阅读说明：本技术 一种肾功能损害风险降低的方法及应用 (Method for reducing risk of renal function damage and application ) 是由 王滨燕 秦献辉 张磊 王念 邢厚恂 于 2020-05-14 设计创作，主要内容包括：本发明提供一种待测个体是否接受降压药与叶酸的联合治疗以降低肾功能损害的风险的方法及其应用；本发明还涉及用于待测个体是否接受上述联合治疗的试剂盒。本发明通过特定实验室检测项目来确定高血压患者的更佳用药方案,即实行个体化用药,从而达到提高临床疗效的目的。(The invention provides a method for reducing the risk of renal function damage by judging whether a subject to be tested receives combined treatment of antihypertensive drugs and folic acid and application thereof; the invention also relates to a kit for determining whether an individual is to receive the combination therapy. The invention determines the better medication scheme of the hypertension patient through a specific laboratory detection project, namely, the individual medication is carried out, thereby achieving the purpose of improving the clinical curative effect.)

1. Use of the following agents (a) or (b) in the manufacture of a kit for determining whether a test individual is to receive a blood pressure lowering drug in combination with folic acid to reduce the risk of renal impairment:

the reagent (a) is used for detecting the concentration of alkaline phosphatase in a plasma or serum sample of the subject to be detected, wherein if the concentration of the alkaline phosphatase in the sample is more than or equal to 110IU/L, the subject to be detected is indicated to receive the combined treatment of the antihypertensive drug and folic acid;

the reagent (b) is used for determining the neutrophil count in the blood sample of the individual to be tested, wherein the neutrophil count of the sample is more than or equal to 4.6 multiplied by 10⁹and/L indicates that the individual to be tested receives the combined treatment of the antihypertensive drug and folic acid.

2. The use of claim 1, wherein the impairment of renal function is an estimated significant or sustained decrease in glomerular filtration rate.

3. Use of an agent (a) for the manufacture of a kit for determining whether a test individual not associated with diabetes is to receive a blood pressure lowering drug in combination with folic acid to reduce the risk of renal dysfunction:

the reagent (a) is used for detecting the concentration of alkaline phosphatase in a plasma or serum sample of the subject to be tested, wherein the alkaline phosphatase concentration of the sample is more than or equal to 110IU/L, and the subject to be tested is indicated to receive the combined treatment of the antihypertensive drug and folic acid.

4. Use of the following agents (a) or (b) in the manufacture of a kit for selecting a suitable control regimen for renal dysfunction in a test subject undergoing combination therapy with a hypotensive agent:

the reagent (a) is used for detecting the concentration of alkaline phosphatase in a plasma or serum sample of the individual to be detected, wherein if the concentration of the alkaline phosphatase in the sample is more than or equal to 110IU/L, the individual to be detected is instructed to continue to receive the combined treatment of the antihypertensive drug and folic acid;

the reagent (b) is used for detecting the neutrophil count in the blood sample of the individual to be detected, wherein the neutrophil count of the sample is more than or equal to 4.6 multiplied by 10⁹and/L indicates that the individual to be tested continues to receive the combined treatment of the antihypertensive drug and folic acid.

5. The use according to any one of claims 1 and 3 to 4, wherein the combination therapy is a co-therapy of a hypotensive agent and folic acid.

6. The use of any one of claims 1 and 3 to 4, wherein the subject has essential hypertension.

7. The use according to any one of claims 1 and 3 to 4, wherein the impairment of renal function is primarily a complication caused by essential hypertension.

8. The use of any one of claims 1 and 3-4, wherein the subject is at risk for developing or has progressed on impaired renal function.

9. The use of any one of claims 1 and 3 to 4, wherein the kit is used to determine whether the subject is to receive or continue the combination therapy.

10. The control population of claim 9 wherein the individuals predicted to be tested to receive combination therapy are from a population of essential hypertension patients who have no history of major cardiovascular events and who are receiving a hypotensive drug therapy.

