Biomarker, analysis method and kit for nephrotic syndrome

Abstract

The invention discloses a biomarker for nephrotic syndrome, an analysis method and a kit, which can be effectively used for diagnosis of nephrotic syndrome and have high sensitivity and accurate and reliable results. The biomarker for nephrotic syndrome comprises the mRNA, the protein and the enzymatic activity level of the following substances: lactate dehydrogenase B.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical testing, and in particular to a biomarker for nephrotic syndrome, an analysis method for the biomarker for nephrotic syndrome, and the use of the biomarker for nephrotic syndrome in a kit for early diagnosis of nephrotic syndrome. Background Art

[0002] Nephrotic syndrome (NS) is a serious kidney disease characterized by heavy proteinuria and hypoproteinemia. It includes primary FSGS, MCD, and MN, which are diagnosed based on clear histopathological changes, as well as secondary diseases with clear etiologies such as diabetic nephropathy. Due to the unclear etiology and low prevalence of biopsies, the incidence of the disease is not uniform worldwide. The incidence of primary NS in children is 1.15-16.9 / 100,000, while the incidence in adults is 3.5-4.3 / 100,000 per year. The incidence of secondary NS is even higher. Due to insufficient understanding of the disease, the only standard treatment for NS is immunosuppression. 20% of NS patients are resistant to immunotherapy. Such patients often have a poor prognosis and may eventually progress to chronic kidney disease or even end-stage renal disease, requiring dialysis or kidney transplantation within 2 to 10 years.

[0003] LDHB (lactate dehydrogenase B) is a subunit of lactate dehydrogenase (LDH). Together with LDHA, it constitutes the LDH isoenzyme, catalyzing the reversible conversion of pyruvate to lactate. Compared to LDHA, LDHB has a higher affinity for lactate, catalyzing the conversion of lactate to pyruvate. Currently known selective inhibitors for LDHB include AXKO-0046, and inhibitors of LDH include LDH-IN-1, Galloflavin, and LDHA-IN-7. Summary of the Invention

[0004] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a biomarker for nephrotic syndrome, which can be effectively used for the diagnosis of nephrotic syndrome with high sensitivity and accurate and reliable results.

[0005] The technical solution of the present invention is: the biomarker for nephrotic syndrome comprises the mRNA, protein and enzyme activity levels of the following substances: lactate dehydrogenase B.

[0006] A method for analyzing biomarkers of nephrotic syndrome, comprising the following steps:

[0007] (1) Detection of LDH B protein level: Fresh kidney samples were frozen, embedded, and sliced, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. The podocyte-specific markers—nephrin from guinea pig and lactate dehydrogenase B from rabbit—were incubated at room temperature for 2 hours, and then incubated at 37°C with secondary antibodies against different fluorescent dyes for 1 hour. DAPI was also detected.

[0008] The dye was incubated at room temperature for 10 min, and then a mounting solution containing an anti-fluorescence quencher was added to the slides for sealing. LDHB was observed under an upright fluorescence microscope, and the area where nephrin and lactate dehydrogenase B were co-expressed represented the expression level of LDHB in podocytes.

[0009] (2) Detection of LDH B mRNA levels: Fresh kidney samples were microdissected under a microscope to remove glomerular tissue. The Illumina TruSeq mRNA Sample Prep v2 kit was used to prepare mRNA samples. Multiplex amplification technology was used to generate 100-base cDNA on the Illumina HiSeq2000 platform and sequenced. After quality assessment, batch effect removal, and annotation of the data, an expression matrix was obtained to characterize the expression level of LDH B. The unpaired Student's t-test was used for analysis. A p-value < 0.05 was considered to be significantly different. Graphpad Prism software was used to perform Pearson correlation analysis on the expression level of LDH B with the patient's urine protein and GFR levels. SPSS software was used to perform ROC curve analysis on LDH B.

