Application of geranyl and geranyl pyrophosphate synthetase in simple renal cyst

Abstract

The invention discloses an application of geranyl-geranyl pyrophosphate synthetase in simple renal cyst, provides a development relationship between the geranyl-geranyl pyrophosphate synthetase and the simple renal cyst, and provides a new direction for targeted therapy of SRC by inducing formation of the renal cyst due to deletion of the geranyl-geranyl pyrophosphate synthetase. The invention further discloses that the variation frequency of the rs3806394 located in the GGPS1 promoter is increased in SRC patients, and the research on the pathological mechanism of SRC occurrence is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of simple renal cyst, in particular to an application of geranylgeranyl pyrophosphate synthase in simple renal cyst. BACKGROUND

[0002] Simple renal cysts (SRCs) are common benign lesions in adult kidneys, SRC is the most common acquired renal cyst in adults, with a prevalence of approximately 7-10%.

[0003] Its incidence increases with age, and men, hypertension and renal insufficiency are at higher risk. SRC is usually asymptomatic, and only when complications such as abdominal discomfort, back pain, hematuria, infection or cyst rupture occur, clinical intervention is required.

[0004] Recent studies have shown that SRC is associated with hypertension, proteinuria, impaired renal function and decreased graft function.

[0005] The size and characteristics of most SRCs can remain stable for a long period of time, but sometimes the cysts can rapidly enlarge, especially in young patients, however, the pathological mechanism of SRC has not been clear so far. One hypothesis is that compensatory hyperfiltration and tubular hypertrophy caused by parenchymal ischemia contribute to the formation of cysts, and another study suggests that SRC originates from distal tubules and tubular diverticula.

[0006] Therefore, the present application is directed to the pathological mechanism of simple renal cysts. SUMMARY

[0007] The technical problem solved by the present application is to overcome the above technical defects, and to provide a pathological study of SRC, which reveals an application of geranylgeranyl pyrophosphate synthase in simple renal cysts.

[0008] To solve the above technical problems, the technical solution provided by the present application is: an application of geranylgeranyl pyrophosphate synthase in simple renal cysts.

[0009] Preferably, the expression of geranylgeranyl pyrophosphate synthase is up-regulated or the activity of geranylgeranyl pyrophosphate synthase is inhibited to inhibit the proliferation of renal cyst epithelial cells.

[0010] In another aspect, the present application discloses an application of geranylgeranyl pyrophosphate synthase expression in the diagnosis of simple renal cysts.

[0011] Preferably, the expression of geranylgeranyl pyrophosphate synthase in simple renal cysts is down-regulated.

[0012] Another aspect of the present application discloses the application of the geranylgeranyl pyrophosphate synthetase GGPS1 promoter in reducing the expression of geranylgeranyl pyrophosphate synthetase, wherein the rs3806394 site variation in the GGPS1 promoter region causes down-regulation of the expression of geranylgeranyl pyrophosphate synthetase.

[0013] Another aspect of the present application discloses a simple renal cyst diagnostic reagent, comprising a combination of one or more of a GGPPS activity detection reagent, and rs3806394 typing primers.

[0014] Compared with the prior art, the present application has the advantages that: the present application provides the relationship between geranylgeranyl pyrophosphate synthetase and the development of simple renal cysts, and the deletion of the enzyme can induce the formation of renal cysts, providing a new direction for the targeted treatment of SRC, and it is also disclosed that the variation frequency of rs3806394 located in the GGPS1 promoter is increased in SRC patients, which is helpful for the study of the pathological mechanism of SRC. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The expression of GGPPS in human SRC tissues is reduced, (A) confocal microscopy observation of the expression and localization of GGPPS in normal kidney and SRC tissues, (B) immunohistochemical detection of the expression difference of GGPPS in normal kidney and SRC tissues (n=5), ***p<0.001;

[0016] Figure 2 Representative immunohistochemical staining images of high expression and low expression of GGPPS in SRC tissues;