11. A kit for predicting whether a test subject will receive a combination therapy of a hypotensive agent and folic acid based on plasma or serum alkaline phosphatase concentration, wherein the kit comprises:

1) an alkaline phosphatase standard; 2) an alkaline phosphatase control; 3) biotin-labeled anti-TXA 2 antibodies; 4) affinity streptomycin-HRP; 5) dilution buffer, washing buffer.

12. The kit of claim 11 for predicting whether a test subject will receive said combination therapy, wherein the concentration of alkaline phosphatase in a sample from the test subject is determined using enzyme-linked immunosorbent assay.

13. A kit for predicting whether a subject is to be treated with a combination of a hypotensive agent and folic acid based on a whole blood neutrophil count, wherein the kit comprises:

1) diluting the solution; 2) hb hemolysin; 3) cleaning fluid; 4) and (3) an anticoagulant.

14. The kit of claim 13, for determining whether a test individual is receiving the combination therapy, wherein a blood routine test (hematology analysis) is performed using an automated hematology analyzer to determine the neutrophil count in a sample from the test individual.

Technical Field

The invention relates to a method for detecting activity of alkaline phosphatase (ALP) and neutrophil count and a new application thereof, which are used for predicting whether a to-be-detected object receives combined treatment of antihypertensive drugs and folic acid to reduce the risk of renal function damage, and belong to the field of medicine.

Background

Essential hypertension is a polygenic hereditary disease which is influenced by genetic background and environmental factors, and is a risk factor for the morbidity and the mortality of cardiovascular and cerebrovascular diseases. The kidney is one of the major target organs for pathological damage of hypertension, and thus a common complication of hypertension includes Chronic Kidney Disease (CKD). Epidemiological data show that the number of people who enter renal function damage due to hypertension tends to increase year by year. End-stage renal disease (ESRD), which evolves gradually from CKD, may be one of the major factors leading to increased mortality in hypertensive patients. Research suggests that the deterioration of indexes such as glomerular filtration rate, urinary microalbumin, insulin resistance and the like is independently related to renal function damage of a hypertensive, and is also a main risk factor of death of the hypertensive due to complications.

The clinical manifestations of renal damage caused by hypertension are mainly proteinuria and glomerular function impairment, most patients show glomerular filtration rate reduction and microalbuminuria in the early stage, renal tubular function impairment is usually seen in the later stage, renal pathological changes are usually shown as renal arteriole or renal parenchyma impairment, benign arteriolar nephrosclerosis is mainly taken, and the occurrence of renal damage is positively correlated with the severity and duration of hypertension. Generally, untreated hypertension, which lasts for 5-10 years, causes renal arteriolosclerosis, thickening of the vessel wall, narrowing of the vessel lumen, and thus, stimulation of ischemic damage to the renal parenchyma. Studies have shown that a significant increase in ESRD risk occurs when systolic blood pressure is increased from 120mmHg to 130 mmHg.

Enalapril is a commonly used blood pressure lowering drug, belongs to angiotensin converting enzyme inhibitors, and is hydrolyzed into enalaprilat in the liver after oral administration to play a role. The latter has over 8 times of inhibiting effect on angiotensin converting enzyme. Enalapril can also lower blood pressure, maintain myocardial contractility, and has no influence on cardiac output, so as to relieve cardiac preload and afterload and improve cardiac function. Enalapril is one of the representative medicaments for clinically treating hypertension, can also increase renal blood flow, and has no obvious influence on blood sugar, uric acid and cholesterol metabolism.

Enalapril folic acid tablet (Eye for short) is a new class 1.5 compound medicine, and is composed of two active components of enalapril and folic acid, wherein the folic acid is used for reducing the concentration of homocysteine in blood plasma or supplementing the shortage of folic acid in the body. The present inventors participated in a clinical study (Huo Y, Li J, Qin X, et al. effectiveness of clinical therapy in primary preservation of stroke amplitude with hypertension in China: the CSPPT randomised clinical trial. JAMA 2015; 313:1325-35.) and found that in more than 2 ten thousand subjects with hypertension in China, 10.8mg of Eben (containing 10mg of Enalapril and 0.8mg of folic acid) was taken per day, and the average treatment period was 4.5 years, and as a result, it was found that Eben could further reduce the risk of stroke by 21% compared with 10mg of Enalapril taken alone. It was also found in this study that the use of Eye helps target organ protection, including improvement of renal function, in hypertensive patients.

