Introduction

Diabetic nephropathy (DN) is a chronic microvascular kidney disease induced by diabetes mellitus (DM) and is clinically characterized by microalbuminuria and/or decreased glomerular filtration rate [1]. It’s reported that DN accounts for 16.3% of end-stage renal diseases in China [2]. Due to the insidious onset of DN, most patients are diagnosed with DN in the clinical stage. And renal injury is so difficult to reverse that it gradually develops into end-stage renal failure. Therefore, early detection, early diagnosis and effective control of DN seem to be of the utmost importance [3]. Data statistics in 2020 revealed that more than 90% of the diabetic population in China had type 2 diabetes mellitus (T2DM) [4]. Hence, the focus of this investigation was on determining the severity of renal injury in patients with type 2 diabetic nephropathy (T2DN).

Urinary albumin excretion rate (UAER) is usually applied to assess the degree of renal injury. However, UAER is susceptible to interference by some other factors because of the time-consuming urine collection and the significantly increased UAER under urinary tract infection and menstrual status [5]. For the assessment of renal impairment in patients with T2DN, it is therefore crucial to develop markers with simple sampling and stable outcomes. Albumin/globulin ratio (AGR) is a new predicting indicator that can comprehensively reflect the inflammation and nutrition in the body [6]. It can’t be ignored that inflammatory response is crucial in the development of DN [7]. Recent studies have reported that, as sensitive indicators to assess the degree of renal injury in T2DN, albumin (ALB) and AGR have advantages like simplicity and speed [8]. Based on the above, we retrospectively analyzed the clinical data of 192 T2DM patients in our hospital. And our study aimed to assess the relationship between serum ALB, globulin (GLB) and AGR and renal impairment in patients with T2DN and provide new ideas for clinical diagnosis and treatment of this condition.

Materials and Methods

Study design

A retrospective analysis was conducted on 110 T2DN patients and 82 T2DM patients with normal kidneys (Control group) who visited the First Affiliated Hospital of Wannan Medical College from October 2019 to April 2022. Our study was approved by the Ethics Committee of the First Affiliated Hospital of Wannan Medical College.

Inclusion criteria were shown as follows. All enrolled patients (1) satisfied the diagnostic criteria of T2DM in the literature of Guidelines for the prevention and treatment of Type 2 diabetes in China: 2017 [9]. The diagnostic criteria included (a) typical symptoms of diabetes mellitus (irritable thirst, polyuria, polyphagia, unexplained weight loss); (b) random blood glucose ≥ 11.1 mmol/L; (c) fasting blood glucose ≥ 7.0 mmol/L; (d) glucose load 2 h blood sugar ≥ 11.1 mmol/L. The (a) one is necessary, and (a) plus any one of the (b), (c) or (d) one will give the patient a diagnosis of diabetes. If there are no typical diabetic symptoms, the diagnosis should be confirmed by re-examination at a later date; (2) met the criteria of UAER ≥ 30 mg/24 h and with hyperglycemia as etiology [10]; (3) aged from 18 to 75 years; (4) had complete clinical data.

Patients were excluded if they suffered from (1) acute diabetic complications such as ketoacidosis, (2) cardiac insufficiency, an autoimmune disorder of connective tissue, infection and respiratory disorder, (3) lupus nephropathy, (4) kidney lesions caused by tumors, (5) type 1 diabetes, gestational diabetes, and other special types of diabetes; (6) severe heart, liver, lung and cerebrovascular diseases, (7) various stress states (e.g. infection, acute myocardial infarction, acute stroke, trauma, surgery, etc.); had (8) long-term application history of steroid hormone therapy, (9) history of previous neuropsychiatric disorders; received (10) angiotensin converting enzyme inhibitors or angiotensin II receptor antagonist antihypertensive drugs or had an uncontrolled hypertension, (11) dialytic treatment.

According to the grading criteria for renal impairment reported by Mogensen et al. [11], UAER of 30–300 mg/24 h indicated mild renal impairment and UAER > 300 mg/24 h represented moderate renal impairment. T2DN patients were divided into the mild renal impairment group (n = 75) and moderate renal impairment group (n = 35) depending on the level of UAER. Then, general information including gender, age, body mass index (BMI), smoking history, drinking history, fasting blood glucose, glycosylated hemoglobin (HbA1c), ALB, GLB, AGR and others were collected from the three groups.

Determination of serum and urine indicators

Venous blood samples were collected in the morning after fasting for 10 h in all the included patients. Next, an automatic chemistry analyzer (Hitachi 7600; Hitachi High-Technologies Corporation, Japan) was applied to detect fasting plasma glucose (FPG), ALB and GLB, followed by the calculation of AGR. Finally, an automatic HbA1c analyzer (ADAMS A1c HA-8180, ARKRAY Factory, Inc., Japan) was adopted for the detection of HbA1c level.