[0017] Figure 3 The GGPS1 rs3806394 variation is related to gene transcription regulation, (A) GGPS1 promoter region tag SNP positioning map, (B) prediction of the binding of the transcription factor FOXA1 to the rs3806394 site by JASPAR software (https: / / jaspar.genereg.net / ), (C) in HK2 cells transfected with GGPS1 promoters containing rs3806394 (MUT) and FOXA1 transcription factor T alleles, the relative activity of luciferase is significantly reduced (n=3), **p<0.01; ***p<0.001. DETAILED DESCRIPTION

[0018] The present application will be further described in detail below with reference to the accompanying drawings.

[0019] GGPPS is a key enzyme in the isoprenoid biosynthesis pathway, responsible for the synthesis of geranyl diphosphate from farnesyl diphosphate. Farnesyl diphosphate is the substrate for protein farnesylation and the source of cholesterol synthesis, while geranyl diphosphate is the substrate for protein geranylation and the precursor of vitamin K2 and ubiquinone. The balance between farnesylation and geranylation affects the membrane localization and function of small G proteins, which play a crucial role in cell biology such as cell proliferation, apoptosis, metabolism, differentiation, and protein transport.

[0020] The present application includes 77 patients who underwent renal cyst decortication, collects their clinical data and tissue sections. The expression and localization of GGPPS in SRC tissue are detected by immunohistochemistry (IHC) and immunofluorescence (IF), and the association between GGPPS expression and cyst volume is evaluated by multivariate logistic regression analysis. The tag single nucleotide polymorphism (SNP) in the promoter region of GGPS1 gene is screened, and the effect of rs3806394 site variation on GGPS1 promoter activity is analyzed by luciferase reporter experiment.

[0021] Immunohistochemistry shows that GGPPS expression in SRC tissue is significantly down-regulated, and the larger the cyst volume, the lower the GGPPS expression in cyst wall epithelial cells (P=0.007). Multivariate logistic regression shows that low GGPPS expression in SRC tissue is an independent risk factor for increased cyst volume (OR=6.447, 95% CI 1.900-21.878, P=0.003). In addition, the variation frequency of rs3806394 site in the promoter region of GGPS1 in SRC patients (T allele frequency 75%) is significantly higher than that in East Asian population (22.8%). Dual luciferase reporter experiment confirmed that T allele of this site can significantly reduce the activity of GGPS1 promoter (P<0.01).

[0022] Specifically, when in use:

[0023] Retrospectively included SRC patients who underwent renal cyst surgery in Nanjing Hospital of Nanjing Medical University from January 2016 to June 2022, excluding those with renal cancer, polycystic kidney or incomplete data. The research program was approved by the Ethics Committee (2021-SR-036), and all patients signed the informed consent form.

[0024] Clinical characteristics, relevant laboratory parameters and comorbidities were retrospectively collected. Preoperative ultrasound or computed tomography was performed to determine the number, location and maximum diameter of the cysts. Hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg or receiving antihypertensive treatment; diabetes mellitus was defined as fasting blood glucose ≥ 7 mmol / L or receiving hypoglycemic treatment; chronic kidney disease (CKD) was defined as estimated glomerular filtration rate (eGFR) < 60 ml / min / 1.73m 2 ;

[0025] The formula for calculating the volume of the cysts was: volume = 4 / 3*π*(length / 2)*(width / 2)*(height / 2).

[0026] Tissue sections were obtained from the residual tissue wax preserved in the Department of Pathology, Yifu Hospital, Nanjing Medical University. Sections were cut into 4 μm sections. For immunohistochemistry and immunofluorescence staining, sections were de-waxed, rehydrated, antigen-repaired, blocked, and then incubated with primary and secondary antibodies according to standard protocols. The following antibodies were used: anti-GGPPS antibody (#sc-271680, Santa Cruz Biotechnology, USA). The percentage of staining results was analyzed using Image J software.