[0010] (3) Lactate dehydrogenase B enzyme activity detection: The tissue sample is glomerular tissue, which is added with tissue lysis buffer and homogenized using a tissue grinder under low temperature conditions. The tissue is then centrifuged at 4°C and 12,000×g for 5 minutes to obtain the supernatant for subsequent detection. The appropriately diluted tissue supernatant is added to a microplate pre-coated with lactate dehydrogenase B monoclonal antibody, and a gradient concentration of lactate dehydrogenase B standard solution is added to the microplate to prepare a standard curve. The plate is incubated at room temperature for 2 hours. The supernatant is discarded and the plate is washed three times with washing buffer. The plate containing NAD is added. + , L-lactic acid, and WST-8 colorimetric solution, and immediately measure the absorbance at 450 nm using an appropriate microplate reader. Record the 0-minute reading as A1; react at 37°C for 20-30 minutes, measure A450, and record it as A2; ΔA = A2-A1, and substitute ΔA into the standard curve to obtain the lactate dehydrogenase B activity.

[0011] The biomarker for nephrotic syndrome and the analysis method of the present invention can be effectively used for the diagnosis of nephrotic syndrome, with high sensitivity and accurate and reliable results.

[0012] Also provided are kits for biomarkers of nephrotic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It was shown that LDHB was significantly decreased in NS glomeruli / podocytes and could be used as a marker for disease diagnosis. Figure 1 A shows the expression level of LDHB mRNA in the glomeruli of NS patients in the NEPTUNE cohort; B shows the ROC curve of LDHB in the NEPTUNE cohort; C shows the Pearson correlation results of LDHB with urine protein and GFR in NS patients in the NEPTUNE cohort; D shows the immunofluorescence co-localization results of LDHB and podocyte marker-Nephrin in the ADR-induced NS rat model.

[0014] Figure 2 Figure 3: ADR-induced reduction in lactate content and net uptake in the NS rat model. A shows spatial imaging of lactate and its expression levels in glomeruli and renal tubules; B shows a scatter plot of net lactate uptake in the kidney (Kid) and circulatory system (Cir).

[0015] Figure 3 Shown are the skeletal morphology and energetic assessment of primary podocytes from Ldhb-knockout mice. A shows representative images of the scratch assay and migration rate results; B shows representative images of primary podocyte morphology; C shows representative results of JC-1 staining to characterize mitochondrial membrane potential.

[0016] Figure 4 Podocyte-specific Ldhb conditional knockout mice (Ldhb ΔPOD ) Renal pathology and renal function assessment. A shows representative images of PAS staining and WT-1 immunohistochemical staining; B shows semiquantitative results of glomerular sclerosis and the number of WT-1-positive podocytes; C shows urine protein levels; D shows renal function indicators such as blood urea nitrogen (BUN) and serum creatinine (sCr). DETAILED DESCRIPTION

[0017] This biomarker for nephrotic syndrome includes mRNA, protein, and enzyme activity levels of the following substance: lactate dehydrogenase B.

