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Introduction

Renal cell carcinoma (RCC), which has the highest mortality rate among all types of genitourinary cancers, represents a serious problem to public health (1). Over 300,000 patients are diagnosed with RCC each year worldwide and the incidence rate of RCC still exhibits an increasing trend (2). Although various drugs, including axitinib, sorafenib, nivolumab and everolimus have been developed to treat patients with RCC (3,4), treatment of advanced RCC has typically failed to provide satisfactory outcomes due to a series of problems, such as drug tolerance and individual difference in response to drugs (5). It has previously been demonstrated that, although recurrence will occur in a minority group of patients, RCC is generally curable in early stages (1). Therefore, early diagnosis and treatment is the key to the treatment of RCC. Nephritis occurs in tubules, glomeruli and interstitial tissue surrounding the tubules and glomeruli. It has been demonstrated that the onset and development of RCC may be associated with certain risk factors of kidney inflammation, including oxidative stress (6). Therefore, proper treatment of nephritis will inhibit the progression of RCC, and therefore potentially reduce the mortality rate.

Phosphatase and Tensin Homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway serves essential roles in the regulation of the balance between survival and apoptosis as well as cell migration (7,8). The pathway also has inhibitory effects on a number of human diseases including different types of cancer, such as prostate cancer (9), breast cancer (10), hepatocellular carcinoma (11) and RCC (12). PTEN-Long, which is a translational variant of PTEN produced by in-frame alternative translation, has been demonstrated to have similar roles to PTEN in inhibiting the growth of tumors by inhibiting PI3K signaling through a phosphatase-dependent manner (13). Compared with PTEN, PTEN-Long protein contains an additional N-terminal region, which gives it the ability to cross the cell membrane (14), and due to its functions, PTEN-Long has been selected as a therapeutic target for the treatment of pancreatic cancer (15,16). A previous study has demonstrated that expression levels of PTEN-Long were increased in patients with clear cell RCC (ccRCC), and that PTEN-Long overexpression can inhibit cell proliferation, migration and invasion, which in turn leads to the inhibition of tumor growth (14), indicating the inhibitory effects of PTEN-Long on ccRCC. Although functions of PTEN-Long have been studied previously in various cancers including RCC, its specific roles in the progression of nephritis have not yet been investigated, to the best of our knowledge. Due to the pathogenesis shared by RCC and nephritis, it may be hypothesized that PTEN-Long also has important functions in the progression of nephritis.

CRISPR/Cas9 is a novel gene-editing tool derived from prokaryotic adaptive immune system against phage/virus and foreign DNA invasion. It is widely used in mammalian genomic editing, including repressing gene expression (17), gene knock-in (18), repairing disease-associated genes (19) and development of animal models of cancer (20). In the present study, effects of PTEN-Long on nephritis have been investigated in patients and animal models. In addition, effects of PTEN-Long on inflammatory factors were also evaluated using PTEN-Long knock-in and knock-out mice established by the CRISPR/Cas9 technique.

Materials and methods

Specimen collection

Blood samples from patients with nephritis, RCC and normal healthy controls (n=60 each) treated at the Institute of Urology of The Affiliated Yantai Yuhuangding Hospital of Qingdao University (Yantai, China) between May 2014 to December 2016 were collected. No patient received chemoradiotherapy or other antitumor therapy prior to surgery. The median age is 58 years (range, 45–77 years) and the ratio of male to female was 0.68 (73/107). The present study was approved by the Ethics Committee of The Affiliated Yantai Yuhuangding Hospital of Qingdao University, and all participants provided written informed consent. All surgery was performed according to the Declaration of Helsinki.

