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

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.

replacement therapy. 6,7 Studies have shown that a range of mediators or physiological pathways, including hyperglycemia, advanced glycation end products, protein kinases C, oxidative stress, and inflammation, are implicated in the pathogenesis and progression of DN. 8,9 During the past two decades, many researchers tried to illustrate the pathogenesis of DN; however, the underlying mechanisms of DN still remained unclear.
Recent studies have shown that DN patients have early morphologic changes in abnormal proliferation, hypertrophy, and aging of renal tubular epithelial cells. 10 The inflammatory response caused by elevated blood sugar is considered an inseparable factor in diabetic complications. 11 More importantly, the levels of inflammatory cytokines in DN patients were increased, and these levels of inflammatory factors increased steadily with the progression of kidney disease. 12,13 However, the pathogenesis of DN remains unclear. Hence, the development of novel treatment targets is essential for understanding the progress of DN. the human body, mainly produced and secreted by testicular interrogating cells. 16 It plays an important role in the testicular decline and functional maintenance and belongs to the insulin family. 17

| Quantitative polymerase chain reaction (qPCR)
According to the manufacturers' protocol, total RNA was extracted were performed at least in triplicate.

| Cell culture and high-glucose treatment
The and high glucose (30 nM glucose) and incubated in a humidified atmosphere at 37°C, 5% CO 2, until subsequent experiments.

| Flow cytometry
The apoptotic cells were detected by flow cytometry. The cells of each group were washed twice with PBS. The cell concentration was adjusted to 1 × 10 5 cells/ml. 5 μ 1 L annexin V and PI was added and mixed, and incubated in dark for 15 min. The apoptosis rate was detected with flow cytometry (Thermo Fisher Technology Co., Ltd., MA, USA).

| Hoechst 33258 staining
To assess nuclear morphology changes during apoptosis, staining

| Animal experiments
The SD rats were provided by Animal Experiment Middle School of Soochow University. The rats were randomly divided into two groups: Control group and Model group. Ten rats in each group.
The rats in Control group were received normal diet, and the rats in the serum of the rats were determined with a kit provided by Nanjing Jiangcheng Bioengineering Institute (Nanjing, China).

| Hematoxylin and eosin (HE) staining
The rats were anesthetized by intraperitoneal injection of chloral hydrate and sacrificed. The left kidneys were collected and fixed with 4% paraformaldehyde for 24 h, and paraffin-embedded sections were made. Then, the sections were stained with hematoxylin for 3 min and eosin for 30 s. The sections were observed and photographed by inverted microscope (Olympus, Tokyo, Japan).

| Western blotting assay
Proteins were extricated with lysis buffer and electrophoretically transferred onto polyvinylidene difluoride membranes (PVDF; Millipore). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies against INSL3 and GAPDH (Cell Signaling Technology Inc, Danvers, MA, USA) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies for 2 h at 37°C. The ECL detection system (Millipore, USA) was employed to visualize the signals.

| Statistical analysis
All the data were shown using mean ± standard deviation (SD). The

| Elevated INSL3 expression in serum samples from DN patients
We summarized the clinical features, serum indexes, and the expression of INSL3 in all the participants (Table 1). High INSL3 expression was considerably correlated with FBG, PPBG, Scr, serum creatinine, disease duration, Hb1Ac, eGFR, IL-1β, IL-6, IL-8, and TNFα. Using qPCR assay, we found that the expression of INSL3 was highly increased in serum samples from DN patients compared to T2DM patients or healthy participants (Figure 1).

| The diagnostic value of INSL3 in DN
Through receiver operating characteristic analysis, we verified the potentials of INSL3 concerning discriminating DN from T2DM patients or healthy controls. The results in Figure 2A,

| Elevated INSL3 was highly expressed in SV40-MES-13 cell after high-glucose stimulation
To investigate the role of INSL3 in DN, expression of INSL3 in SV40-

MES-13 cells under high-glucose treatment was measured using
qPCR and Western blot assay. As shown in Figure 3A-C, INSL3 was up-regulated in mRNA and protein levels after being treated with high-glucose compared with normal glucose treatment conditions ( ** p < 0.01).

| INSL3 silencing promoted the proliferation while inhibited apoptosis in SW40-MES130 cells under high-glucose conditions
To

| INSL3 expression was up-regulated in the kidney of the DN rats
Finally, we further explored the expression of INSL3 in the DN rats. We found that compared with the Control group, the mRNA were also significantly increased in the Model group ( Figure 5D).
Moreover, the HE staining showed that in the Control group, the glomerular epithelial cells were arranged orderly; in the Model group, the volume of glomerulus and extracellular matrix was increased, the mesangial area was widened, the basement membrane was thickened, and the renal interstitium was infiltrated by inflammatory cells ( Figure 5E). Hence, a larger sample sizes of DN patients were required.

| DISCUSS ION
In summary, it has been identified that INSL3 expressions were significantly elevated in DN serum samples and cells.
Mechanism studies further illustrated the diagnostic value of INSL3 in DN, which may promote further exploration for DN clinical treatment.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.