In view of the above research results, the long-term combination of antihypertensive drug and folic acid for treating hypertension can further exert the advantage of target organ protection, and is also suitable for patients with concurrent renal impairment, and is expected to reduce the risk of occurrence and progression of renal impairment in hypertensive patients. However, this clinical value finding is derived from the above-mentioned rigorously designed clinical trial studies, with actual efficacy varying among individuals in the "real world" population of hypertensive patients. It would be of great importance to individualized medical practice if such differences could be ascertained by some means, i.e., to predict the magnitude of the renal benefit of a hypertensive individual receiving a combination of a hypotensive drug and folic acid, to determine whether the individual is eligible for such combination, or to determine the patient population characteristics (certain parameters) that would be most clinically beneficial.

Alkaline phosphatase (ALP) belongs to phosphomonoesterase, is a group of specific phosphoesterases, is widely distributed in human tissues and body fluids, has high content in bones, livers, mammary glands and small intestines, is mostly generated by bone cells, and is discharged into intestinal tracts from the livers to detect the ALP of serum clinically, and is mainly used for auxiliary diagnosis of diseases of the livers, the gallbladders and the bone tissues; neutrophil is one of the leukocyte classifications in circulating blood, and neutrophil count is mainly used clinically for diagnosis or auxiliary diagnosis of bacterial infectious diseases, inflammation, granulocytic leukemia or agranulocytosis. The inventor unexpectedly finds that the activity level of the blood ALP and the neutrophil count can predict the protective effect of the combined treatment of the antihypertensive drug and the folic acid on the kidney target organ of the hypertensive, so that the medicine can be used for guiding the individualized medication of the hypertensive and is a significant improvement of the prior technical scheme.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects of the existing theory and technology of clinical combined treatment of antihypertensive drugs and folic acid and providing a new method for predicting the curative effect of the kidney target organ protection of the hypertension patients by applying the combined treatment.

According to the invention, after-study analysis of a kidney subgroup study of a Chinese cerebral apoplexy first-level prevention test (CSPPT), a certain cut-off value of a baseline ALP level and a neutrophil count is found to be closely related to proteinuria and renal function damage occurrence or progress of a hypertensive patient after the combination of a blood pressure lowering drug and folic acid. The novel application of the ALP level and the neutrophil count in laboratory detection for predicting the prevention or improvement effect of renal function damage after the hypertension patient receives combined treatment of antihypertensive drugs and folic acid means that whether the patient can receive the combined treatment or not is judged by detecting the ALP level and the neutrophil count of the hypertension patient, a reasonable medication suggestion is given, the individualized treatment is guided, and the purposes of improving the clinical curative effect and improving the life quality of the patient are achieved.

In one aspect, the invention provides a method for evaluating the effect of the combination therapy of ALP and neutrophils on the prevention or improvement of renal function damage by applying antihypertensive drugs and folic acid to an individual to be tested; in another aspect, the present invention also provides the use of an agent for detecting ALP levels, neutrophil counts, in the manufacture of a test agent or test system for predicting the clinical efficacy of a combination therapy.

According to a specific embodiment of the present invention, the subject to be tested is a patient with essential hypertension.

According to an embodiment of the present invention, when the ALP level or the neutrophil count is increased in a sample from a test subject, the renal protection effect of the test subject using the antihypertensive drug in combination with folic acid is better.

According to a specific embodiment of the present invention, the level of ALP is serum, plasma level of ALP.

According to an embodiment of the present invention, the method for detecting the level of ALP in the present invention may be any feasible method known in the art, for example, an enzyme-linked immunosorbent assay, a PNP microplate assay, a PNP colorimetric assay, a disodium phenyl phosphate microplate assay, a disodium phenyl phosphate colorimetric assay.