[0027] Selection of GGPS1 promoter single nucleotide polymorphisms (SNPs)

[0028] SNPs in the GGPS1 promoter were identified using data from northern Han Chinese (CHB) and southern Han Chinese (CHS) in the 1000 Genomes Project (https: / / www.internationalgenome.org / ). Next, tag SNPs that met the following quality control criteria were selected: minimum minor allele frequency (MAF) > 0.05, minimum genotype > 95%, Hardy-Weinberg equilibrium (HWE) P value > 0.001. Then, Haploview 4.2 software was used for pairwise linkage disequilibrium (LD) analysis (R 2 ≥ 0.8), and tag SNPs were selected. The reference allele and variable allele frequencies of East Asian SNPs based on the 1000 Genomes Project were obtained from dbSNP Overview (https: / / www.ncbi.nlm.nih.gov / snp / ).

[0029] SNP genotyping identification

[0030] Genomic DNA was extracted from the venous blood of all patients using the DNeasy Blood & Tissue Kit (Beijing Tiangen Biotech Co., Ltd.). The SNP genotyping was identified by quantitative real-time polymerase chain reaction (qRT-PCR) using the qRT-PCR kit of Shanghai Boyicheng Biotechnology Co., Ltd. The primer sequences were as follows: rs6688441, F1 5'-AGCCACAGCTTTTACCTTCTACAAA-3', F2 5'-AGCCACAGCTTTTACCTTCTACAAC-3', and R 5'-CAAATTAAAATTTGTAATACTTGTG-3'; rs3806394, F1 5'-TGATGAAAGAGAATCTTATTTATTA-3', F2 5'-TGATGAAAGAGAATCTTATTTATTG-3', and R 5'-CTAAAATTTTACAGTACTGTTCACA-3'.

[0031] Plasmid construction and luciferase reporter assay

[0032] The transcription factor FOXA1 can bind to the rs3806394 site, which was predicted by JASPAR software (https: / / jaspar.genereg.net / ). The recombinant plasmids pGL4.10-GGPS1 promoter (WT), pGL4.10-GGPS1 promoter (MUT), and FOXA1-plasmid were constructed by GenScript and Genechem Co., Ltd. in Shanghai, China. pGL4.10-Empty was used as a control. The luciferase reporter assay was used to determine the promoter activity difference caused by the GGPS1 gene rs3806394 allele C>T variation. The human renal tubular epithelial cell line HK2 was cultured in RPMI-1640 medium (Shanghai Thermo Fisher Scientific) containing 10% fetal bovine serum (Shanghai Saybio Biotech Co., Ltd.) and 1% penicillin-streptomycin at 37°C with a CO2 concentration of 5%. In the luciferase reporter assay, HK2 cells were seeded in a 12-well plate and transfected with 0.1 μg / ml of pGL4.10-GGPS1 promoter (WT) or promoter (MUT) plasmid at a confluence of 60% using Lipofectamine 2000 (Shanghai Thermo Fisher Scientific) for 48 h. Meanwhile, 0.1 μg / ml of FOXA1 plasmid and 0.01 μg / ml of Renilla plasmid were transfected. Then, the transfected cells were collected, and the firefly luciferase activity was detected using the Dual-Glo Luciferase Assay System (Beijing Promega Biotechnology). The Renilla luciferase activity was used as an internal control.

[0033] Statistical methods

[0034] Continuous variables with normal distribution were expressed as mean ± standard deviation and compared using t-test. Variables with skewed distribution were expressed as median and interquartile range and compared using non-parametric test. Categorical variables were expressed as number and frequency and compared using Chi-square test. The optimal cut-off value of GGPPS expression was determined using receiver operating characteristic (ROC) curve. Binary logistic regression was used for univariate and multivariate analysis, and the results were expressed as OR and 95% CI. All statistical tests were performed using SPSS Statistics 23.0 software (International Business Machines Corporation, New York, USA). Differences were considered statistically significant at a two-sided P < 0.05.