Preparation of mouse nephritis model

All mice used in the present study were 8 weeks old with a body weight of 20–25 g and were housed in a pathogen-free environment with 12-h light/dark cycles at room temperature (20±2°C) with a relative humidity of 50–70%. Animals had free access to food and water. PTEN-Long knock-in and knock-out mice were constructed using CRISPR-Cas9 technique by Cyagen Biosciences, Inc. (Santa Clara, CA, USA). The efficiency of knock-in and knock-out was detected by western blotting. Female transgenic BALB/c mice (Cyagen Biosciences), including 10 knock-in mice and 10 knock-out mice, were used in the present study. A total of 20 transgenic mice (10 knock-in mice and 10 knock-out mice) were treated with intraperitoneal injection of lipopolysaccharide (30 mg/kg, 50 µl; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and renal homogenate (50 µl; renal tissue:saline, 1:6 g/ml; Cyagen Biosciences) and wild type BALB/c mice (n=10) in the control group were administered 100 µl saline. The present study was approved by the Ethics Committee of The Affiliated Yantai Yuhuangding Hospital of Qingdao University.

Determination of serum levels of inflammatory factors

Following intraperitoneal injection, blood samples (0.2 ml) were extracted from the fundus vein at 12, 24 and 48 h later. Blood samples were injected into EDTA anticoagulant tubes and maintained at 4°C for 30 min, followed by centrifugation at 800 × g for 10 min at 4°C to collect supernatant. A Bio-Plex system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to compare the secretion of tumor necrosis factor (TNF)-α (cat. no. ab6671), interleukin (IL)-6 (cat. no. 100712), IL-1β (cat. no. ab100704) and IL-18 (cat. no. ab216165; all Abcam, Cambridge, UK) in serum of different groups. All kits used to detect inflammatory markers were purchased from Abcam and the manufacturer's protocols were followed.

Detection of inflammatory factors' gene expression in renal tissue by reverse transcription semi-quantitative polymerase chain reaction (PCR)

Mice were sacrificed at 48 h following intraperitoneal injection. Left kidneys were harvested and cut, added into normal saline prior to preparing a renal tissue homogenate using an electric homogenizer. Total RNA was extracted from tissue homogenate using TRIzol (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Purity and integrity of total RNA were detected and cDNA was synthesized via reverse transcription using PrimeScript™ RT reagent kit (cat. no. RR037Q; Takara Biotechnology Co., Ltd., Dalian, China). SYBR Green Master Mix kit (Thermo Fisher Scientific, Inc.) and 1 µl cDNA were used to prepare the PCR reaction system. All primers used for PCR reaction are listed in Table I. PCR reaction conditions were as follows: 95°C for 5 min, and 40 cycles of 95°C for 20 sec, 60°C for 30 sec and 72°C for 30 sec. Subsequently, PCR product was subjected to 1.2% agarose gel horizontal electrophoresis. Ethidium bromide was visualized using the Gel Doc XR gel imaging system (Bio-Rad Laboratories, Inc.) that allowed images to be captures, and ImageJ software 1.48u (National Institutes of Health, Bethesda, MD, USA) was used to analyze the results. The relative expression level of TNF-α, IL-6, IL-1β and IL-18 mRNA were quantified according to the endogenous control (β-actin) using the aforementioned software.

Table I. Primer sequences.

Table I.

Primer sequences.

Gene	Forward	Reverse
TNF-α	5′-TACTCCCAGGTTCTCTTCAAGG-3′	5′-GGAGGCTGACTTTCTCCTGGTA-3′
IL-6	5′-GAGTTGTGCAATGGCAATTC-3′	5′-ACTCCAGAAGACCAGAGCAG-3′
IL-1β	5′-CACCTCTCAAGCAGAGCACAG-3′	5′-GGGTTCCATGGTGAAGTCAAC-3′
IL-18	5′-AAACCCGCCTGTGTTCGA-3′	5′-TCAGTCTGGTCTGGGATTCGT-3′
β-actin	5′-TTGTTACCAACTGGGACG-3′	5′-GGCATAGAGGTCTTTACGG-3′

[i] TNF, tumor necrosis factor; IL, interleukin.