According to a particular embodiment of the invention, the neutrophil count refers to the count of neutrophils in the circulating blood.

According to a specific embodiment of the present invention, the method for detecting neutrophil count in the present invention may be any feasible method known in the art.

According to a specific embodiment of the present invention, when plasma or serum ALP is not less than 110IU/L or neutral granulocyte count is not less than 4.6X 10⁹And when the blood pressure of the patient is/L, the patient to be tested is recommended to receive the combined treatment of the antihypertensive drug and the folic acid, and the prevention or improvement effect on the renal function damage of the hypertensive is expected to be better.

In another aspect, the present invention provides a detection system (detection apparatus) for guiding a subject to be tested whether to receive a combination therapy of an antihypertensive drug and folic acid, the detection system comprising:

a detection unit including reagents for detecting ALP level and neutrophil count;

and the analysis unit is used for analyzing the detection result of the detection unit and evaluating whether the individual to be detected receives the combined treatment.

According to the specific embodiment of the invention, the detection system for evaluating whether the test individual receives the combination therapy of the antihypertensive drug and the folic acid is used for evaluating the effect of the test individual on reducing the risk of renal function damage when the test individual receives the combination therapy of the antihypertensive drug and the folic acid according to the ALP level and the neutrophil count in a sample from the test individual. Wherein elevated ALP levels and increased neutrophil counts are present in a sample from a test subject eligible to receive such combination therapy.

In a specific embodiment of the present invention, the present invention is used in a test system for evaluating whether a subject to be tested receives a combination therapy of an antihypertensive drug and folic acid, and the analysis unit performs the analysis evaluation according to the following operations (evaluation principle):

when serum or plasma ALP level is not less than 110IU/L and neutrophil count is not less than 4.6 × 10⁹On the condition of/L, the individual to be tested is recommended to receive antihypertensive drugs and folic acidThe medicine has better effect of preventing or improving the renal function damage of the hypertension patient by combined treatment.

The detection system for evaluating whether the individual to be detected receives the combined treatment of the antihypertensive drug and the folic acid can be a virtual device as long as the functions of the detection unit and the analysis unit can be realized. The detection unit can comprise various detection reagents, kits or detection instruments; the data analysis unit may be any operation instrument, module or virtual device capable of analyzing and processing the detection result of the detection unit to obtain the effect of the combined therapy of the antihypertensive drug and folic acid, for example, a data chart which is convenient for comparison and reference is prepared by the ALP level, the neutrophil count and whether the corresponding individual receives the combined therapy of the antihypertensive drug and folic acid according to the above evaluation principle in advance, and the reasonable prediction of whether the individual to be detected receives the combined therapy can be obtained by comparing the detection result of the detection unit with the data chart.

In another aspect, the present invention provides a method for predicting whether a subject is treated with a combination of a hypotensive agent and folic acid, the method comprising: detecting ALP level detection and neutrophil count in an individual (which may be a sample from an individual to be tested); whether the individual receives the combination therapy is evaluated according to the ALP level and the neutrophil count of the individual.

In conclusion, the invention takes the detection of the blood ALP level and the neutrophil count as a new method and application for predicting whether the hypertension patient receives the combination therapy of the antihypertensive drug and the folic acid to reduce the occurrence risk of renal function damage, and provides a basis for individualized medical treatment of the hypertension patient in clinical practice.

Detailed Description

EXAMPLE 1 analysis of the relationship between plasma ALP levels and renal function impairment in hypertensive patients treated with antihypertensive drugs in combination with folic acid

The method comprises the following steps:

A. chinese cerebral apoplexy first-level prevention study (CSPPT) and kidney subgroup study:

CSPPT is a random double-blind control test, which is implemented in 32 communities of Anhui and Jiangsu provinces in China in 2008-2013, and is incorporated into 20702 male and female hypertension patients with the age of 45-75 years, wherein the hypertension is defined as the systolic pressure of a sitting position being more than or equal to 140mmHg and/or the diastolic pressure being more than or equal to 90 mmHg. Patients were treated with a 1: the ratio of 1 was randomly assigned to one of 2 treatment groups: 1 tablet taken orally per day of a compound tablet (Egyptian group) containing 10mg of enalapril and 0.8mg of folic acid, or 1 tablet taken orally per day of a single drug tablet (Enalapril group) containing only 10mg of enalapril; follow-up every 3 months for the patients; the use of other antihypertensive drugs (mainly calcium channel blockers or diuretics) was allowed at the same time during the trial, but the use of B vitamins was not allowed. The results after an average of 4.5 years of treatment showed that the Eyela group reduced the risk of first stroke by 21% compared to the enalapril group.