Subsequently, the 24-h urine was collected from all the included patients, and the total amount of ALB in urine was detected by immunoturbidimetric assay. UAER could be obtained after calculation of urinary excretion of ALB per unit time.

Methods for 24-h urine collection: the patients were instructed to prepare a clean plastic bucket (4000 ml of size and with a lid) by themselves. The patients should urinate once in the morning at a certain time (e.g., 7:00 a.m.), and this time the urine should not be poured into the bucket. From this discharge, all discharged urine within 24 h should be poured into the plastic bucket. The last urine discharge was at 7:00 am the next morning, and this time the discharged urine should also be introduced into the plastic bucket. When filling the first urine into the plastic bucket, the patients need to pour the antiseptic provided by the nurse into the plastic bucket and shake it well. Shaking of the bucket should be performed after each introduction of the discharged urine, followed by covering the bucket to prevent evaporation of urine. Twenty-four hours later, the bucket will be picked up by the specialized nursing staff. Notably, collection of samples from female patients during menstruation should be avoided; patients ate and moved normally during urine collection.

Statistical analysis

The data from this study were analyzed and processed using SPSS 22.0 statistical software. To be specific, measurement data conforming to normal distribution were expressed as mean ± standard deviation (SD). One-way ANOVA was used for comparison between multiple groups and then last significant difference (LSD) test was used for comparison between two groups. Enumeration data were expressed as n (%) and the chi-square test was used for comparison between multiple groups. Pearson correlation was used to analyze the correlation between UAER levels with serum ALB, GLB and AGR levels in the T2DM group. Receiver operating characteristic (ROC) curves were plotted to analyze the diagnostic value of ALB, GLB, and AGR for moderate renal injury in T2DN. p < 0.05 was considered statistically significant.

Results

Comparison of clinical baseline characteristics among the patients in the three groups

As shown in Table 1, there was no significant difference in gender, age, BMI, smoking history, and drinking history among the patients in the three groups (p > 0.05). However, the duration of T2DM, history of hypertension, FPG, and HbA1c in the mild renal impairment and moderate renal impairment groups showed significant difference with those in the Control group (p < 0.05). The comparisons among the three groups disclosed the longest duration of T2DM and the highest proportion of hypertension history, FPG level, and HbA1c level in the moderate renal impairment group.

Table 1 Comparison of clinical baseline characteristics among the three groups
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Comparison of albumin, globulin and albumin/globulin ratio levels among the three groups

As displayed in Table 2, the ALB and AGR levels in the mild renal impairment group and moderate renal impairment group were lower while GLB levels were higher than those in the Control group, and the differences were statistically significant (p < 0.05). Besides, compared with the mild renal impairment group, the moderate renal impairment group presented lower ALB and AGR levels, but there was no significant difference in GLB level between the two groups (p > 0.05).

Table 2 Comparison of albumin, globulin and albumin/globulin ratio levels among the three groups
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Correlation of albumin, globulin, and albumin/globulin ratio levels with urinary albumin excretion rate in patients with T2DN

Pearson correlation analysis revealed (Fig. 1A–C) that ALB and AGR levels had a significantly negative correlation with the UAER level in patients with T2DN (p < 0.05), while GLB levels had no obvious correlation with the UAER level (p > 0.05).

Fig. 1
figure 1

Correlation analysis of albumin, globulin, and albumin/globulin ratio levels with urinary albumin excretion rate in patients with type 2 diabetes mellitus (T2DM). –C: scatter plots of albumin (ALB), globulin (GLB), albumin/globulin ratio (AGR) and urinary albumin excretion rate (UAER)

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Diagnostic value of albumin, globulin, albumin/globulin ratio and urinary albumin excretion rate levels in moderate renal injury in patients with T2DN

ROC curves showed that the AUCs for ALB and AGR levels in predicting moderate renal injury in patients with T2DN were 0.880 (95% CI: 0.817–0.943) and 0.714 (95% CI: 0.608–0.820), respectively. The above findings indicated that ALB and AGR levels were of high value in assessing moderate renal injury in patients with T2DN (p < 0.05). As for GLB level, it had no significant diagnostic value for moderate renal injury in patients with T2DN (p > 0.05) (Fig. 2 and Table 3).

Fig. 2
figure 2

ROC curves for the diagnostic value of albumin (ALB), globulin (GLB), and albumin/globulin ratio (AGR) in moderate renal injury in patients with type 2 diabetes mellitus (T2DM)

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Table 3 Diagnostic value evaluation for albumin, globulin, and albumin/globulin ratio in moderate renal injury in patients with T2DN
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Discussion

As a disease with an insidious onset, DN leads to an aggressive renal deterioration that worsens with time, and even progresses to renal insufficiency and uremia [12]. Some studies have pointed out that DN can be induced by multiple factors. Among these factors, glucose metabolism disorders, changes in renal hemodynamics, a variety of cytokines and genetic background play crucial roles [13]. Hence, more novel predictors have also been applied for the assessment of renal impairment in patients with T2DN. This study was designed to explore the correlation of ALB, GLB and AGR with renal injury in T2DM patients.

