal I / R can cause certain damage to the liver function of mice and iron sucrose can alleviate the liver damage caused by renal I / R in mice.

[0054] Figure 2 C is the H&E staining result of the renal tissue pathological section of mice after I / R injury after pretreatment with sucrose iron. It shows that renal I / R can cause tubular damage such as vacuolar degeneration of tubular epithelial cells, tubular dilation, and brush border shedding in the renal tissue of mice, while pretreatment with sucrose iron can significantly protect the renal tubules to maintain normal morphology.

[0055] Figure 2 D is Figure 2 C shows the statistical results of MDA-positive tubules, a lipid peroxidation component, in mouse kidney tissue sections.

[0056] Figure 2 E is the result of the MDA concentration test in the kidney. Figure 2 CE results showed that renal I / R increased the MDA level in the renal tissue of mice, causing oxidative damage to renal tissue cells; while pretreatment with sucrose iron could significantly reduce the MDA concentration in renal tissue and protect the renal tissue cells of mice from oxidative damage.

[0057] Figure 2 F is Figure 2 The statistical results of TUNEL-positive cells in C show that renal I / R can cause apoptosis of renal tissue cells in mice, while pretreatment with sucrose iron can significantly inhibit apoptosis of renal tissue cells in mice;

[0058] The above results show that intravenous injection of 3.0 mg (150 mg / kg body weight) of iron sucrose one day before surgery can significantly reduce the inflammatory response of I / R kidneys, reduce the levels of blood creatinine and urea nitrogen in mice, and effectively and safely reduce the renal damage caused by I / R. This provides an important basis for the clinical use of iron sucrose in renal transplant recipients before surgery to reduce ischemia-reperfusion injury during transplantation, and provides a new strategy to reduce the delayed recovery of transplanted kidney function and improve the long-term survival of transplanted kidneys.

[0059] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of iron sucrose in preventing ischemia-reperfusion injury in transplanted kidneys.

2. The use of iron sucrose according to claim 1 in preventing ischemia-reperfusion injury in transplanted kidneys, characterized in that: The intravenous injection dose of iron sucrose is 150 mg / kg body weight.

3. The use of iron sucrose according to claim 1 or 2 in preventing ischemia-reperfusion injury in transplanted kidneys, characterized in that: The time for intravenous injection of iron sucrose is one day before the operation.

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