Increased urinary Smad3 is significantly correlated with glomerular hyperfiltration and a reduced glomerular filtration rate and is a new urinary biomarker for diabetic nephropathy

Subjects with T2DM (n = 245) and their respective age- and sex-matched healthy volunteers (n = 82) were recruited from the out-patient clinic at the Shanghai Clinical Centre for Diabetes (Shanghai, China). The diagnosis of diabetes mellitus was performed according to the World Health Organization (WHO) study group (1999) criteria. Patients and controls were interviewed personally by the investigators to exclude acute complications of diabetes mellitus, a history of non-diabetic renal disease, urinary tract infection, symptoms or history of heart disease, and acute or severe chronic liver disease. All patients with T2DM were treated with oral antidiabetic drugs. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. All study participants provided written informed consent prior to enrolment.

Demographic and clinical data, including age, sex, diabetes duration, weight and height, were recorded. Blood pressure was measured twice using a Hawksley sphygmomanometer after 10 min of supine rest, and the average blood pressure was calculated using the formula: (systolic blood pressure [SBP] + 2 (diastolic blood pressure)/3. Venous blood samples were collected between 08:00 and 09:00 h after a 12-h fast. Serum samples were separated by centrifugation at 4 °C at 4000 g for 10 min. Fasting blood samples were collected to determine fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c) and serum lipid, lipoprotein and creatinine levels. Body mass index (BMI) was calculated as weight divided by height squared (kg/m²). Plasma glucose levels were measured in the fasting state (i.e., FPG) and in the 2-h postprandial state (2hPG) using the glucose oxidase method. HbA1c was determined using the Bio-Rad Variant II analyser (Bio-Rad Laboratories Inc., Hercules, CA, USA). Serum urea nitrogen, creatinine, uric acid, and lipid profiles, including measurements of total cholesterol (TC), triglycerides, high density lipoprotein and low density lipoprotein (LDL) were measured on a Hitachi 7600 analyser using an enzymatic assay (Hitachi Inc., Tokyo, Japan). Serum high-sensitive C-reactive protein was measured by a particle-enhanced immunoturbidimetric assay (Dade Behring Inc., Newark, NJ, USA). Urinary albumin was determined using a Dade Behring BN II analyser by nephelometry (N antiserum in the Human Albumin Assay, Dade Behring, Glasgow, DE, USA). Serum creatinine and urinary creatinine concentration were measured on a Hitachi 7600 analyser using the sarcosine oxidase-PAP method. The urinary albumin:creatinine ratio (UACR) was computed and reported in mg/g (1 mg/g = 0.131 mg/mmol). The albuminuria categories included normoalbuminuria, microalbuminuria and macroalbuminuria defined as an ACR <30, 30–299, and ≥300 mg/g, respectively. The eGFR was estimated using the Modification of Diet in Renal Disease equation: 186 × [serum creatinine (mg/dL)]^−1.154 × (age)^−0.203 × (0.742 if female). The eGFR categories included ≥131, 90–130, 60–89, and <60 ml/min/1.73 m².

Hyperfiltration has largely been considered a dichotomous variable in the academic literature, with a threshold of 125–140 ml/min/1.73 m², depending on the population evaluated and the formula used to estimate kidney function. We defined hyperfiltration as an eGFR ≥131 ml/min/1.73 m² (mean ± 2 standard deviations) in accordance with age- and sex-specific healthy subjects, and an eGFR of 90–130 ml/min/1.73 m² was defined as normofiltering.

Freshly voided urine was collected from patients during their routine visits to Shanghai Clinical Centre for Diabetes. During the same period, samples were obtained from healthy volunteers with no history of diabetes, hypertension, or renal disease. Urine samples were maintained at −80 °C and were later thawed, centrifuged, and assayed directly by enzyme-linked immunoassay (ELISA). The usmad3 measurements were performed using a commercially available ELISA kit (USCN Life Science and Technology Co., Wuhan, Hubei, PRC) according to the manufacturer’s protocol. The detection limit in the human usmad3 assay was 0.159 ng/ml, and the intra- and interassay coefficients of variations were 6.7 % and 8.5 %, respectively. Duplicate measurements were obtained for all samples. The usmad3 level was expressed as a ratio relative to the creatinine level (ng/mmol creatinine). Serial dilutions of recombinant smad3 were included in all assays as standards.