Observation of pathological changes by hematoxylin and eosin staining

At room temperature, mouse renal tissue was fixed in 4% paraformaldehyde for 12–24 h, followed by paraffin-embedding for 1 h. The embedded tissue was then cut into 4-µm sections. Dewaxing was performed twice with xylene for 15 min at 24°C. Following hydration by passing a series of graded ethanol concentrations (95% ethanol for 5 min, 90% ethanol for 5 min, 70% ethanol for 2 min and distilled water for 5 min), hematoxylin staining was performed for 10 min, followed by washing with water for 10 min at room temperature. Following treatment with 95% ethanol for 5 sec, eosin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) staining was performed for 2 min at 24°C. Following staining, dehydration was performed by passing a series of graded ethanol concentrations (70% ethanol for 2 min, 80% ethanol for 2 min, 90% ethanol for 5 min and twice with 100% ethanol for 5 min). Then, tissue sections were then treated twice with xylene, 10 min each. Neutral gum was used to seal the sections and optical microscopy (magnification, ×200; Olympus Corporation, Tokyo, Japan) was used to observe the signals.

Western blotting

Total protein was extracted from human blood samples and mice renal tissue by TPE™ kit (Beijing Solarbio Science & Technology Co., Ltd.) and quantified using the bicinchoninic acid protein assay method. Protein samples were boiled for 3 min and then loaded into 10–20% Ready Gels (40 µg/lane; Bio-Rad Laboratories, Inc.) for electrophoresis, followed by transferring to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk for 2 h at room temperature. Following washing, membrane was incubated with PTEN-Long primary antibody (1:500; cat. no. ABM-2052; Cascade Bioscience, Inc., Winchester, MA, USA), β-actin polyclonal antibody (1:500; cat. no. orb129534; Biorbyt Ltd., Cambridge, UK) overnight at 4°C. Following washing with TBS-Tween-20 (3X), the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1,000; cat. no. ab150084; Abcam) at room temperature for 2 h. Enhanced chemiluminescence (cat. no. 34580; Thermo Fisher Scientific, Inc.) was used to detect the signals. ImageJ software 1.48u was used to analyze the data and the relative expression level of PTEN-Long was quantified relative to the endogenous control, β-actin.

Statistical analysis

Statistical analysis was performed using SPSS 19.0 statistical software (IBM, Corp., Armonk, NY, USA). Data are presented as mean + standard deviation. All data were analyzed using one-way analysis of variance with Bonferroni's correction for multiple group comparisons and Student's t-test for the comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Serum levels of PTEN-Long protein in serum of patients with nephritis, RCC and normal controls

As presented in Fig. 1, Serum levels of PTEN-Long in patients with nephritis and RCC carcinoma were significantly reduced by 47 and 66% compared with normal controls (P<0.01). In addition, serum levels of PTEN-Long in patients with nephritis was significantly higher than that of patients with RCC (P<0.01). These data suggest that serum level of PTEN-Long was negatively associated with the severity of renal disease (Fig. 1).

Figure 1. Serum levels of PTEN-Long protein in serum of patients with NEP, RCC and controls. *P<0.01 vs. C; #P<0.01 vs. Nep. C, control group; NEP, nephritis; RCC, renal cell carcinoma; PTEN, phosphatase and tensin homolog.

Expression of PTEN-Long protein in serum and rental tissue of different groups

As presented in Fig. 2, expression levels of PTEN-Long protein were significantly lower in serum than in rental tissue. Expression levels of PTEN-Long protein in serum and renal tissue of the model group were significantly reduced by 64 and 36%, respectively, compared with the control group (P<0.01). Compared with the model group, expression levels of PTEN-Long protein were significantly increased in serum and rental tissue of knock-in group, but remained lower than those of control group (68 and 92% of the control group, respectively; P<0.01). Compared with the model group, expression levels of PTEN-Long protein were significantly reduced in knock-out group (P<0.01). The data suggested that low expression of PTEN-Long protein may promote the progression of nephritis, whereas PTEN-Long protein overexpression may inhibit it (Fig. 2).

Figure 2. Expression of PTEN-Long protein in serum and rental tissue of different groups. (A) Expression of PTEN-Long protein in serum; (B) Expression of PTEN-Long protein in renal tissue. K-I, knock-in; K-O, knock-out; **P<0.01 vs. control; ##P<0.01 vs. model; ••P<0.01 vs. K-I. PTEN, phosphatase and tensin homolog; K-I, knock-in; K-O, knock-out.

