Primary glomerulonephritis (PGN) is a common glomerular disease caused by infectious immunity, genetic tumors, and other factors [1, 2]. The main clinical manifestations of PGN are proteinuria, hematuria, edema, and hypertension. Late-stage PGN can have serious consequences, such as renal parenchymal damage and heart failure. PGN is a leading cause of chronic kidney disease, which progresses to end-stage kidney disease in approximately 40% of patients within 20 yrs of diagnosis, which seriously affects patient safety [3, 4]. Currently, the only means of diagnosing PGN is through a kidney biopsy, as no biomarker with adequate specificity and sensitivity is available. Patients exhibit considerable diversity in terms of epidemiology, clinical manifestations, renal deterioration, and long-term consequences across various racial and ethnic backgrounds [5, 6].

Common renal function (such as blood urea nitrogen and serum creatinine) indicators change significantly only when renal function is moderately or severely impaired [7]. These indicators offer low sensitivity in diagnosing early renal injury, hindering the diagnosis of early glomerular injury [8]. Identifying better indicators of early renal injury is important for earlier clinical intervention. Serum cystatin C (CYSC), serum β2-microglobulin (β2-MG), and urine transferrin (TRF) expression levels differed significantly between healthy individuals and patients with PGN, suggesting their value for assessing the degree of kidney injury [9, 10]. To ascertain the clinical significance of these PGN indicators, we evaluated their expression in patients with PGN and their correlations with renal function indicators. We also explored the utility of CYSC, β2-MG, and TRF in clinical PGN diagnosis and monitoring.

This retrospective study was conducted at the Department of Nephrology and Rheumatology (Associated Hospital; Beihua University) from May 2021 to May 2023. We included 105 patients with PGN in the observation group (70 men and 30 women, aged 35–72 yrs [56.2±3.6 yrs]). For the control group, we recruited 50 healthy individuals (35 men and 15 women, aged 33–70 yrs [55.3±3.7 yrs]) from the physical examination center during the same period.

We used the following inclusion criteria: 1) met the diagnostic criteria of PGN described in the Clinical Practice Guidelines for the Treatment of Glomerulonephritis Formulated by the Global Organization for Improving the Prognosis of Kidney Diseases [11], accompanied by symptoms such as proteinuria or hematuria, edema, and hypertension; 2) an age of ≥ 18 yrs; 3) did not participate in other clinical studies before enrollment. The protocol for collecting clinical samples was approved by the Associated Hospital, Beihua University ethics committee (Protocol Number 2020-10), and written informed consent was obtained from all study participants.

In both groups, the recommended daily allowance for protein intake on a low-protein diet was 0.55–0.60 g/kg/day (observation group) or 0.83 g/kg/day (control group) per the 2020 Kidney Disease Outcomes Quality Initiative Clinical Practice Guidelines for Nutrition. This diet was followed for 3 days before sample collection. All participants were advised to avoid processed and high-calorie meals. Soft drinks, alcoholic beverages, and caffeine were to be avoided in the morning before sample collection. We collected 3–5 mL fasting venous blood samples from patients in the morning. The samples were centrifuged at 3,000 rpm for 5 min, and the serum was separated before subsequent analysis. Blood samples showing hemolysis or chylous reactions were discarded. We also collected 5 mL of first-morning urine from patients, centrifuged them at 3,000 rpm for 10 min, and collected the supernatants for TRF testing.

The serum CYSC levels were assessed via immunoturbidimetry using an AU5821 analyzer (Beckman Coulter, Brea, CA, USA) and a kit provided by Zhongshan Beikong Biotechnology Co., Ltd (Zhongshan, China). The serum β2-MG and urinary TRF levels were detected using an IMMAGE 800 Specific Protein Analyzer (Beckman Coulter) and kits and reagents from the manufacturer. Following the manufacturer’s instructions, the normal reference intervals for CYSC, β2-MG, and TRF were set to 0.66–1.22, 0–2.2 mg/L, and 0–2.2 mg/L, respectively. The serum CYSC and β2-MG levels and urinary TRF levels were compared between both groups and between different clinicopathological types. The positive rates for detecting all three indicators and each indicator individually were compared. Statistical analysis was performed using SPSS 20.2 (IBM Corp., Armonk, NY). Measurement data were expressed as the mean±standard deviation, and the group comparisons were conducted by performing t-tests. The chi-square test was used for categorical data. Binary regression was used to assess the prediction probability function of PGN, and an ROC curve and area under the curve (AUC) were plotted to evaluate the diagnostic efficiency of PGN with various indicators and prediction functions. P<0.05 was considered statistically significant.

