Study objectives

The present study adopts a randomized controlled clinical trial design, strictly adhering to the principles of the Helsinki Declaration and standards of clinical trial research. Prior to the inclusion of each participant, research personnel systematically introduce the study’s objectives, procedures, and potential risks. Written informed consent is obtained from each participant before the commencement of the study. The research protocol has been reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Jinan University.

In this randomized controlled clinical trial targeting patients with type 2 diabetes, the study participants underwent at least 12 weeks of stable baseline treatment before enrollment, ensuring the homogeneity and reliability of the study. This baseline treatment comprised three aspects. Firstly, diabetes education was provided to emphasize the importance of a diabetes diet, regular exercise, regular blood glucose monitoring, and consistent medication adherence to ensure the self-management capacity of the patients throughout the entire study period. Secondly, based on the patient’s blood glucose levels, an appropriate hypoglycemic regimen was selected, consisting of a stable dose of a single medication or a combination of two medications(Górriz et al. 2015) (including insulin, metformin, α-glucosidase inhibitors, or insulin secretagogues, but excluding hypoglycemic medications that may have independent renal protective effects such as empagliflozin, thiazolidinediones, GLP-1 receptor agonists, DPP-4 inhibitors, or SGLT2 inhibitors). The target for blood glucose control was a fasting blood glucose level between 4.4 mmol/L and 7.0 mmol/L and a postprandial blood glucose level of ≤ 10.0 mmol/L after 2 h. For at least 4 weeks prior to enrollment, the type and dose of oral hypoglycemic drugs were kept unchanged, and if the patient was using insulin, the total insulin dose was adjusted by ≤ 10%. Lastly, for patients with comorbid hypertension and dyslipidemia, dietary guidance was provided, and antihypertensive and lipid-lowering drugs were used for treatment. ACE inhibitors or ARBs were the preferred options for blood pressure control, and if blood pressure control was inadequate despite using the maximum tolerated dose, other antihypertensive drugs, except diuretics, were added. For patients with dyslipidemia, dietary guidance or combination therapy using lipid-lowering drugs was implemented, with statins used for elevated cholesterol levels and fibrates for elevated triglyceride levels, and both drugs used if necessary. The blood pressure and lipid control targets were based on the 2017 edition of “Guidelines for the Prevention and Treatment of Type 2 Diabetes Mellitus in China”. For at least 4 weeks prior to enrollment, the aforementioned types and doses of oral medication remained unchanged.

Through this study, we hope to further explore the potential of SGLT2 inhibitors in early tubular protection, providing scientific support and new treatment strategies for the early prevention and treatment of DKD.

Inclusion and exclusion criteria

Screening will be conducted on type 2 diabetes patients who received basic treatment at the outpatient or inpatient department of The First Affiliated Hospital of Jinan University from July 2019 to July 2023.

Inclusion criteria

(1) Glycated hemoglobin level between 6.5% and 9.0%. (2) UACR less than 30 mg/g and eGFR equal to or greater than 60 ml/min/1.73 m2. (3) Meeting the diagnostic criteria for diabetes according to the 1999 World Health Organization (WHO) guidelines. (4) Age above 18 and below 70 years. (5) No alcohol or drug dependency, no severe psychiatric or intellectual impairments. (6) Obtaining informed consent and cooperation from the patient and their family, with the signing of an informed consent form.

Exclusion criteria

(1) Significant increase in fasting blood glucose (≥ 11.1 mmol/L) or substantial rise in blood pressure (≥ 180/110 mmHg) after initial treatment, with evident clinical symptoms of hyperglycemia or hypertension. (2) Severe hyperlipidemia (low-density lipoprotein cholesterol ≥ 4.9 mmol/L or total cholesterol ≥ 7.2 mmol/L or triglycerides ≥ 5.6 mmol/L) or severe hyperuricemia (serum uric acid ≥ 540 µmol/L) following initial treatment. (3) If ACE inhibitors (ACEI) or angiotensin receptor blockers (ARB) were not used during initial treatment, subsequent use may be required due to the patient’s condition. (4) Use of corticosteroids or other medications that significantly affect blood glucose levels, or the necessity of using drugs such as prednisone or diuretics that affect renal metabolism due to the patient’s condition. (5) Recent occurrence of acute cardiovascular or cerebrovascular events within the past 3 months, major gastrointestinal surgeries within the past 2 years, or a history of cancer within the past 5 years. (6) Coexistence of primary or secondary renal diseases, such as gouty nephropathy, kidney stones, renal cysts, renal transplantation, urinary tract infections, etc. (7) Presence of severe infections, diabetic ketoacidosis, diabetic ketoacidosis coma, or hyperosmolar hyperglycemic state. (8) Severe organic abnormalities in the heart, liver, kidney, brain, or other organs. (9) Pregnant or breastfeeding women. (10) Type 1 diabetes and other specific types of diabetes.

