Application of DUSP26 as biomarker in preparation of prediction product for transformation from acute kidney injury to chronic kidney disease

Abstract

The invention provides application of DUSP26 as a biomarker in preparation of a product for predicting transformation from acute kidney injury to chronic kidney disease. Specifically, the invention relates to application of a reagent for detecting the expression quantity of a biomarker DUSP26 in preparation of a product for predicting transformation from acute kidney injury to chronic kidney disease. It is found for the first time that DUSP26 is remarkably up-regulated in the AKI-CKD transformation process, and especially is highly expressed in proximal renal tubules of patients suffering from renal fibrosis and CKD; therefore, the DUSP26 can be used as a biomarker for transformation from acute kidney injury AKI to chronic kidney disease CKD, and is used for early recognition of high-risk AKI patients, so that AKI prognosis prediction is assisted, and a new way is provided for prediction of AKI-CKD transformation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of DUSP26 as a biomarker in the preparation of a prediction product for the transformation of acute kidney injury to chronic kidney disease. Background Art

[0002] Acute Kidney Injury (AKI) is a severe syndrome characterized by a sharp decline in renal function. Its high incidence and poor prognosis pose a major clinical challenge. About 40% of AKI survivors will further develop chronic kidney disease (CKD), which not only increases the risk of end-stage renal disease by 3.8 times, but also significantly increases the risk of cardiovascular disease and all-cause mortality, leading to long-term medical dependence and impaired work ability in patients, greatly increasing the dual burden of medical care and the economy and society. At present, clinical intervention after AKI is mainly based on supportive treatment to maintain renal function, and there is still a lack of targeted strategies to effectively block the transformation of AKI to CKD.

[0003] As the core unit of renal tubular function, the energy supply of proximal tubular epithelial cells for their physiological activities is highly dependent on adenosine triphosphate (ATP) generated by the fatty acid oxidation (FAO) pathway to maintain cellular metabolic homeostasis and antioxidant capacity. FAO is the core metabolic pathway for breaking down fatty acids to generate energy, which mainly occurs in the mitochondrial matrix and can provide more than 70% of the basic energy requirements for proximal tubular cells (PTCs). However, the unique microvascular environment and high energy demand of the kidney make PTCs susceptible to stimulation such as ischemia and nephrotoxic drugs, leading to FAO inhibition and lipid metabolism imbalance. FAO disorders in the proximal tubules have been observed in a variety of AKI-CKD transformation models, such as ischemia-reperfusion, folic acid induction, and ureteral obstruction. This phenomenon is also widely present in patients with acute kidney disease and chronic kidney disease, suggesting that FAO disorders are a common event in AKI-CKD transformation.

[0004] The dual-specificity phosphatase (DUSP) family has the ability to dephosphorylate protein serine / threonine (Ser / Thr) and tyrosine (Tyr) residues, and plays an important role in cell stress, inflammation and fibrosis by regulating the phosphorylation signaling network. As an atypical DUSP family member, DUSP26 lacks the classic MAPK-specific binding domain, and has a wider substrate spectrum. It can not only regulate MAPK signaling molecules such as ERK and JNK, but also target non-MAPK substrates such as p53 and AK2, showing important pleiotropic regulatory functions in tumors, metabolic diseases and cardiovascular diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a new biomarker DUSP26 for predicting the transformation of acute kidney injury to chronic kidney disease, and to provide a new approach for assisting the prognosis prediction of acute kidney injury and the prediction of AKI-CKD transformation.

[0006] In order to achieve the above-mentioned object, the present invention provides the use of a reagent for detecting the expression amount of the biomarker DUSP26 in preparing a prediction product for the transformation of acute kidney injury to chronic kidney disease.

[0007] In a specific embodiment, the product comprises a kit or a reagent.

[0008] In a specific embodiment, the test sample of the subject is kidney tissue.

[0009] In a specific embodiment, the expression level of DUSP26 in the renal tissue of the subject is positively correlated with the degree of renal interstitial fibrosis.

[0010] In a specific embodiment, the expression level of DUSP26 in the kidney tissue of the subject is negatively correlated with the glomerular filtration rate value, an indicator for evaluating renal function.

[0011] The beneficial effects of the present invention include at least: 1. The present invention discovered for the first time that DUSP26 is significantly upregulated during the AKI-CKD transformation process, especially highly expressed in the proximal tubules of patients with renal fibrosis and CKD; thus, DUSP26 can be used as a biomarker for the transformation of acute kidney injury (AKI) to chronic kidney disease (CKD), for early identification of high-risk AKI patients, thereby assisting in the prediction of AKI prognosis and providing a new approach for the prediction of AKI-CKD transformation.