We included 105 patients with PGN and classified them into four stages based on the glomerular filtration rate (GFR), per the 2020 Kidney Disease: Improving Global Outcome Clinical Practice Guidelines. These stages included: G1, kidney damage with a normal GFR (>90 mL/min); G2, mild reduction in the GFR (60–89 mL/min); G3a, moderate reduction in the GFR (45–59 mL/min); and G3b, moderate reduction in the GFR (30–44 mL/min). No statistical differences were observed in terms of age, sex distribution, or disease duration between the groups (P>0.05; Table 1).

The detection rates for CYSC, β2-MG, and TRF in the observation group were 80%, 70%, and 70%, respectively, whereas that for one of all three indicators was significantly higher at 90% (P<0.05). The CYSC, β2-MG, and TRF levels in the observation group were 2.53±0.52, 6.99±1.74, and 3.46±1.65 mg/L, respectively. The serum levels of CYSC and β2-MG and urinary levels of TRF were higher in the observation group than in the control group (P<0.05; Table 2). The serum levels of CYSC and β2-MG and urinary levels of TRF gradually increased in G2, G3a, and G3b (P<0.05) relative to those in G1. CYSC and β2-MG showed higher diagnostic efficiency for PGN than TRF, with respective AUCs of 0.856 and 0.819, sensitivities of 71.43% and 92.86%, specificities of 87.67% and 65.75%, and Youden indices of 0.678 and 0.656. The combined diagnostic efficiency of the three indicators was better than that of the individual indicators. The AUC, sensitivity, specificity, and Youden index were 0.903, 78.57%, 89.66%, and 0.775, respectively (Fig. 1).

The clinical manifestations of PGN mainly involve hypertension, edema, and other symptoms. As routine indicators of renal function do not significantly change in the early stage of renal injury, diagnosis is challenging or susceptible to misdiagnosis, thereby delaying its diagnosis and culminating in renal failure [12, 13]. Therefore, identifying early diagnostic indicators of renal injury is important for the early clinical detection, intervention, and treatment of PGN [14].CYSC is a secreted cysteine protease that is widely distributed in human nucleated cells. CYSC production is stable and unaffected by age, sex, diet, and other factors. Thus, CYSC is an ideal indicator for determining the GFR with high sensitivity and specificity. When renal function is mildly impaired, serum CYSC levels increase, and their levels gradually increase with disease aggravation, which correlates positively with the degree of injury. Serum CYSC levels are useful indicators of early renal injury [15].

The single-chain polypeptide amino acid, β2-MG, is widely distributed in bodily fluids. β2-MG can freely pass through the glomerular basement membrane, after which almost all of it is reabsorbed, degraded within the renal tubules, and does not return to the bloodstream. Normal human blood β2-MG levels are substantially low [16, 17]. β2-MG can reflect the filtration function of the kidney and is considered a sensitive indicator of early impairment of renal function. TRF is a single-chain glycoprotein synthesized by the liver, is composed of 679 amino acids, and is the main transporter of Fe. TRF reflects the sensitivity index for glomerular filtration membrane damage [18].

In this study, the single-index detection rates were 80% for CYSC, 70% for β2-MG , and 60% for TRF. The positivity rate for detecting one of the three indicators in the observation group was significantly higher, at 90%. The sensitivity and accuracy of nephritis diagnosis were significantly improved by the combined detection of these three indicators. The serum levels of CYSC and β2-MG and the urinary level of TRF increased significantly over time after renal injury. Statistical analysis showed a positive correlation between CYSC, β2-MG, and TRF levels, suggesting that the three indices increased with PGN progression, which has important clinical value for diagnosing and monitoring PGN. The importance of these predictive biomarkers was consistent with previous findings [19, 20].

This study had several limitations. The data were not population-based, and the number of patients was insufficient to determine the predictive power of the biomarkers. Large multicenter-cohort analysis is necessary to validate these biomarkers as combined prognostic indicators for PGN.

In summary, detecting serum CYSC and β2-MG levels and urinary TRF levels provides a reliable laboratory basis for early PGN diagnosis. This diagnostic capability has important clinical value for assessing the degree of renal damage and monitoring the disease. The sensitivity and accuracy of PGN diagnosis can be improved via the combined detection of CYSC, β2-MG, and TRF.