[0018] As shown in Table 1, preferably, one of the following natural drug monomers targets and binds to LDHB: tannic acid, aconitum biflavonoids, astaxanthin, irinotecan, diosmin, hesperidin, erbuliside, deacetyldilan, grape seed extract, lanolin C, daurine, forsythiaside A, methyl hesperidin, sultamicillin, privet flavonoids, hypericin, salvianolic acid C, salvianolic acid B, eriocitrin, isocarpus glycosides, citrus glycosides, sennoside A, linaloside, spinosad, forsythiaside B, chrysanthemoside, neohesperidin, dioscin, coelenterazine, anemarrhena saponin A3, oroxylin B, naringin, neodiosmin, narcissin, australine, fennel glycosides, parsnip saponin VII-1, isoliensinine, cyperus rotundus A, salinomycin, psoralea corylifolia. Lenkast, Dioscorea slender saponin, Crocin, 1,2,3,4,6-O-pentagalloylglucose, Scrophulariaceae IA, Solanine, Kaempferol-3-O-rutinoside, Parisopsin B, Withaferin A, Rutin, Hederacoside C, Epicatechin gallate, Salvianolic acid A, Parisopsin I, Liensinine, Pinesterol diglucoside, Menghuain, 9"-salvianolic acid B monomethyl ester, Methyl protodioscin, Dipsacoside B, Tomatine hydrochloride, Epigallocatechin gallate, Asiaticoside, Methyl neferine, Typhaneroglucosid, Cynarin, Paliperidone, Neomangiferin, Echinacea glycoside, Scutellaria baicalensis, Akebia saponin D, 4'-O-vitexin glucoside, Ligustrum lucidum glycoside, Trillium, Lithospermic acid, Isochlorogenic acid A, Hubeisuan , cinnamomum tobacillus venom base, Paris polyphylla saponin II, tomatine, proanthocyanidin B2, rifaximin, silybin, silymarin, veratramide, thiostrepton, tea polyphenols, bevirima, epigallocatechin gallate, dauricine, irinotecan hydrochloride, luteolin-7-O-glucuronide, jujube saponin A, icariin, neohesperidin dihydrochalcone, verbascose glycosides, isochlorogenic acid C, barleysonide A, wild sumac glycosides, periwinkle glycosides, parisin VI, barleysonide B, gentian ester glycosides, angiotensin, pseudoprotodioscin, pulsatilla saponin B4, madecassoside, estradiol benzoate, tedizolid phosphate, doramectin, hesperidin methyl chalcone, bicuculline, plantagoside, complanatine, schaftoside, polygalaconone III, isochlorogenic acid B, amaranthone, privet glycosides, vitexin glucoside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, dihydroquercetin 7-rhamnoside, crocin II, apigenin-7-glucuronic acid, diserpine, folic acid, bilirubin, daidzein, genistein, Gynostemma pentaphyllum extract, naringin, astragaloside A, Liquorice glycoside, efodipidine, luteolin, chelidonol glycoside, flavonoid glycosides of Vaccaria segetalis, isosilybin, catalpa toxin, jujube saponin B, soybean saponin Bb, pirarubicin, flax lignan, digoxin, rebaudioside C, ganoderic acid A, difloxacin hydrochloride, trichloroacetic acid, chaenomelanin A, epimedin I, acetyl corydaline, epimedin C, leptomycin B, astragaloside I,Gynostemma pentaphyllum saponin XLIX, flavopisate hydrochloride, kaempferol-3-glucorhamnoside, citronellol, paclitaxel, cefoperazone, hyodeoxycholic acid, troxerutin, astragaloside IV, deguelin, carnosol, akebia phenylethanoid glycoside B, isorhamnetin-3-O-neohesperidoside, kaempferol-3-O-neohesperidoside, daphneol, anemarrhena saponin BII, ruscosaponin, oleandrin, solanum saccharum, lovastatin, baicalin, fisetin, naringin dihydrochalcone, honeysuckle saponin B, estradiol cypionate, cepharanthin, safflower yellow, solvent red 23, tedizolid, glycoursodeoxycholic acid, kaempferol, spinosum , benzoylpeoniflorin, psoralen, oleanolic acid, bardoxolone, plantagoside, gossypol, honeysuckle saponin A, epimedium B, pulsatilla saponin A3, 7-ketolithocholic acid, raloxifene hydrochloride, chlorhexidine hydrochloride, tauroursodeoxycholic acid, chlorothione, isoquercetin, ginsenotriol, tauroursodeoxycholic acid, glycocholic acid, 5-O-methylvisamidoside, aminophenacidine hydrochloride, chicoric acid, peucedanum rosin, parfum saponin VII-2, ruscosaponin, rubicin, sanguisorbamen II, 7-xylosyl-10-deacetylpaclitaxel, minocycline hydrochloride, triptolide, glycyrrhetinic acid, myricetin, dihydroquercetin, sesamin, Dihydromyricetin, paeoniflorin, sophora flavescens, brassinolide, α-hedera saponin, licorice root, dicoumarol, sulfinpyrazone, nicergoline, milbemycin oxime, cimicifuga glycosides, asarone, pepstatin-21, cucurbitacin B, sanguinarine, isovitexin, wogonin, apigenin-7-glucoside, oroxylin A, isoliquiritigenin, trifoliate glycoside, isoorientin, esterbufogenin, deoxyrhubarb glycoside, epibrassinolide, thymosin, itraconazole, lupeol, luteolin, rotenone, lappaconitine hydrobromide, quercetin, avosidib hydrochloride, chrysanthene, baohuoside I, protopine, bupavaquinone, cycloastragenol, avosidic acid Vermectin B1, 7-ethylcamptothecin, α-naphthoflavone, 7-dehydrocholesterol, sofarone, vitexin-2-O-rhamnoside, cefotiam hydrochloride, glycochenodeoxycholic acid, oleuropein, corosolic acid, phylloside, eleutheroside E, rubusoside, cyperifolia pine, cyperifolia glycoside, isopsoralea corylifolia dihydroflavone, 7-epitaxel, alismatol B, alismatol A, schisandra chinensis ester B, eriodictyol, scrophulariaceae glycoside IB, cimicifuga aglycone glucoside, purpurogenol, levulinic acid, acetylated epigallocatechin gallate, pullulan, carminic acid, 25-hydroxycholesterol are used for preparing a drug for treating nephrotic syndrome NS.