[0012] 2. The expression level of DUSP26 is negatively correlated with the glomerular filtration rate (eGFR), an indicator for evaluating renal function, and positively correlated with the degree of renal interstitial fibrosis. Therefore, DUSP26 can be used as a biomarker for the progression of acute kidney injury (AKI) to chronic kidney disease (CKD), which helps to identify high-risk patients with AKI progressing to CKD at an early stage, so that timely intervention measures can be taken to improve the patient's prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The expression of DUSP26 in the renal tissue of AKI-CKD transformation model mice, among which, Figure 1 a in the figure shows the results of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the sham operation group 21 days after surgery. Figure 1 b shows the results of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the uIR group 7 days after surgery. Figure 1 c in the figure shows the results of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the uIR group 14 days after surgery. Figure 1 The d in the figure is the result of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the uIR group 21 days after surgery. Figure 1 e in the figure shows the results of DUSP26 immunohistochemical staining in the cortex and outer medulla of the renal tissue of mice in the sham operation group. Figure 1 f in the figure is the result of DUSP26 immunohistochemical staining in the cortex and outer medulla of the renal tissue of mice in the uIR group 7 days after surgery. Figure 1 g in the figure shows the results of DUSP26 immunohistochemical staining in the cortex and outer medulla of the renal tissue of mice in the uIR group 14 days after surgery. Figure 1 h in the figure shows the results of DUSP26 immunohistochemical staining in the cortex and outer medulla of renal tissue of mice in the uIR group 21 days after surgery; Figure 2 The results of correlation analysis between DUSP26 expression and renal interstitial collagen fiber deposition in the sham operation group and uIR group; Figure 3 The results of correlation analysis between DUSP26 expression and mouse GFR in the sham operation group and uIR group; Figure 4 Western blotting results of the cortex and outer medulla of the renal tissues of mice in the sham operation group 21 days after surgery and in the uIR group 7 days, 14 days, and 21 days after surgery; Figure 5 Statistical analysis results of Western blotting of the cortex and outer medulla of renal tissues of mice in the sham operation group 21 days after surgery and in the uIR group 7 days, 14 days, and 21 days after surgery; Figure 6The results of DUSP26 and immunofluorescence staining of renal biopsy tissues in the CKD group and the control group (MCD) are shown in Figure 2. Figure 6 a in the figure is the result of DUSP26 immunofluorescence staining of the control group (MCD). Figure 6 b is the result of LTL immunofluorescence staining of the control group (MCD). Figure 6 c in the figure is the Merge immunofluorescence staining result of the control group (MCD). Figure 6 The d in the figure is the Magnified immunofluorescence staining result of the control group (MCD). Figure 6 e in the figure is the result of DUSP26 immunofluorescence staining in the CKD group. Figure 6 f in the figure is the result of immunofluorescence staining of LTL in the CKD group. Figure 6 g in the figure is the Merge immunofluorescence staining result of the CKD group. Figure 6 h in the figure is the Magnified immunofluorescence staining result of the CKD group; Figure 7 The statistical results of the percentage of DUSP26-positive cells in LTL-negative and LTL-positive renal tubules by immunofluorescence co-staining in the CKD group and the control group (MCD); Figure 8 The results of the correlation analysis between the percentage of DUSP26-positive cells in LTL-positive renal tubules and the patients' eGFR in the CKD group and the control group; Fig. 9 The results of correlation analysis between the proportion of DUSP26-positive cells in LTL-positive renal tubules and renal interstitial fibrosis in the CKD group and the control group. DETAILED DESCRIPTION

[0014] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0015] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Specifically: polyacrylamide gel (SDS-PAGE) kit was purchased from abiowell; protease inhibitor cocktail (P8340), BCA detection kit, protein ladder, anti-DUSP26 antibody (PA-5-22013) were purchased from Invitrogen; anti-GAPDH antibody (10494-1-Ig) was purchased from Proteintech; Sirius red staining reagent (GC307014) was purchased from Servicebio; immunohistochemical staining reagent (PV-9000) was purchased from Zhongshan Jinqiao; immunohistofluorescence staining reagent (TSA-520) was purchased from abiowell, LTL staining solution (RFL-1321) was purchased from Vector Laboratories; transdermal monitoring fluorescein isocyanate (FITC) was purchased from MediBeacon.