Criteria for withdrawal

(1) Withdrawal must be initiated if there is a significant change in the patient’s condition that requires urgent intervention and affects the continuity of the study. (2) Withdrawal should be considered if the patient develops severe complications such as severe infection, ketoacidosis, hyperosmolar non-ketotic diabetic coma, or significant organ dysfunction (e.g., heart, liver, brain) during the treatment period. (3) If the participant experiences serious adverse events or reactions, such as ketoacidosis or genitourinary tract infections, withdrawal from the clinical trial is necessary. (4) Withdrawal may be necessary if the participant requires treatment for other illnesses during the study, which may interfere with the trial.

Exclusion criteria

(1) Participants who fail to adhere to the prescribed medication regimen and significantly deviate from the trial protocol. (2) Participants with incomplete clinical data. (3) Participants who voluntarily withdraw from the clinical trial. (4) Participants who are unable to cooperate with follow-up procedures.

Randomization and drug intervention in group assignment

Patients who meet the inclusion criteria will be randomly assigned into two groups, the control group and the intervention group (empagliflozin group), using a random number table. The randomization process will be conducted by experienced professionals who will not be involved in the follow-up and statistical analysis of the patients.

After randomization, drug treatment will be implemented as follows:

(1)

Intervention Group (Empagliflozin Group): In addition to the standard treatment, the patients in this group will receive empagliflozin, an SGLT2 inhibitor, also known as empagliflozin tablets (trade name: Jardiance/Ou Tangjing, Shanghai Boehringer Ingelheim Pharmaceuticals Ltd., Drug Registration Certificate: H20170351, China Drug Approval Number: J20171073). Empagliflozin will be administered orally at a dose of 10 mg once daily on an empty stomach in the morning. The dose of empagliflozin will remain unchanged, but other antidiabetic drugs (of the same class as the baseline therapy) may be adjusted based on blood glucose levels.

(2)

Control Group: In the control group, patients will receive the standard treatment without the addition of empagliflozin. Similar to the intervention group, the dose of antidiabetic drugs (of the same class as the baseline therapy) may be adjusted based on blood glucose levels.

The target for glycemic control in both groups is fasting blood glucose between 4.4 mmol/L and 7.0 mmol/L and postprandial blood glucose below 10.0 mmol/L. The maximum recommended dose of oral antidiabetic drugs should not be exceeded, and combining two different types of oral antidiabetic drugs is not recommended. It is also advised to avoid using four or more different types of oral antidiabetic drugs concurrently. The total dose of insulin should not exceed 1 U/kg. During follow-up, the dosage of ACE inhibitors or ARBs should remain unchanged, while other antihypertensive and lipid-lowering regimens should follow the methods and targets of the baseline therapy stage.

Research method

Prior to the intervention, we recorded basic patient information, including contact details, age, gender, height, weight, BMI, blood pressure, duration of diabetes, history of hypertension, cardiovascular disease history, smoking status, family history, and concurrent medication usage. Before the intervention, we conducted laboratory tests to observe the following indicators: (1) Routine laboratory tests: Fasting blood glucose, lipid profile, blood uric acid, liver function, blood creatinine, blood urea nitrogen, and postprandial blood glucose (measured using an automated biochemical analyzer, Model 7600 Series, Hitachi) under fasting conditions. We also used fresh morning urine samples to measure urinary microalbumin (measured using an automated luminescent system, MAGLUMI 4000, Shenzhen New Industries Biomedical Engineering Co., Ltd.) and urinary creatinine (measured using an automated biochemical analyzer, Model 7600 Series, Hitachi), and calculated the UACR. Furthermore, we used high-performance liquid chromatography (D-10 kit, Bio-Rad, USA) to measure glycated hemoglobin (HbA1c). (2) We calculated the eGFR using the modified diet in renal disease (MDRD) equation for the Chinese population (Ma et al. 2006).

