Extracellular status of thrombospondin-2 in type 2 diabetes mellitus and utility as a biomarker in the determination of early diabetic kidney disease

Levels of serum TSP-2 are significantly increased in T2DM patients and correlate closely with a cluster of early DKD risk factors

TSP-2 assay was used to measure serum concentrations in 494 subjects with T2DM and 148 healthy controls included in this study as shown in Table 1. Serum TSP-2 levels ranged from 0.11 to 52.12 ng/mL. Unexpectedly, T2DM subjects had significantly higher serum TSP-2 levels than healthy [7.03 (3.48–12.91) vs. 6.86 (5.11–8.36) ng/mL, p = 0.014], in addition, the levels were significantly increased in these T2DM patients with early DKD compared to those without [10.48 (7.19–15.73) vs. 7.03 (3.48–12.91) ng/mL, p < 0.001], suggesting that TSP-2 may be related to the progress of early DKD in these patients with T2DM.

Table 1 Baseline characteristics of the study participants with early-stage renal damage stratified UACR (if ≥ 30 mg/g)

Consistent with the change of age, a higher prevalence of hypertension was observed in these subjects, following a longer duration of T2DM [(102.63 ± 80.04) vs. (39.08 ± 68.80) months ng/mL, p < 0.001], and a lower eGFR [100.1 (106.9–114.7) vs. 93.6 (101.6–111.4) mL/min/1.73m2, p = 0.002]. However, no obvious differences in FPG and Albumin, as well as blood lipid parameters including TG, TC, HDL-c and LDL-c, and renal function parameters including SUA, BUN and SCR, were observed in these T2DM subjects with or without early DKD (all p > 0.05).

To investigate whether TSP-2 is related to the pathogenesis of early DKD in patients with T2DM, we next investigated the relationship between serum TSP-2 levels and a cluster of anthropometric parameters and renal function parameters. Correlation analysis showed a significant positive association of serum TSP-2 levels with SBP, TG, platelet,and renal function parameters including SUA, SCR, and UACR, respectively (all p < 0.05), but a negative association with eGFR (p < 0.05), after adjusting for age. Furthermore, the positive correlation of serum TSP-2 with these parameters, except for SBP, remained significant even after adjusting for age, sex, and T2DM duration (Table 2). Although there was a significant difference in anti-diabetic treatments (Table 1), no correlation was found between the anti-diabetic treatments and serum TSP-2 levels (Table 2). These results suggested that TSP-2 is closely ralated to renal function in patients with T2DM.

Table 2 Correlation between serum TSP-2 (log transformed) with various parameters (n = 494)Serum TSP-2 is independently associated with early DKD

To further evaluate the potential clinical values of TSP-2 for early-stage renal damage in patients with T2DM, multivariable logistic regressions with different models on the basis of variant risk factors were performed. The results of the logistic regression analysis for predicting early DKD were shown in Table 3. Logistic regression analysis included age, sex, T2DM duration, BMI, drug use of statins, ACEIs/ARBs, and TSP-2 levels at baseline. We found that the serum TSP-2 level was independently associated with development of early DKD [OR 2.26 (95% CI 1.63–3.14), p < 0.001], together with age [OR 1.06 (95% CI 1.03–1.09), p < 0.001], T2DM duration [OR 1.00 (95% CI 1.00–1.01), p = 0.008], and baseline BMI [OR 1.09 (95% CI 1.02–1.17), p = 0.015]. Results were similar after adjustment for baseline plus TG, SUA, SCR, eGFR, Platelet, and UACR levels. Serum TSP-2 level remained independently associated with early DKD [OR 1.94 (95% CI 1.24–3.04), p = 0.004], together with platelets [OR 1.66 (95% CI 1.06–2.60), p = 0.026], and UACR, a recently commonly used clinical laboratory marker [OR 2.52 (95% CI 1.82–3.49), p < 0.001].

Table 3 Multivariable logistic regression analysis showing the association of serum TSP-2 level with DN (n = 494)

For the lack of sensitivity and specificity in UACR (commonly used for the diagnosis of DKD), we next analysed the area under the receiving operator curve (AUROC) of both TSP-2 and UACR. Data from receiving operator characteristics (ROC) analysis indicated that the absolute value of AUROC for serum TSP-2 was 0.66 (95% CI 0.61–0.71), and for the UACR was 0.78 (95% CI 0.73–0.83), these results remained stable with a combined analysis for both TSP-2 and UACR, which reached to 0.80 (95% CI 0.75–0.84), furthermore, a larger AUROC for both TSP-2 and UACR after the addition of age at baseline was produced (Fig. 1). Taken together, serum TSP-2 levels may be a good predictor for the incidence of early DKD in patients with T2DM, and measurement of serum TSP-2 levels may be beneficial for identifying the high and low risk of DKD in patients who have undergone T2DM.

Fig. 1figure 1

Serum TSP-2 levels and other clinical risk factors for the identification of DN in study participants with T2DM (n = 494)

TSP-2 expression is increased in kidney tissues of db/db mice and positively associated with UACR

Serum levels of TSP-2 were significantly increased in db/db mice (p < 0.05), (Fig. 2A). The db/db mice had significantly higher serum TSP-2 levels than the db/m mice [(468.56 ± 62.34) vs. (384.27 ± 51.44) ng/mL, p = 0.048], and m/m mice [(468.56 ± 62.34) vs. (357.88 ± 36.36) ng/mL, p = 0.012]. In addition, we also detected the levels of FPG, SCR, and UACR, consistent with the development of diabetes, higher FPG level, but lower levels of SCR were observed in the db/db mice. Similarly, the level of UACR was markedly elevated in db/db mice compared with db/m mice [(73.43 ± 34.66) vs. (17.78 ± 2.69) mg/g, p = 0.004] (Table 4). In addition, correlation analysis revealed a significant positive correlation between UACR and its serum protein concentration (Fig. 3). These data are in agreement with our clinical observations showing increased serum levels of TSP-2 in T2DM individuals.

Fig. 2figure 2

TSP-2 is expressed in kidney tissue and is elevated in db/db mice. A: Serum level of TSP-2 in different mice. B: Detection and comparison of TSP-2 protein expression in various tissue between db/db mice and their lean littermates. C: Comparison of TSP-2 protein expression in the kidney of m/m, db/m, and db/db mice. **p < 0.01, *p < 0.05. SAT, subcutaneous adipose tissue; WAT, white adipose tissue; BAT, brown adipose tissue

Table 4 Baseline characteristics of miceFig. 3figure 3

Correlation between serum levels of TSP-2 and its UACR (log transformed) in mice

To further observe the physiological relevance of TSP-2 above in clinical findings, we next compared TSP-2 expression between various tissues and renal tissue in different 16-week-old male mice by Western blotting. Interestingly, TSP-2 expression was increased only in renal tissues of both db/m and db/db mice (Figs. 2B). The observation that TSP-2 was expressed in the renal tissue of db/m led us to examine whether TSP-2 expression was altered in diabetes. To this end, we compared the TSP-2 expression in renal tissues between 16-week-old male db/db mice and db/m mice. Notably, TSP-2 expression in renal tissue was markedly elevated in diabetic mice compared with db/m mice (Fig. 2C). These data suggest that renal tissue contributed, at least in part, to the elevated circulating TSP-2 levels observed in diabetic mice.

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