In our study, there were significant differences in the duration of T2DM, incidence of hypertension, FPG and HbA1c levels among the three groups. Moreover, long T2DM duration, a history of hypertension and poor glycemic control may affect the occurrence and development of T2DN. It’s reported that long-term hyperglycemic infiltration is a major determinant in the development of diabetic microangiopathy, and diabetic patients with long disease duration and poor glycemic control are prone to microangiopathy like nephropathy. Briefly, the duration of disease and glycemic control have a great impact on renal injury in T2DM patients. Hypertension is also closely related to renal impairment. Diabetic patients with hypertension suffer from more severe insulin resistance and hyperinsulinemia and are prone to renal injury [14, 16]. The above findings are consistent with the results of our study.

Pearson correlation analysis in this study revealed a significantly negative correlation between the ALB and AGR levels and the UAER level in patients with T2DN. Patients with UAER > 30% suggest the presence of mild renal impairment [11], i.e., early renal disease. If patients suffer from massive albuminuria, the early renal disease will develop into clinical kidney disease. The clinical kidney disease will lead to thicker glomerular basement membranes than the early kidney disease, and even results in compensatory glomerular hypertrophy. The resulting symptoms will seriously impair the renal functions of patients. In this case, early diagnosis of kidney disease and timely treatment are crucial to improving the prognosis of the patients [17]. Low ALB, AGR level and high UAER level in patients with T2DN in our study suggested severe renal injury. Accordingly, the relationship between ALB, AGR and the degree of renal impairment in T2DN patients can be established by UAER.

After comparative analysis, ALB and AGR levels were much lower in the moderate renal impairment group than those in the mild renal impairment group and the Control group; the mild renal impairment group exhibited lower ALB and AGR levels than the Control group. In addition, GLB level was higher in the Control group than that in both the moderate renal impairment group and the mild renal impairment group; there was no significant difference in GLB level between the moderate renal impairment group and the mild renal impairment group. The above results demonstrated a certain sensitivity of ALB and AGR levels to the judgement of renal injury. Studies have reported that ALB is negatively correlated with C-reactive protein (CRP) [18], so the up-regulation of plasma ALB level has a positive effect on the improvement of chronic inflammatory diseases. Li Jie et al. [19] claimed that, on the basis of conventional treatment, increasing the intake of compound α-keto acids could effectively regulate the proteinuria in patients with chronic kidney disease (CKD), significantly increase serum ALB level, and finally delay CKD progression. ALB has antioxidant effects on carcinogens and can stabilize cell growth and DNA replication [20]. Hypoproteinemia may be a sign of malnutrition, which raises the risk of sepsis and can impair the immune system. Anyhow, hypoproteinemia is effective in predicting morbidity and mortality of patients with renal diseases [21]. GLB is a low molecular weight protein synthesized by immune organs, and its rate of synthesis and release is constant in healthy humans. Specifically, GLB is present in cells other than placental trophoblast cells and mature erythrocytes. The relatively small molecular mass of GLB allows it to freely traverse capillary walls, be excreted and decomposed by the kidneys, and be measured with ease [22, 23]. When glomerular filtration membrane is damaged, GLB level in urine is significantly increased; GLB can serve as a sensitive indicator to detect early renal damage in diabetic patients [24, 25]. However, several studies have stated that despite high sensitivity, GLB has poor specificity and is easily affected by many factors such as tumor, inflammation, and immunity [26]. Hence, GLB level alone do not accurately reflect the degree of renal impairment.

AGR is a comprehensive indicator reflecting all non-ALB proteins and ALB. In recent years, research has pointed out some benefits for AGR in predicting the prognosis of colon cancer, liver cancer, and lung cancer [6, 27, 28]. For instance, Tsai C C et al. reported [29] that AGR was superior to ALB in predicting mortality in peritoneal dialysis because AGR could comprehensively reflect the inflammation and nutritional status of the body. Compared with serum ALB, AGR combined with ALB and GLB is a relatively more stable and reliable indicator because it is less affected by inflammation, hepatic synthesis, catabolism, and changes in hydration status. Similar to the findings of previous studies, the results of ROC curve analysis in this study showed that ALB and AGR had high diagnostic value for moderate renal injury in patients with T2DM. All in all, ALB and AGR are valuable and practical biomarkers for assessing renal function impairment in patients with T2DN clinically.

There are still some limitations in this study. For example, the retroactive nature of our article and the small sample size could lead to outcome biases. In other words, the conclusions above require further verification through large-scale prospective studies.

Conclusion

Taken together, the expression level of ALB and AGR are decreased in T2DN patients. Moreover, ALB and AGR may be valuable in clinical application as a sensitive indicator for the diagnosis of T2DN.