[0019] Table 1. Binding energy results of 286 natural compound monomers and LDHB virtual docking

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] A method for analyzing biomarkers of nephrotic syndrome, comprising the following steps:

[0031] (1) LDHB protein level detection: Fresh kidney samples were frozen, embedded, and sliced, and 4%

[0032] After fixation with paraformaldehyde, permeabilization with 0.1% Triton X-100, and blocking with 3% BSA, the cells were incubated with podocyte-specific markers—guinea pig-derived primary antibodies for nephrin and rabbit-derived primary antibodies for LDHB—at room temperature for 2 hours, followed by incubation with corresponding secondary antibodies against different fluorescent dyes at 37°C for 1 hour, and DAPI dye incubated at room temperature for 10 minutes. The sections were then sealed with mounting solution containing an anti-fluorescence quencher, and LDHB was observed under an upright fluorescence microscope. The area where nephrin and LDHB co-expressed represented the expression level of LDHB in podocytes.

[0033] (2) Detection of LDHB mRNA level: Fresh kidney samples were microdissected under a microscope to remove glomerular tissue, and the Illumina TruSeq mRNA Sample Prep v2 kit was used to prepare mRNA samples. Multiple amplification technology was used to generate 100 base cDNA on the Illumina HiSeq2000 platform and sequenced. After quality assessment, batch effect removal, and annotation of the data, an expression matrix was obtained to characterize the expression level of LDHB. The unpaired Student's t-test was used for analysis. A p-value < 0.05 was considered to be significantly different. At the same time, the GraphPad Prism software was used to compare the expression level of LDHB with that of the patient's urine protein,

[0034] Pearson correlation analysis was performed for GFR levels, and ROC curve analysis was performed for LDHB using SPSS software;

[0035] (3) LDHB enzyme activity detection: The tissue sample is glomerular tissue, which is added with tissue lysis buffer and homogenized using a tissue grinder under low temperature conditions. The supernatant is then centrifuged at 4°C and 12,000×g for 5 minutes to obtain the supernatant for subsequent detection. The appropriately diluted tissue supernatant is added to a microplate pre-coated with LDHB monoclonal antibody, and a gradient concentration of lactate dehydrogenase B standard solution is added to the microwells to prepare a standard curve. The solution is incubated at room temperature for 2 hours; the supernatant is discarded and the sample is washed three times with washing buffer, and a solution containing NAD is added. + , L-lactic acid, and WST-8 colorimetric solution, and immediately measure the absorbance at 450 nm using an appropriate microplate reader. The reading at 0 minutes is recorded as A1. Incubate at 37°C for 20-30 minutes, and measure A450, which is recorded as A2. ΔA = A2-A1.