The CSPPT Kidney Subsubgroup study included 15104 patients in the CSPPT cohort with the standard for eGFR (estimated glomerular filtration rate) of not less than 30mL/min/1.73m²Of these 1671 cases, renal function impairment was observed. As a result, it was found that the probability of progression of renal function impairment did not differ significantly in the Eyela group compared to the enalapril group in the total population, but eGFR was found at baseline<60mL/min/1.73m²Or proteinuria patients, treatment with added folic acid significantly reduced the rate of progression of renal impairment by as much as 55%.

Posterior analysis of CSPPT Kidney study

The CSPPT renal subgroup study was followed by post hoc analysis with a baseline ALP test for 12734 patients with a median treatment period of 4.4 years. These patients were from 20 rural communities (areas with low folate levels) in Jiangsu province.

And (3) laboratory detection: (1) patient serum and urine samples were collected at baseline and at the time of exit visit, sent to the Guangzhou national Kidney disease clinical research center laboratory, and serum creatine, homocysteine (Hcy), blood lipids, and fasting glucose were measured using a fully automated biochemical analyzer (Beckman Coulter). The eGFR was calculated using the renal impairment epidemiological co-ordination (renal impairment-EPI) equation. (2) ALP detection: 1) after the blood sample is kept still for 4 hours, the blood sample is centrifuged for 5 minutes at the normal temperature at 6000 revolutions, and the supernatant is taken. 2) The kit comprises an ELISA plate, a plastic film cover plate, a standard substance, a reference substance, a standard substance dilution buffer solution, a biotin-labeled anti-TXA 2 antibody, an affinity streptomycin-HRP, a washing buffer solution, a substrate A, a substrate B, a stop solution and a specimen dilution solution. 3) The detection method comprises the following steps: the solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) is characterized in that a standard substance with known concentration and a sample with unknown concentration are added into a microporous ELISA plate for detection. The test substance and the biotin-labeled antibody are incubated simultaneously. After washing, avidin-labeled HRP was added. After incubation and washing, unbound enzyme conjugate is removed, and then substrate A, B is added and acted upon simultaneously with the enzyme conjugate. A color is produced. The shade of the color is proportional to the concentration of the substance to be detected in the sample.

The ending index: (1) the main outcome indexes are as follows: progression of renal impairment, defined as a decrease in eGFR of 30% or greater, or a baseline eGFR of 60mL/min/1.73m or greater²When the assembly is finished, the temperature is reduced to<60mL/min/1.73m²(ii) a Or if the baseline eGFR<60mL/min/1.73m²When the group is formed, the eGFR is reduced by more than or equal to 50 percent; or renal failure (eGFR)<15mL/min/1.73m²) (ii) a Or require dialysis. (2) Secondary outcome indicator: 1) renal function impairment occurs, defined as a baseline eGFR ≥ 60mL/min/1.73m²When out of group eGFR<60mL/min/1.73m²And the annual rate of decline of eGFR>1mL/min/1.73m²(ii) a 2) The rapid decrease of eGFR is defined as the average decrease of eGFR per year being more than or equal to 5mL/min/1.73m²(ii) a 3) The annual rate of decline of eGFR, estimated asWhere t is the time of year the group was detected from baseline.