[0035] Results

[0036] Down-regulation of GGPPS expression in SRC tissues

[0037] In previous studies, the inventors found that Ggps1 gene deletion in mouse model of renal tubular epithelial cells could induce the formation of kidney cysts. In HK2 cells, knockdown of GGPSS could increase the proliferation ability of cells.

[0038] Firstly, the expression of GGPPS in SRC tissues and normal kidney tissues was detected by IF and IHC. GGPPS was specifically expressed in human renal tubular epithelial cells Figure 1 A).

[0039] It was also found that GGPPS staining in SRC tissues was significantly weaker than that in normal kidney tissues Figure 1 B). These results suggest that GGPPS may be involved in the development of SRC.

[0040] Low expression of GGPPS is associated with large volume SRC

[0041] To further detect the role of GGPPS in the development and size of SRC,

[0042] A total of 77 SRC patients were retrospectively collected. According to the median value of the volume of the largest cyst as the cut-off value, these patients were divided into the larger cyst group (n = 38) and the smaller cyst group (n = 39). The clinical characteristics of the patients are summarized in Table 1. Among them, 40 cases were male (51.9%), and 37 cases were 60 years old and above (48.1%). The median age was 59 years (21-78 years). The average age was 57.6 years. The median volume of the largest cyst was 86.7 cm 3 .

[0043] Patients with larger volume of the largest cyst in Table 1 were relatively older (P = 0.049). The larger the cyst, the lower the expression of GGPPS in SRC tissues (P = 0.007). There was no difference in gender, BMI, blood pressure, number and location of cysts, hypertension, diabetes, CKD, history of kidney stone disease, etc. between the two groups.

[0044] Low GGPPS expression increases the risk of large volume SRC

[0045] For the continuous variable of GGPPS expression in SRC tissues, the optimal cutoff value derived from the ROC curve divided the patients into low expression group and high expression group. Figure 2 ) Subsequently, the relationship between GGPPS expression in SRC tissues and cyst volume was further observed (Table 2).

[0046] In the univariate analysis, low GGPPS level significantly increased the risk of large cysts (OR = 4.853, 95% CI 1.777-13.250; P = 0.002).

[0047] In the multivariate model adjusted for age, gender, BMI, systolic blood pressure, diastolic blood pressure, cyst number and location, hypertension, diabetes, CKD and kidney stone history, GGPPS expression was still negatively correlated with cyst size (OR = 6.447, 95% CI 1.900-21.878; P = 0.003). The above results suggest that low GGPPS expression in SRC tissues is an independent risk factor for the enlargement of kidney cysts.

[0048] rs3806394 variation frequency in the promoter region of GGPS1 increased in SRC patients

[0049] To explore the potential possibility of GGPPS expression down-regulation, 6 venous blood samples from SRC patients were collected in the present invention, and the SNP located in the GGPS1 promoter was identified. 11 variable SNPs located in the GGPS1 promoter were identified from the 1000 Genomes Project. After LD analysis filtering, rs6688441 and rs3806394 were determined for further study Figure 3 A) The results showed that the rs6688441 phenotypes of CC, CA and AA were 4, 1 and 1 cases respectively (Table 3). The frequencies of C and A alleles were 0.75 and 0.25 respectively. At the same time, in the 1008 East Asian participants in the 1000 Genomes Project study, the frequency of A allele was 0.251.

[0050] The rs3806394 phenotypes of CC, CT and TT were 1, 1 and 4 cases respectively. The frequencies of C and T alleles were 0.25 and 0.75 respectively. However, in the 1008 East Asian participants reported in the 1000 Genomes Project study, the frequency of T allele was 0.228. This means that the variation frequency of rs6688441 in SRC patients is similar to that of East Asia, while the variation frequency of rs3806394 in SRC patients is significantly higher than that of East Asia. The variation of rs3806394 may lead to the down-regulation of GGPPS expression in SRC patients.