[0036] LDHB activity was obtained by substituting ΔA into the standard curve.

[0037] The biomarker for nephrotic syndrome and the analysis method of the present invention can be effectively used for the diagnosis of nephrotic syndrome, with high sensitivity and accurate and reliable results.

[0038] Preferably, the mRNA level of LDHB is decreased in the glomeruli of NS patients.

[0039] Preferably, the expression of LDHB protein is reduced in NS podocytes.

[0040] Preferably, the LDHB-mediated lactate utilization disorder in NS glomeruli.

[0041] Preferably, podocyte-specific knockout of LDHB results in overt proteinuria, and primary podocytes have energy disorders and skeletal damage.

[0042] Also provided are kits for biomarkers of nephrotic syndrome.

[0043] The present invention is described in detail below.

[0044] 1. The mRNA level of LDHB in the glomeruli of NS patients decreased, and its AUC value as a diagnostic indicator was 0.804. It is significantly negatively correlated with the clinical indicator of the disease - urine protein, and significantly positively correlated with GFR .

[0045] The present invention is based on the transcriptomic data of the glomerular part of NS patients in the NEPTUNE cohort (LD=8, NS=220). Differential gene expression analysis was performed and it was found that compared with the normal control (LD group), LDHB was significantly reduced in NS patients ( Figure 1 A); To evaluate the performance of LDHB as a disease diagnosis marker, ROC curve analysis was performed, and the AUC value was 0.804, indicating that LDHB performed well as a diagnostic model ( Figure 1B). Pearson correlation analysis of LDHB expression levels in glomeruli and NS disease-related indicators revealed a significant negative correlation between LDHB and urine protein, and a significant positive correlation with GFR ( Figure 1 C).

[0046] 2. In the ADR-induced NS rat model, it was found that the expression of LDHB protein in NS podocytes was reduced.

[0047] A single tail vein injection of doxorubicin (ADR) was used to establish a NS rat model, which exhibits disease characteristics such as massive proteinuria, localized glomerulosclerosis, and podocyte loss. Based on this model, immunofluorescence co-localization of LDHB with the podocyte-specific marker nephrin was performed. The results showed that the co-localization positive area of ​​nephrin and LDHB staining decreased, indicating that LDHB expression in podocytes of NS rats was significantly reduced ( Figure 1 D).

[0048] 3. The glomerular lactate content of NS rats decreased, and the kidneys converted from net lactate uptake to net lactate release, indicating that NS kidney Impaired LDHB-mediated lactate utilization in glomeruli.

[0049] LDHB uses lactate as a substrate, catalyzing the production of pyruvate, which then enters the TCA cycle for complete oxidation to provide energy for tissue and cellular life. Therefore, the accumulation and utilization of lactate in the kidneys can reflect LDHB activity.

[0050] The DESI-MSI platform was used to detect the spatial metabolites of frozen NS rat kidney tissues. It was found that compared with the CON group, the lactic acid in the glomeruli of NS rats was significantly reduced ( Figure 2 A). The decrease in lactate expression may be due to an increase in the amount of pyruvate converted into the TCA cycle, or it may be due to a large amount of lactate being transported outside the cells. Therefore, the carotid artery, renal vein, and jugular vein blood were specifically drained for targeted metabolic testing. The renal absorption / release rate was calculated based on the lactate concentration difference between the renal vein and artery (>0, net uptake; <0, net release), and the jugular vein-artery concentration difference was used as a circulatory system control. The results showed that the kidneys of normal rats net absorbed lactate, while the kidneys of NS rats changed from net lactate absorption to net lactate release ( Figure 2 B) This indicates that lactate is transported to the extracellular space and lactate utilization is reduced in the kidneys of NS rats, confirming LDHB dysfunction.

[0051] 4. Podocyte-specific LDHB knockout mice developed dominant proteinuria, and primary podocytes were extracted and found to have energy barriers. Obstruction and skeleton damage.

