Clinical Value of Plasma Humanin in Acute Kidney Injury
Conditions: Acute Kidney Injury
Sponsor: Guangdong Provincial People's Hospital

Title: Clinical Value of Plasma Humanin in Acute Kidney Injury
Date: Augest 1,2022

The research plan
1. Clinical Value of Plasma Humanin in Acute Kidney Injury
1.1 Study design
This study is a single-center cross-sectional study, observed and analyzed patients with acute and
chronic kidney disease from August 2022 to June 2024, according to the following criteria: adult
patients aged 18 years, both male and female; AKI patients: acute kidney injury patients meeting
the definition of KDIGO guidelines; control group: people from healthy examination, with normal
blood routine physical examination, urine routine, liver function, renal function, glycosylated
hemoglobin; patients had complete baseline medical records, including renal and kidney function,
urinary protein level, blood creatinine value; agreed to join the group and signed informed
consent;
Population was excluded by the following criteria: patient age <18 years; patient baseline data was
missing.
1.2 Relevant Definitions
According to the 2012 Global prognosis (KDIGO) criteria, acute kidney injury (Acute Kidney
Injury, AKI) was defined as: blood creatinine level increased 0.3 mg/dl (26.5 μ mol / L) or more
than 1.5 times or above the base value, and clearly or inferred to occur within 7d; or continuous 6h
urine volume <0.5ml kg- ¹ h- ¹;
1.3Data collection
Observational analysis of patients with acute kidney injury from August 2022 to June 2024 in our
hospital, and collected plasma from patients meeting the inclusion criteria and divided into AKI
group and control group
Clinical baseline data of patients meeting the inclusion criteria, including gender, age, blood
pressure, underlying diseases (hypertension, diabetes, hyperuricemia), serum creatinine, eGFR,
serum albumin, hemoglobin concentration, glycated hemoglobin, cholesterol, triglycerides,
high-density lipoprotein, low density lipoprotein, uric acid, blood phosphorus, blood calcium,
potassium, serum iron, 24h urinary protein / urinary albumin, urinary protein / urinary albumin
creatinine ratio.
2.plasma Humanin concentration detection
2.1 Samples to be taken
Patients who met the inclusion criteria were retained for 4ml of blood / sterile urinary urine with
an EDTA tube during hospitalization
Within 2h, after centrifugation (3000 r/min, 10min), take
the supernatant at 300-600 μ in the-80℃refrigerator.
2.2 Experimental reagent consumables and instruments
Human humanin peptide (MT-RNR 2) ELISA kit, centrifuge, EP tube, different specifications of
enzyme-free gun head, different specifications of pipete gun and exhaust gun, plasma water,
microplate reader, 4℃refrigerator, -20℃refrigerator, -80℃refrigerator, 37℃incubator, etc
2.3 Experimental method

（1）Reagent reheating: Transfer all reagents to room temperature (18-25C) to equilibrate for
at least 30 minutes
（2）Reagent equipped with:
①Standard: Remove a standard from the kit at 6000-10000rpm for 30 seconds. Dissolve
with 1ml sample dilution, pump the gun at the bottom of the cold storage tube for 5
times to dissolve the standard S7; reserve 71.5ml centrifuge tubes with different
concentrations (use the following concentrations: 1800,900,450,225,112.5,56.25,28,0 pg
/ ml);
②Washing liquid working liquid: the concentrated washing liquid should be diluted by
1:25 times with de-ionized water, and qualified before use;
③Biotin-labeled antibody working solution: biotin-labeled antibody solution is diluted
1:100 times and consummated within 10 minutes before use;
④
Horseradish peroxidase labeled avidin working solution: horseradish peroxidase
labeled avidin by 1; 100 times with horseradish peroxidase labeled avidin dilution, within
10 minutes before use;
⑤Sample dilution: Samples are diluted 1:5 times for testing;
（3）Add samples: set up standard holes and sample holes to be tested respectively. Add
100u1 to each well, shake the well gently, and warm 37C for 2 hours.
（4）Leave the liquid and dry without washing.
（5）100u1, covered with new plates, and incubated at 37C for 1 hour.
（6）Remove the liquid in the hole, throw it on, and wash the plate for 3 times. Soak for 2
minutes, 200u / each well and dry.
（7）Horseradish peroxidase-labeled avidin working solution was 100u1 per well, covered
with new plate paste and incubated at 37°C for 1 hour.
（8）Remove the liquid in the hole, shake it dry, and wash the plate for 5 times. Soak for 2
minutes, 200ul / per well and dry.
（9）Add substrate solution 90u1,37C for 15-30 min.
（10）The reaction was terminated by adding 50u1 of the termination solution per well.
（11）The optical density (OD) of each well was measured at 450nm within 5 minutes of the
reaction
（12）Calculated
concentration:
According
to
the
standard
concentration
and
the
corresponding OD value, the standard curve of Humanin is analyzed by Curve Expert 1.4
software, and the concentration of Humanin to be measured in the sample is calculated by using
the equation of the standard curve.
3. Statistical methods
SPSS software. Measurement data following normal distribution are described by (x ± s) and t
test was used for comparison between groups. Count data are represented by frequency,
percentage (%), and chi-square test. The receiver working characteristic curve (ROC) was drawn
to analyze the diagnostic value of Humanin for AKI. A P <0.05 was considered as a statistically
significant difference.