[0016] The specific operation steps of Western blotting detection, immunofluorescence staining, picrosirius red staining, immunohistochemical staining and glomerular filtration rate determination in the present invention are as follows: Western blotting After total protein was extracted from kidney tissue or cells by lysis, protein concentration was determined by BCA method. 30 μg protein was added to each well and separated by electrophoresis in 10% or 12% SDS-PAGE gel. Then, the membrane was wet-transferred to PVDF membrane at 120 mA constant current for 1.5 h. After the membrane was blocked with blocking solution at room temperature for 1 h, TBST was eluted and the primary antibody was added and incubated overnight at 4 °C. The membrane was washed with TBST three times the next day for 10 min each time; then the corresponding HRP-labeled secondary antibody was added and incubated at room temperature for 1 h, and the membrane was washed with TBST again for 3 times for 5 min each time. The antigen-antibody complex was developed by enhanced chemiluminescence (ECL) luminescence method, exposed and scanned using darkroom X-ray film. Protein quantification was performed by gray value analysis of the target band, and the relative expression of the target protein was expressed as the gray value of the target band / GAPDH gray value.

[0017] Sirius red staining Weigh 0.1g of Sirius red and add it to 100 mL of saturated picric acid solution. Mix thoroughly until completely dissolved to prepare 0.1% Sirius red staining solution. Dewax the paraffin sections routinely to water (after dewaxing in xylene, sequentially through 100%, 95%, 80%, and 70% ethanol, 2-3 minutes for each step). Rinse with distilled water twice, 1 minute each time. Stain the sections in 0.1% Sirius red staining solution for 8-10 minutes (can be extended to 15 minutes depending on the thickness of the tissue). Rinse the sections quickly with distilled water twice to remove excess stain. Differentiate the sections in acidic ethanol (0.5% glacial acetic acid and 95% ethanol) twice, each for about 30 seconds, to remove non-specific background. Dehydrate with anhydrous ethanol twice, 2 minutes each time. After being transparentized with xylene, seal the sections with neutral gum.

[0018] Immunofluorescence staining and LTL staining After paraffin renal tissue sections were dewaxed and hydrated, the sections were placed in a microwave oven for antigen retrieval. After being washed 3 times with 1×PBS for 5 minutes each time, they were blocked with 5% BSA at room temperature for 1 hour and incubated with anti-DUSP26 (1:200) at 4°C overnight. The next day, they were washed 3 times with 1×PBS for 5 minutes each time, incubated with the corresponding fluorescent-labeled secondary antibody at room temperature for 1 hour; washed 3 times with 1×PBS again, incubated with LTL staining solution (1:100) at room temperature for 1 hour; washed 3 times with 1×PBS again, and then sealed with a sealing agent with DAPI, and pictures were taken under a fluorescence microscope. The quantitative analysis of fluorescent positive staining was performed using ImageJ software to quantify the percentage of DUSP26-positive cells in LTL-positive and LTL-negative renal tubules.

[0019] Immunohistochemical staining After paraffin renal tissue sections were dewaxed and hydrated, the sections were placed in a microwave oven for antigen retrieval. The slides were then exposed to 3% H 2 O 2 To block endogenous peroxidase activity, 0.1% Triton X-100 buffer and 5% BSA blocking buffer were used to reduce nonspecific binding, and the slides were incubated with anti-DUSP26 (1:200) at 4°C overnight, washed 3 times with 1× PBS for 5 minutes each, and incubated with the corresponding HRP-conjugated secondary antibody for 45 minutes at room temperature. The signal of the antigen-antibody complex was detected using the DAB kit according to the manufacturer's protocol. Finally, the slides were counterstained with hematoxylin for nuclei. Immunohistochemical positive staining was quantified using ImageJ software to quantify the percentage of DUSP26-positive cells.

[0020] Determination of Glomerular Filtration Rate (GFR) in Mice The GFR of mice was measured by transdermal monitoring of the clearance of fluorescein isocyanate (FITC)-labeled sinistrin. Briefly, the skin was prepared one day in advance (the hair on the left side of the mouse's back was shaved and fully depilated with a depilatory cream), and the transdermal GFR monitor was attached to the skin using a double-sided adhesive patch (MediBeacon, Germany), and the device was fixed to the mouse with medical tape. The mouse was then injected with 75 mg / kg FITC-sinistrin (15 mg / mL, dissolved in 0.9% sterile saline) via tail vein injection. The mouse was returned to the cage, and the GFR was monitored for 1-2 hours, after which the device was removed and the data were analyzed using the elimination kinetic curve of FITC-sinistrin.

[0021] Example 1 Construction of AKI-CKD transformation mouse model and detection of DUSP26 expression in kidney tissue of AKI-CKD transformation mouse model In this example, a uIR-AKI mouse model was constructed, i.e., a unilateral ischemia-reperfusion injury (uIR) in the kidney.