And (3) data analysis: treatment groups calculated mean ± standard deviation and ratio of population characteristics based on baseline ALP bipartite (cut at 110 IU/L). By t-test or X²Testing for differences in overall characteristics, Logistic or linear regression models were used to assess the effect of baseline ALP bipartite on the association between hypotensive plus folate therapy and primary or secondary kidney outcome, confounders including age, sex, body mass index, smoking, alcohol consumption, albumin-corrected calcium, phosphate, uric acid, eGFR, systolic blood pressure, fasting blood glucose levels, total cholesterol levels, proteinuria, treatment periodUse of meta Calcium Channel Blockers (CCBs) and diuretics, etc.; the interaction of ALP and folate was examined by incorporating the interaction term into a multivariate correction model. P<0.05 was considered statistically significant and all statistical analyses used R software (version 3.4.3).

(II) results:

first, in patients with higher baseline ALP levels (≧ 110IU/L), treatment with Etopone was significantly effective in both preventing progression of renal impairment (OR, 0.54; 95% CI, 0.35-0.85) and reducing incidence of renal impairment (OR, 0.62; 95% CI, 0.42-0.91) compared to enalapril, indicating that there was significant interaction between ALP levels and Etopone treatment (see Table 1).

TABLE 1 Effect of baseline serum ALP levels (<110 or ≧ 110IU/L) on the onset or progression of renal function impairment in two groups of patients

^*Adjusted for age, sex, body mass index, smoking, drinking, albumin corrected calcium, phosphate, uric acid, total cholesterol, eGFR, systolic blood pressure, proteinuria, diabetes, CCB during treatment, and diuretic use at baseline.

This analysis only included baseline eGFR ≧ 60mL/min/1.73m²Of the participant(s).

Second, in patients with higher baseline ALP levels (> 110IU/L), treatment with Etopodium was more effective than enalapril in preventing the rapid decline in eGFR (OR, 0.63; 95% CI, 0.36-1.11) but in preventing the rate of decline in eGFR (%) (OR, -0.30; 95% CI, -0.55-0.06) (see Table 2).

TABLE 2 Effect of baseline serum ALP levels on eGFR in two groups of patients

^*Adjusted for age, sex, body mass index, smoking, drinking, albumin corrected calcium, phosphate, uric acid, total cholesterol, eGFR, systolic blood pressure, proteinuria, diabetes, CCB during treatment, and diuretic use at baseline.

Diabetes is also an important risk factor for renal function impairment. Therefore, we performed a hierarchical analysis of the presence of diabetes, and the results indicated that: when the patient is not accompanied by diabetes and the baseline ALP level is higher (more than OR equal to 110IU/L), the effect of enalapril alone and epeiba treatment on renal function impairment is obviously different (OR, 0.49; 95% CI, 0.28-0.83); when the patient had diabetes and the baseline ALP levels were higher (. gtoreq.110 IU/L), there was no significant difference in the effect of enalapril alone versus enalapril alone on the impairment of renal function (OR, 0.66; 95% CI, 0.29-1.50) (see Table 3).

TABLE 3 Effect of baseline ALP levels on the onset or progression of renal function impairment in patients (not accompanied by diabetes)

Adjustment based on baseline age, gender, body mass index, smoking, drinking, albumin-corrected calcium, phosphate, uric acid, total cholesterol, eGFR, Systolic Blood Pressure (SBP), time-averaged SBP, proteinuria, diabetes, Calcium Channel Blocker (CCB) during treatment, and diuretic use

Insufficient folate intake is a ubiquitous phenomenon in most countries (including china) where folic acid is not mandatory for food addition. Research proves that the low folate level or the high Hcy level of blood in a human body is a risk factor for cardiovascular diseases on average, folic acid supplementation not only can compensate folic acid deficiency in the body, but also can obviously reduce the Hcy level of the blood, and folic acid has the effects of resisting oxidation, improving endothelial function, enhancing oxidative stress and the like, so kidney injury can be relieved or prevented, and the risk of renal function injury is reduced.