[0051] To demonstrate the effect of rs3806394 SNP on the activity of the GGPS1 promoter in renal tubular epithelial cells, a GGPS1 promoter reporter carrying the C (WT) or T (MUT) allele of the SNP was used and the expression of the reporter was evaluated. The transcription factor FOXA1 is predicted to bind to the rs3806394 site, and this binding would be affected by the T allele of the SNP Figure 3 B). It was also observed that HK2 cells transfected with the GGPS1 promoter (MUT) plasmid had lower luciferase activity than cells transfected with the GGPS1 promoter (WT) plasmid Figure 3 C). This result indicates that the T allele of rs3806394 is associated with reduced GGPS1 promoter activity in SRC patients.

[0052] The studies in the present application show that GGPPS is downregulated in SRC tissue, and low levels of GGPPS in SRC are an independent risk factor for SRC size. The reduction of GGPPS in the epithelium of the capsule membrane can promote the growth of SRC. In addition, it is reported for the first time that the frequency of the rs3806394 variation located in the GGPS1 promoter is increased in SRC patients.

[0053] The variation in the rs3806394 site reduces the activity of the GGPS1 promoter. This can be one of the reasons for the downregulation of GGPPS expression in most SRC patients.

[0054] Table 1. Analysis of the clinical characteristics of patients with simple renal cysts.

[0055]

[0056]

[0057] Abbreviations: BMI, Body mass index; CKD, Chronic kidney disease.

[0058] Table 2. Odds ratio of the correlation between GGPPS expression and the size of simple renal cysts

[0059]

[0060] Model 1 was not adjusted for any covariate;

[0061] Model 2 was adjusted for covariates including age, sex, BMI, systolic blood pressure, diastolic blood pressure, number and location of cysts, hypertension, diabetes, CKD, and history of kidney stones;

[0062] Abbreviations: OR, odds ratio; CI, confident interval; BMI, Body mass index; CKD, Chronic kidney disease.

[0063] Table 3. SNP genotype-phenotype of GGPS1 gene promoter in patients with simple renal cysts

[0064]

[0065] The content not described in detail in the specification belongs to the prior art known to the person skilled in the art.

[0066] The basic principles and main features of the present application and the advantages of the present application are shown and described above. The person skilled in the art should understand that the present application is not limited by the above examples, and the above examples and the description in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application.

[0067] The scope of protection of the present application is defined by the appended claims and their equivalents, therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the claimed present application, but only to represent selected embodiments of the present application.

[0068] Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor fall within the scope of protection of the present application.

[0069] The present application and its embodiments are described above, which is not restrictive, and the embodiments shown in the accompanying drawings are only one of the embodiments of the present application, and the actual structure is not limited thereto. In summary, if the person skilled in the art is inspired by it, without departing from the purpose of the present application, without creative design, similar structure and embodiments to the technical solution should belong to the protection scope of the present application.

Claims

1. Use of geranylgeranyl pyrophosphate synthase in simple renal cysts.

2. The use of a geranylgeranyl pyrophosphate synthase in simple renal cysts according to claim 1, characterized in that: Inhibits the proliferation of renal cyst epithelial cells by upregulating the expression or activity of geranylgeranyl pyrophosphate synthase.

3. Application of geranylgeranyl pyrophosphate synthase expression in the diagnosis of simple renal cysts.

4. The use of geranylgeranyl pyrophosphate synthase expression in the diagnosis of simple renal cysts according to claim 3, characterized in that: The expression of geranylgeranyl pyrophosphate synthase is downregulated in simple renal cysts.

5. Use of the geranylgeranyl pyrophosphate synthase GGPS1 promoter in reducing the expression of geranylgeranyl pyrophosphate synthase, characterized in that: The rs3806394 site mutation in the GGPS1 promoter region causes downregulation of geranylgeranyl pyrophosphate synthase expression.

6. A diagnostic reagent for simple renal cysts, characterized in that: The method comprises a combination of one or more of a GGPPS activity detection reagent and an rs3806394 typing primer.

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