[0022] The specific construction method is as follows: 8-12 week old male C57BL / 6 mice were used, and pentobarbital (50 mg / kg) was injected intraperitoneally to anesthetize the mice. A 2 cm midline incision was made on the back, and the abdominal wall was cut layer by layer at the location of the left kidney. The kidney was found and the renal pedicle was fully exposed. The renal pedicle was clamped with a vascular clamp that did not damage the blood vessels. After 30 minutes, the vascular clamp was removed to restore the blood supply to the kidney. After the kidney was returned, the back incision was sutured layer by layer. During the operation, the mice were placed on a 37°C constant temperature pad to maintain the body temperature of the mice at 36.5±0.3°C. The control mice underwent sham surgery, that is, except for not clamping the renal pedicle, the rest of the surgical procedures were the same as those of the uIR mice. The left kidney tissues of the mice were collected 7 days, 14 days, and 21 days after the surgery for picrosirius red staining, and the mouse GFR was measured percutaneously to identify the successful construction of the AKI-CKD transformation mouse model. The contralateral kidney was surgically removed one day before the kidney was collected to avoid interference from the contralateral healthy kidney. Western blotting was further performed to detect the expression of DUSP26. The test results are detailed in Figures 1 to 5 shown.

[0023] See also Figure 1 , Figure 1The expression of DUSP26 in the renal tissue of AKI-CKD transformation model mice, among which, Figure 1 a in the figure is the result of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the sham operation group 21 days after surgery. Figure 1 b shows the results of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the uIR group 7 days after surgery. Figure 1 c in the figure shows the results of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the uIR group 14 days after surgery. Figure 1 The d in the figure is the result of Sirius red staining of the cortex and outer medulla of the renal tissue of mice in the uIR group 21 days after surgery. Figure 1 e in the figure shows the results of DUSP26 immunohistochemical staining in the cortex and outer medulla of the renal tissue of mice in the sham operation group. Figure 1 f in the figure is the result of DUSP26 immunohistochemical staining in the cortex and outer medulla of the renal tissue of mice in the uIR group 7 days after surgery. Figure 1 g in the figure shows the results of DUSP26 immunohistochemical staining in the cortex and outer medulla of the renal tissue of mice in the uIR group 14 days after surgery. Figure 1 Figure h shows the results of DUSP26 immunohistochemical staining in the cortex and outer medulla of renal tissue of mice in the uIR group 21 days after surgery.

[0024] from Figure 1 As can be seen from b, c, and d, as the kidney repair time increases, typical renal interstitial fibrosis appears in the kidney tissue, indicating that the AKI-CKD transformation mouse model was successfully constructed.

[0025] See also Figures 2 to 5 , Figure 2 The results of correlation analysis between DUSP26 expression and renal interstitial collagen fiber deposition in the sham operation group and uIR group; Figure 3 The results of correlation analysis between DUSP26 expression and mouse GFR in the sham operation group and uIR group; Figure 4 Western blotting results of the renal cortex and outer medulla of mice in the sham group 21 days after surgery and in the uIR group 7 days, 14 days, and 21 days after surgery; Figure 5 Statistical analysis results of Western blotting of renal cortex and outer medulla of mice in sham operation group 21 days after surgery and in uIR group 7 days, 14 days, and 21 days after surgery.

[0026] from Figures 1 to 5It can be seen that the expression of DUSP26 gradually increased in the AKI-CKD transformation model, and was positively correlated with the deposition of collagen fibers in the renal interstitium and negatively correlated with the glomerular filtration rate (GFR). This suggests that in the AKI-CKD animal model caused by unilateral renal ischemia-reperfusion model, DUSP26 expression is significantly upregulated and is closely related to the degree of renal interstitial fibrosis.

[0027] It should be noted that in Example 1 of the present invention, in order to scientifically set up a control group and take animal ethics into consideration, the mice in the sham operation group were uniformly sampled on the 21st day after surgery, which corresponds to the time point when renal interstitial fibrosis and DUSP26 expression were most significant in the uIR group. The selection of this time point can effectively control the interference of time factors, ensure that the observed differences are mainly derived from the surgical operation itself rather than natural changes in the postoperative recovery process, and have a good scientific control basis.