[0052] Podocyte-specific LDHB knockout mice were constructed, and primary podocytes were extracted from 1-3 day old rats. The scratch test showed that compared with wild-type podocytes, the absence of LDHB led to a decrease in the 24h migration area and migration ability of podocytes ( Figure 3A); 12 hours after podocyte plating, imaging analysis was performed, and the results showed that LDHB knockout led to a decrease in podocyte lamellipodia and filopodia, and damage to the skeletal system ( Figure 3 B); The results of JC-1 fluorescence staining, a specific dye for mitochondrial membrane potential, showed that the membrane potential of podocytes with LDHB knockout was significantly reduced, and the energy function was insufficient ( Figure 3 C).

[0053] The renal function of podocyte-specific LDHB knockout mice was further evaluated in vivo. PAS pathological staining showed that LDHB ΔPOD The glomerular basement membrane of mice was thickened and localized glomerular sclerosis was present; WT-1 immunohistochemical staining showed that LDHB ΔPOD The number of podocytes in mouse glomeruli decreased and podocytes were lost ( Figure 4 AB). Disease-related blood and urine biochemical indicators were tested and it was found that adult LDHB ΔPOD Mice developed overt proteinuria and significantly elevated serum creatinine (sCr) levels.

[0054] 5. Targeted binding of 286 natural drug monomers including Tannic acid, Amentoflavone and Astaxanthin LDHB, treatment of NS.

[0055] The present invention used AXKO-0046, a known LDHB-selective inhibitor, as a positive control and performed virtual molecular docking with 3,092 natural compounds from a natural product compound library (PDB ID: 7DBK). The results showed that AXKO-0046 had an affinity for LDHB of -9.0. 286 compounds in the natural product compound library (including tannic acid, amentoflavone, and astaxanthin) had an affinity for LDHB greater than or equal to that of the positive control (see Table 1), suggesting potential for targeted LDHB therapy.

[0056] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of protection of the technical solution of the present invention.

Claims

1. A biomarker for nephrotic syndrome, characterized by: It contains mRNA, protein, and enzyme activity levels of: Lactate dehydrogenase B.