Example 2 analytical study of the relationship between neutrophil count and renal function impairment in hypertensive patients taking Eye for a prolonged period of time

The method comprises the following steps:

A. chinese cerebral apoplexy first-level prevention study (CSPPT) and kidney subgroup study:

(same as example 1, but not shown)

Posterior analysis of CSPPT Kidney study

The CSPPT renal subgroup study was followed post hoc and was analyzed on a baseline neutrophilic granulocyte count assay in a total of 9448 (4678 in the egyptian group and 4770 in the enalapril group) patients from 20 rural communities (lower folate levels) in Jiangsu province with a median course of treatment of 4.4 years.

And (3) laboratory detection: (1) patient serum and urine samples were collected at baseline and at the time of exit visit, sent to the Guangzhou national Kidney disease clinical research center laboratory, and serum creatine, Hcy, blood lipids, blood glucose, etc. were measured using a fully automated biochemical analyzer (Beckman Coulter). The eGFR was calculated using the renal impairment epidemiological co-ordination (renal impairment-EPI) equation. (2) And (3) detecting the neutrophil count: blood samples are taken, either by finger tip bleeding or by elbow vein bleeding. Gently shake the blood to mix it with the anticoagulant. The ratio of the sample amount of the blood examination to the anticoagulant is proper. Then, an automatic blood analyzer was used for routine blood tests (hematology analysis), and the kit composition included diluent (Sandem sodium chloride 6.38g/L, boric acid 1.0g/L, sodium tetraborate 0.2g/L, EDTA-2K0.2g/L), Hb hemolysin (SULFOLYSER (sL S-200A), sodium lauryl sulfate 1.7g/L), cleaning solution (SANDEM CELLCLEAN, STACCLEANER-SYS, hypochlorite 5%), anticoagulant (KEDTA.2H0(1.5-2.2mg per ml blood)).

The ending index: (1) the main indexes are as follows: progression of renal impairment, defined as a decrease in eGFR of 30% or greater, or a baseline eGFR of 60mL/min/1.73m or greater²When the assembly is finished, the temperature is reduced to<60mL/min/1.73m²(ii) a Or if the baseline eGFR<60mL/min/1.73m²When the group is formed, the eGFR is reduced by more than or equal to 50 percent; or renal failure (eGFR)<15mL/min/1.73m²) (ii) a Or need to penetrateAnd (6) analyzing. (2) Secondary indexes are as follows: 1) the rapid decrease of eGFR is defined as the average decrease of eGFR per year being more than or equal to 5mL/min/1.73m²(ii) a 2) The annual rate of decline of eGFR, estimated asWhere t is the time of year the group was detected from baseline.

And (3) data analysis: treatment groups were bi-fractional based on baseline neutrophil counts (4.6X 10 split value)⁹/L) calculating the mean + -standard deviation and ratio of population features. By t-test or X²Testing for differences in overall characteristics, Logistic or linear regression models were used to assess the effect of baseline neutrophil count dichotomies on the association between hypotensive treatment with additional folate supplementation and primary or secondary kidney outcome, confounders including age, gender, body mass index, smoking, alcohol consumption, uric acid, albumin, eGFR, proteinuria, systolic blood pressure, fasting plasma glucose, total cholesterol, triglycerides, high density lipoprotein cholesterol, folic acid, vitamin B₁₂Hcy, systolic pressure, average systolic pressure, etc.; the interaction of neutrophil count and folate was examined by incorporating the interaction term into a multivariate correction model. P<0.05 was considered statistically significant and all statistical analyses used R software (version 3.4.3).

(II) results:

first, the neutrophil count was high (. gtoreq.4.6X10)⁹/L), as compared to enalapril alone, the treatment and prevention of progression of renal function impairment (OR, 0.44; 95% CI, 0.26-0.77) (see table 4).

TABLE 4 Effect of Baseline neutrophil count on the progression of renal function impairment in two groups of patients

^*Adjusting: age, sex, body mass index, smoking, fasting plasma glucose, total cholesterol, triglycerides, high density lipoprotein cholesterol, albumin, eGFR, proteinuria, uric acidFolic acid, vitamin B₁₂Hcy, systolic pressure, mean systolic pressure.

Secondly, the neutrophil count was high (. gtoreq.4.6X10)⁹/L), as compared to enalapril alone, the treatment with ependrox prevents and delays a rapid decrease in eGFR (OR, 0.65; 95% CI, 0.47-0.91) (see table 5).