[0028] Example 2 Analysis of the expression of DUSP26 in renal tissue of clinical patients and its correlation with renal function Renal tissue specimens (n=7) from patients diagnosed with chronic kidney disease (CKD) by renal puncture were collected clinically, and the control group consisted of renal tissue specimens (i.e., renal tissue without tubular atrophy) from patients with minimal change disease (MCD) (n=5) and their clinical data. Immunofluorescence staining was used to detect the expression of DUSP26 in the renal tissues of patients, and the correlation between the expression and the estimated glomerular filtration rate and renal interstitial fibrosis of the patients was analyzed. The clinical and pathological information of the 7 CKD patients is shown in the following table.

[0029] See also Figures 6 to 9 ,in, Figure 6 The results of DUSP26 and immunofluorescence staining of renal biopsy tissues in the CKD group and the control group (MCD) are shown in Figure 2. Figure 6 a in the figure is the result of DUSP26 immunofluorescence staining of the control group (MCD). Figure 6 b is the result of LTL immunofluorescence staining of the control group (MCD). Figure 6 c in the figure is the Merge immunofluorescence staining result of the control group (MCD). Figure 6 The d in the figure is the Magnified immunofluorescence staining result of the control group (MCD). Figure 6 e in the figure is the result of DUSP26 immunofluorescence staining in the CKD group. Figure 6 f in the figure is the result of immunofluorescence staining of LTL in the CKD group. Figure 6 g in the figure is the Merge immunofluorescence staining result of the CKD group. Figure 6h in the figure is the Magnified immunofluorescence staining result of the CKD group; Figure 7 The statistical results of DUSP26 expression in LTL-negative and LTL-positive cells by immunofluorescence co-staining in the CKD group and the control group (MCD); Figure 8 The results of the correlation analysis between the percentage of DUSP26-positive cells in LTL-positive tubules and the patients' eGFR in the CKD group and the control group; Fig. 9 The results of correlation analysis between the proportion of DUSP26-positive cells in LTL-positive tubules and renal interstitial fibrosis in the CKD group and the control group.

[0030] from Figure 6 and Figure 7 It can be seen that DUSP26 is highly positively expressed in the renal biopsy tissues of CKD patients, and the expression of DUSP26 in the nuclei of LTL-positive tubules is more significantly increased, while in the renal biopsy tissues of patients with minimal change disease (MCD) in renal tissue pathology, DUSP26 expression is relatively low.

[0031] from Figure 8 and Fig. 9 It can be seen that the proportion of DUSP26-positive tubular cells in LTL-positive tubules is negatively correlated with the patient's eGFR level and positively correlated with the degree of renal fibrosis, which is similar to the animal experimental results provided in Example 1. This suggests that the level of DUSP26 in renal tissue can reflect the severity of renal fibrosis.

[0032] Combining the experimental results of Example 1 and Example 2, it can be seen that during the AKI-CKD transformation process, the expression of DUSP26 in the renal puncture specimens of CKD patients and the renal tissue of the AKI-CKD transformation mouse model is differentially expressed, specifically, compared with the control group, DUSP26 is significantly highly expressed in the proximal tubules of renal fibrosis and CKD renal tissue, and its expression is negatively correlated with the renal function assessment index eGFR (estimated glomerular filtration) and positively correlated with the degree of renal interstitial fibrosis. This shows that DUSP26 is closely related to proximal tubular injury, can be used as a biomarker for AKI-CKD transformation, and can be used to assist in the prognosis prediction of patients with acute kidney injury, providing a new way for the auxiliary diagnosis and prediction of the transformation of acute kidney injury to chronic kidney disease.

[0033] It should be noted that chronic kidney disease and chronic kidney disease are two terms for the same disease, that is, chronic kidney disease refers to chronic kidney disease.

[0034] It should be noted that Glomerular Filtration Rate (GFR) is an indicator that truly measures or approximately reflects the glomerular filtration function. It can be measured directly or indirectly through a variety of methods in animal experiments. Estimated Glomerular Filtration Rate (eGFR) is the glomerular filtration rate "estimated" by indicators such as serum creatinine and is commonly used in human clinical practice.

[0035] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. Application of reagents for detecting the expression of the biomarker DUSP26 in the preparation of predictive products for the transformation of acute kidney injury to chronic kidney disease.

2. The use according to claim 1, characterized in that: The predicted product includes a kit or a reagent.

3. The use according to claim 1 or 2, characterized in that: The test sample of the subject is kidney tissue.

4. The use according to claim 3, characterized in that: The expression level of DUSP26 in the renal tissue of the subjects was positively correlated with the degree of renal interstitial fibrosis.

5. The use according to claim 3, characterized in that: The expression level of DUSP26 in the renal tissue of the subjects was negatively correlated with the glomerular filtration rate, an indicator for evaluating renal function.

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