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. 2021 Sep;35(9):e23898.
doi: 10.1002/jcla.23898. Epub 2021 Jul 7.

Clinical value of INSL3 in the diagnosis and development of diabetic nephropathy

Jing Zhu  1 , Xiang Zheng  1
Affiliations

Clinical value of INSL3 in the diagnosis and development of diabetic nephropathy

Jing Zhu et al. J Clin Lab Anal. 2021 Sep.

Abstract

Background: Insulin-like factor 3 (INSL3) was stated to be an essential regulator in many diseases. This present study aimed to explore the underlying mechanisms of INSL3 in diabetic nephropathy (DN).

Methods: The serum samples were obtained from 121 DN patients, 67 T2DM patients, and 44 healthy controls. Twenty SD rats were used to establish the DN model in vivo. Quantitative PCR (qPCR) and Western blot were completed to analyze the INSL3 expression in cells, serum samples, and kidney of the rats. The structure of kidney was analyzed by HE staining. The diagnostic values of INSL3 in DN were determined by receiver operating characteristic (ROC) assay. Then, Spearman's correlation analysis was executed to verify the association between INSL3 and glomerular filtration rate (eGFR). Finally, the proliferation and apoptosis status of transfected cells were analyzed by MTT, flow cytometry, and Hoechst33258 staining assay.

Results: We found that INSL3 expression was up-regulated in DN patients and SV40-MES-13 cells. Furthermore, the correlation analysis elucidated that INSL3 expression was negatively correlated with DN diagnosis golden criterion eGFR. INSL3 knockdown promoted the proliferation rate and inhibited the apoptosis rate of SV40-MES-13 cells after high-glucose treatment. Finally, the INSL3 expression and fast blood glucose were up-regulated in DN rats.

Conclusions: Collectively, this study demonstrated the clinical significance of INSL3 in diagnosing and developing DN.

Keywords: INSL3; diabetic nephropathy; diagnosis; glomerular membrane epithelial cells.

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Conflict of interest statement

None.

Figures

FIGURE 1
FIGURE 1
The expression of INSL3 was significantly increased in serum samples from DN compared with those from T2DM patients and healthy controls. p < 0.01, DN vs. healthy, DN vs. T2DM. DN, diabetic nephropathy; T2DM, type 2 diabetes mellitus; healthy, healthy controls
FIGURE 2
FIGURE 2
The diagnostic significance of INSL3 in DN. (A) The AUC of INSL3 concerning discriminating DN from T2DM patients. (B) The AUC of INSL3 concerning discriminating DN from healthy controls. (C) The correlation between INSL3 and eGFR. DN, diabetic nephropathy; T2DM, type 2 diabetes mellitus; healthy, healthy controls; eGFR, glomerular filtration rate
FIGURE 3
FIGURE 3
The expression of INSL3 was markedly up‐regulated in SW40‐MES130 cells after high‐glucose stimulation compared with normal glucose. (A) INSL3 mRNA expression in SW40‐MES130 cells under different glucose conditions. (B) INSL3 protein expression in SW40‐MES130 cells under different glucose conditions. p < 0.01, high glucose vs. normal. Normal cells were treated with normal glucose
FIGURE 4
FIGURE 4
Insulin‐like factor 3 knockdown inhibited SW40‐MES130 cell proliferation under high‐glucose conditions. (A) The transfection efficacy of INSL3 was determined by qPCR assay. p < 0.01, siRNA‐INSL3 vs siRNA‐NC or HG group. (B) MTT assay was employed to measure the cell viability in different groups. (C–D) Flow cytometry was employed to measure the cell apoptosis in different groups. (E) Hoechst33258 staining assay. ** p < 0.01, * p < 0.05, HG +siRNA‐INSL3 vs HG +siRNA‐NC group, HG vs normal group. HG, high glucose
FIGURE 5
FIGURE 5
Insulin‐like factor 3 expression was up‐regulated in DN rats. (A) The expression of INSL3 was determined by qPCR assay. (B–C) The INSL3 protein expression was determined by Western blot assay. (D) The FBG levels of the DN rats. (E) HE staining of the DN rats. *** p < 0.001, Model group VS Control group
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