2. The biomarker for nephrotic syndrome according to claim 1, characterized in that: One of the following 286 natural drug monomers targets lactate dehydrogenase B: tannic acid, schizonepetalum biflavonoids, astaxanthin, irinotecan, diosmin, hesperidin, erbuliside, deacetylated dilanid, grape seed extract, lanolin C, daurine, forsythiaside A, methyl hesperidin, sultamicillin, privet flavonoids, hypericin, salvianolic acid C, salvianolic acid B, eriocitrin, isocarpus glycosides, citrus glycosides, sennoside A, linaloside, spinosad, forsythiaside B, chrysanthemoside, neohesperidin, dioscin, coelenterazine, anemarrhena saponin A3, oroxylin B, naringin, neodiosmin, narcissin, australine, fennel glycosides, parsnip saponin VII-1, isoliensinine, cyperus rotundus A, salinomycin, pranlukast, slender Dioscin, crocin, 1,2,3,4,6-O-pentagalloylglucose, scrophulariaceae IA, solanine, kaempferol-3-O-rutinoside, paridisin B, withaferin A, rutin, hederacoside C, epicatechin gallate, salvianolic acid A, paridisin I, liensinine, pinesterol diglucoside, mongoside, 9"-salvianolic acid B monomethyl ester, Methyl protodioscin, Dipsacoside B, Tomatine Hydrochloride, Epigallocatechin Gallate, Centella asiatica, Neferine, Typhanoside, Cynarin, Paliperidone, Neomangiferin, Echinacea glycoside, Scutellaria baicalensis, Akebia saponin D, 4'-O-vitexin glucoside, Ligustrum lucidum, Trillium, Lithospermic acid, Isochlorogenic acid A, Lake shellfish A, Cinotoxin, Paris polyphylla saponin II, tomatine, proanthocyanidin B2, rifaximin, silybin, silymarin, veratramide, thiostrepton, tea polyphenols, bevirima, epigallocatechin gallate, dauricine, irinotecan hydrochloride, luteolin-7-O-glucuronide, jujube saponin A, icariin, neohesperidin dihydrochalcone, verbascoside, isochlorogenic acid C, barisonoside A, scutellaria glycoside, periwinkle glycoside, Paris polyphylla saponin VI, barisonoside B, gentianine, angiotensin, pseudoprotodioscin, pulsatilla saponin B4, madecassoside, estradiol benzoate, tedizolid phosphate, doramectin, hesperidin methyl chalcone, bicuculline, plantagoside, astragaloside, schaftoside, polygala tenuifolia saponin III, isochlorogenic acid B , cyperasterone, privet glycoside, vitexin glucoside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, dihydroquercetin 7-rhamnoside, crocin II, apigenin-7-glucuronic acid, diserpine, folic acid, bilirubin, daidzein, genistein, Gynostemma pentaphyllum extract, naringin, astragaloside A, liquiritin, efodipine, Luteolin, chelidonol, flavonoid glycosides from Vaccaria segetalis, isosilybin, catalpa toxin, jujube saponin B, soybean saponin Bb, pirarubicin, flax lignans, digoxin, rebaudioside C, ganoderic acid A, difloxacin hydrochloride, trichloroacetic acid, chaenomeles glycoside A, epimedium I, acetyl corydaline, epimedium C, leptomycin B, astragaloside I, gypenosides XLIX,Flavonopsate hydrochloride, kaempferol-3-glucorhamnoside, cirsin, paclitaxel, cefoperazone, hyodeoxycholic acid, troxerutin, astragaloside IV, deguelin, carnosol, akebia phenylethanoid glycoside B, isorhamnetin-3-O-neohesperidoside, kaempferol-3-O-neohesperidoside, daphnehol, anemarrhena saponin BII, ruscosaponin, oleandrin, solanum saccharum, lovastatin, baicalin, fisetin, naringin dihydrochalcone, honeysuckle saponin B, estradiol cypionate, cepharanthin, safflower yellow, solvent red 23, tedizolid, glycoursodeoxycholic acid, kaempferol, spinosum, benzoylpeoniflorin , psoralen, oleanolic acid, bardoxolone, plantagoside, gossypol, honeysuckle saponin A, epimedium B, pulsatilla saponin A3, 7-ketolithocholic acid, raloxifene hydrochloride, chlorhexidine hydrochloride, tauroursodeoxycholic acid, chrysopone, isoquercetin, ginsenotriol, taurochenodeoxycholic acid, glycocholic acid, 5-O-methylvisaminol glycoside, aminophenacidine hydrochloride, chicoric acid, peucedanum rosin, parfum saponin VII-2, ruscosaponin, rubicin, sanguisorbaside II, 7-xylosyl-10-deacetylpaclitaxel, minocycline hydrochloride, triptolide, glycyrrhetinic acid, myricetin, dihydroquercetin, sesamin, dihydromyricetin , paeoniflorin, sophora flavescens, brassinolide, α-hedera saponin, liquiritigenin, dicoumarol, sulfinpyrazone, nicergoline, milbemycin oxime, cimicifuga glycosides, asarone, pepstatin-21, cucurbitacin B, sanguinarine, isovitexin, wogonin, apigenin-7-glucoside, oroxylin A, isoliquiritigenin, trifoliate glycoside, isoorientin, esterbufogenin, deoxyrhuga glycoside, epibrassinolide, thymosin, itraconazole, lupeol, luteolin, rotenone, lappaconitine hydrobromide, quercetin, avosidib hydrochloride, chrysanthene, baohuoside I, protopine, bupavaquinone, cycloastragenol, avermectin B1, 7-ethylcamptothecin, α-naphthoflavone, 7-dehydrocholesterol, sofarone, vitexin-2-O-rhamnoside, cefotiam hydrochloride, glycochenodeoxycholic acid, oleuropein, corosolic acid, phylloside, eleutheroside E, rubusoside, cyperifolia pine, cyperifolia glycoside, isopsoralea corylifolia dihydroflavone, 7-epipaclitaxel, alismatinol B, alismatinol A, schisandra chinensis ester B, eriodictyol, scrophulariaceae glycoside IB, cimicifuga aglycone glucoside, purpurogenol, levulinic acid, acetylated epigallocatechin gallate, pullulan, carminic acid, 25-hydroxycholesterol, as a medicament for the treatment of nephrotic syndrome NS.