ELISA detection of IL8, A1M, B2M, L-FABP, KIM-1, and NGAL

The first step involves the collection and storage of urine samples. Fresh morning urine specimens should be collected on an empty stomach, with each sample volume being 10 ml. Immediately after collection, the samples should be centrifuged at 3000 rpm for 10 min at 4℃ to separate sediment from the urine. Following centrifugation, the supernatant should be transferred to EP tubes and stored at -80℃ for subsequent analysis. When performing the experimental measurements, the frozen samples should be thawed and used immediately to avoid the impact of repeated freeze-thaw cycles.

For the ELISA detection of KIM-1, the Human Urinary TIM-1/KIM-1/HAVCR Quantikine ELISA Kit (catalog number DKM100) from R&D Systems, USA, was utilized. As for NGAL, the Human Lipocalin-2/NGAL Quantikine ELISA Kit (catalog number DLCN20) from R&D Systems, USA, was employed. IL-18 was detected using the Human IL-18 ELISA Kit (catalog number EK118–48, Sencken Biotech, China), L-FABP was analyzed using the Human Liver-Type Fatty Acid-Binding Protein (L-FABP) ELISA kit from Wuhan Merck Biomaterials Co., Ltd. (catalog number 69-75621), A1M was measured using the α1-Microglobulin (A1M) detection kit from Wuhan Yunke Long Biotechnology Co., Ltd. (catalog number: SCA217Hu), and B2M was quantified with the β2-Microglobulin (B2M) detection kit from Wuhan Yunke Long Biotechnology Co., Ltd. (catalog number: SEA260Hu). Before conducting the experiment, the reagents need to be prepared. This includes allowing the kit to reach room temperature from the recommended storage temperature of 2–8℃, as well as preparing washing solution, color reagent, diluent, and KIM-1 standards.

The experimental procedure involves retrieving the microwell plate from the sealed aluminum bag and adding assay diluent, standard curve samples, as well as test samples for incubation. This is followed by plate washing and incubation with Human TIM-1 Conjugate. Subsequently, the plate is washed again, and a color reagent is added for the reaction. Finally, a stop solution is added, and the absorbance is measured using an ELISA reader (ELx808, BIOTEK). The concentration of KIM-1 in the samples is calculated based on the standard curve, and then the results are corrected using the urine creatinine ratio to obtain the final KIM-1 or NGAL concentration levels. The detection methods for IL-8, A1M, B2M, and L-FABP were strictly carried out in accordance with the instructions provided by the respective assay kits.

Follow-up observation of treatment progress

During the treatment period, the patients will be followed up through weekly phone calls to observe their treatment progress. Diabetes education will continue to be provided, emphasizing the importance of medication adherence. Good adherence is defined as the actual medication intake being more than 80% of the prescribed dosage. The patients will be instructed to perform self-monitoring of blood glucose, with a frequency of 3 days per week and 5–7 times per day. Self-monitoring of blood pressure will also be performed, with a frequency of at least 3 times per day for 3 days per week. If necessary, the monitoring frequency will be increased based on the blood glucose and blood pressure levels. The patients will be asked to record their blood glucose and blood pressure levels, and if needed, they will be advised to return to the hospital for treatment adjustment. Adverse reactions during the treatment process will be monitored, and any adverse events, especially ketoacidosis genital and urinary tract infections, will be systematically investigated and recorded. Patients will be reminded to maintain personal hygiene of the external genitalia, drink an appropriate amount of water, and ensure smooth urination.

Follow-up observational indicators

After 6 weeks of treatment, the patients were readmitted for a follow-up assessment, where the baseline observational indicators were re-examined under the same conditions. The patient’s systolic and diastolic blood pressure were recorded, and fasting blood glucose, blood lipid profile, uric acid levels, hepatic function, blood creatinine, UACR, urinary kidney injury molecule-1 (KIM-1), urinary neutrophil gelatinase-associated lipocalin (NGAL), and postprandial blood glucose at 2 h were measured under fasting conditions. The testing methods used for these indicators remained consistent with those employed prior to the intervention. The primary focus of observation was on urinary KIM-1 and NGAL.

Statistical analysis

For continuous variable data, if it follows a normal distribution, the mean and standard deviation are used to represent it. If it follows a skewed distribution, the median (interquartile range) is used. For categorical data, frequency (percentage) is used. To compare the data before and after treatment, a repeated-measures analysis of variance is used for the pre-and post-test data in a controlled design. For comparing differences between two groups, the two-sample t-test, the Mann-Whitney U test, and the chi-square test are used for normally distributed data, skewed data, and categorical data, respectively. All statistical analyses are performed using SPSS 19.0 software, and a significance level of p < 0.05 is considered statistically significant.