TABLE 5 Effect of baseline neutrophil counts on eGFR in two groups of patients

^*Adjusting: age, sex, body mass index, smoking, fasting plasma glucose, total cholesterol, triglycerides, high density lipoprotein cholesterol, albumin, eGFR, proteinuria, uric acid, folic acid, vitamin B₁₂Hcy, systolic pressure, mean systolic pressure.

Example 3 analysis of the relationship between neutrophil count and New albuminuria in hypertensive patients taking Eye for a Long period of time

The method comprises the following steps:

A. chinese cerebral apoplexy first-level prevention study (CSPPT) and kidney subgroup study:

(same as example 1, but not shown)

Posterior analysis of CSPPT Kidney study

The CSPPT renal subgroup study was followed by post hoc analysis with baseline neutrophil counts and in-and out-of-group cases with urine protein determinations, with 8208 (4101 in the leaf group and 4107 in the enalapril group) patients from 20 rural communities (with lower folate levels) in Jiangsu province for a median treatment period of 4.4 years.

And (3) laboratory detection: (1) patient blood and urine samples were collected at baseline and at exit visit (group), sent to the Guangzhou national Kidney disease clinical research center laboratory, serum creatine, Hcy, blood lipids, blood glucose, etc. were measured using a fully automated biochemical analyzer, eGFR was calculated using the renal impairment epidemiological cooperation (renal impairment-EPI) equation, and proteinuria was determined using the protein dipstick method. (2) And (3) detecting the neutrophil count: the same as in example 2.

The ending index: new proteinuria, i.e. change of negative at baseline to positive at group exit (at least 1 + indicated by dipstick)

And (3) data analysis: treatment groups were bi-fractional based on baseline neutrophil counts (4.8X 10 split value)⁹/L) calculating the mean + -standard deviation and ratio of population features. By t-test or X²The difference in overall characteristics was compared and Logistic or linear regression models were used to assess the effect of baseline neutrophil count bipartite on the association between the addition of folate therapy in addition to hypotension and outcome measures, confounding factors including age, gender, Body Mass Index (BMI), smoking, drinking, uric acid, Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), fasting plasma glucose, Total Cholesterol (TC), Triglycerides (TG), high density lipoprotein cholesterol (HDL-C), eGFR, folic acid, Hcy, etc. P<0.05 was considered statistically significant and all statistical analyses used R software (version 3.4.3).

(II) results:

the subject population stratified by the baseline neutrophil count bipartite number is characterized below in table 6. As can be seen from the table, the characteristics of the crowd between the enalapril group and the empyema group are basically consistent and have comparability in the baseline state; after treatment, compared with the enalapril group, the blood folic acid level of the etifolin group is obviously increased, the Hcy level is obviously reduced, and the effect of supplementing folic acid is obvious.

TABLE 6 cut-off values for neutrophils: (<4.8×10⁹/L) stratified subject population characteristics

Proteinuria, which is rare and often indicative of early renal function impairment, is higher in neutrophil counts (. gtoreq.4.8X 10) as seen by statistical analysis in Table 7⁹/L), new proteinuria events were less frequent in the group of leafy (folate supplemented) than in the group of enalapril alone (OR, 0.53; 95% CI, 0.32-0.88, P ═ 0.044), adjustmentThis effect is more pronounced after other interference factors (OR, 0.48; 95% CI, 0.29-0.82, P ═ 0.033). The results show that hypertension patients with higher baseline neutrophil counts can prevent renal function impairment by receiving combined treatment of the antihypertensive drug and the folic acid, and have more clinical benefits compared with the antihypertensive drug alone.

TABLE 7 relationship of baseline neutrophil counts to fresh proteinuria in two groups of patients

Note: adjustment factors include: group, age, gender, body mass index, smoking, fasting plasma glucose, total cholesterol, triglycerides, eGFR, uric acid, folic acid, Hcy, Systolic Blood Pressure (SBP), mean SBP during treatment, etc.