3. The method for analyzing biomarkers of nephrotic syndrome according to claim 1, wherein: It includes the following steps: (1) Detection of LDH B protein level: Fresh kidney samples were frozen, embedded, and sliced, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. The podocyte-specific markers—nephrin from guinea pig and lactate dehydrogenase B from rabbit—were incubated at room temperature for 2 hours, and then incubated at 37°C with secondary antibodies against different fluorescent dyes for 1 hour. DAPI was also detected. The dye was incubated at room temperature for 10 minutes, and then a mounting solution containing an anti-fluorescence quencher was added to the slides for sealing. Lactate dehydrogenase B was observed under an upright fluorescence microscope. The area where nephrin and lactate dehydrogenase B were co-expressed represented the expression level of LDHB in podocytes. (2) Detection of LDH B mRNA levels: Fresh kidney samples were microdissected under a microscope to remove glomerular tissue. The Illumina TruSeq mRNA Sample Prep v2 kit was used to prepare mRNA samples. Multiplex amplification technology was used to generate 100-base cDNA on the Illumina HiSeq2000 platform and sequenced. After quality assessment, batch effect removal, and annotation of the data, an expression matrix was obtained to characterize the expression level of LDH B. The unpaired Student's t-test was used for analysis. A p-value < 0.05 was considered to be significantly different. Graphpad Prism software was used to perform Pearson correlation analysis on the expression level of LDH B with the patient's urine protein and GFR levels. SPSS software was used to perform ROC curve analysis on LDH B. (3) Lactate dehydrogenase B enzyme activity detection: The tissue sample is glomerular tissue, which is added with tissue lysis buffer and homogenized using a tissue grinder under low temperature conditions. The tissue is then centrifuged at 4°C and 12,000×g for 5 minutes to obtain the supernatant for subsequent detection. The appropriately diluted tissue supernatant is added to a microplate pre-coated with lactate dehydrogenase B monoclonal antibody, and a gradient concentration of lactate dehydrogenase B standard solution is added to the microplate to prepare a standard curve. The plate is incubated at room temperature for 2 hours. The supernatant is discarded and the plate is washed three times with washing buffer. The plate containing NAD is added. + , L-lactic acid, and WST-8 colorimetric solution, and immediately measure the absorbance at 450 nm using an appropriate microplate reader. Record the 0-minute reading as A1; react at 37°C for 20-30 minutes, measure A450, and record it as A2; ΔA = A2-A1, and substitute ΔA into the standard curve to obtain the lactate dehydrogenase B activity.

4. The method for analyzing biomarkers of nephrotic syndrome according to claim 3, wherein: Lactate dehydrogenase B mRNA levels are decreased in the glomeruli of NS patients.

5. The method for analyzing biomarkers of nephrotic syndrome according to claim 3, wherein: Lactate dehydrogenase B protein expression was decreased in NS podocytes.

6. The method for analyzing biomarkers of nephrotic syndrome according to claim 3, wherein: Lactate dehydrogenase B-mediated lactate utilization impairment in nephrotic syndrome glomeruli.

7. The method for analyzing biomarkers of nephrotic syndrome according to claim 3, wherein: Podocyte-specific knockout of lactate dehydrogenase B resulted in overt proteinuria, and primary podocytes showed energy disorders and skeletal damage. The kit for biomarkers of nephrotic syndrome according to claim 1 .

Images