Effects of PTEN-Long knock-in and knock-out on serum levels of inflammatory factors

In order to investigate the effects of PTEN-Long on serum levels of inflammatory factors, PTEN-Long knock-in and knock-out mice were constructed, and serum levels of several selected inflammatory factors were determined. As presented in Fig. 3, serum levels of TNF-α, IL-6, IL-1β and IL-18 in the model group were 51.07±4.18, 33.64±3.12, 21.63±2.0 and 32.75±3.19 pg/ml, respectively, which were significantly higher than those in the control group (22.15±1.69, 11.98±2.64, 8.65±1.09 and 13.46±2.03 pg/ml, respectively; P<0.05). Compared with the model group, PTEN-Long knock-out further significantly increased serum levels of TNF-α, IL-6, IL-1β and IL-18 to 55.17±4.23, 38.59±3.46, 22.76±2.09 and 36.62±3.21 pg/ml, respectively (P<0.05). Compared with PTEN-Long knock-out, PTEN-Long knock-in significantly decreased serum levels of TNF-α, IL-6, IL-1β and IL-18 to 24.72±2.36, 14.61±2.79, 10.76±2.05 and 17.93±2.46, respectively (P<0.01). These results suggested that, PTEN-Long can inhibit inflammation caused by nephritis through its interactions with inflammatory factors (Fig. 3).

Figure 3. Comparison of serum levels of inflammatory factors between mice in different groups. (A) TNF-α; (B) IL-6; (C) IL-1β; (D) IL-18. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P 0.01 vs. model; ••P<0.01 vs. K-I. TNF, tumor necrosis factor; IL, interleukin; K-I, knock-in; K-O, knock-out.

Expression of inflammatory factors in renal tissue of mice with different backgrounds

As presented in Fig. 4, expression levels of TNF-α, IL-6, IL-1β and IL-18 in the model group were 1.92±0.13, 2.02±0.14, 1.63±0.12 and 1.47±0.11, respectively, which were significantly higher than those of the control group (P<0.05). Compared with the model group, expression levels of TNF-α and IL-6 mRNA in the knock-out group were significantly increased to 2.17±0.16 and 2.29±0.16, respectively, and no significant differences were found in expression levels of IL-1β and IL-18 between those two groups. In contrast, compared with the model group, expression levels of TNF-α, IL-6, IL-1β and IL-18 in PTEN-Long knock-in group were significantly reduced to 1.12±0.07, 1.21±0.13, 1.09±0.09 and 1.03±0.08, respectively (P<0.05). Expression levels of TNF-α and IL-6 mRNA were significantly higher in the PTEN-Long knock-in group than in the control group, but no significant differences in expression levels of IL-1β and IL-18 mRNA were observed between these groups. These data demonstrated that PTEN-Long can inhibit the expression of inflammatory factors to protect kidney from the injuries caused by nephritis (Fig. 4).

Figure 4. Expression levels of inflammatory factors in mice in different groups. (A) TNF-α; (B) IL-6; (C) IL-1β; (D) IL-18. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P 0.01 vs. model; ••P<0.01 vs. K-I. TNF, tumor necrosis factor; IL, interleukin; K-I, knock-in; K-O, knock-out.

Histological changes of kidney of mice in different groups

As presented in Fig. 5, the structure of glomerular cells in the control group is clear, no interstitial inflammatory cell infiltration and edema were exhibited and no proliferation of mesangial cells and matrix was observed. In the model group, glomerular adhesions were observed, glomerular blood vessels and basement membrane were markedly thickened, and inflammatory cell infiltration was also observed; compared with the model group, structure of the glomerular was improved, the thickening of glomerular blood vessels and basement membrane and inflammatory cell infiltration were reduced in PTEN-Long knock-in group; in PTEN-Long knock-out group, renal tissue structure was destroyed, no clear glomerular was observed and severe inflammatory cell infiltration was observed. The results indicate that PTEN-Long can protect renal tissue structure in patients with nephritis.

Figure 5. Histological changes of kidney of mice in different groups (original magnification, ×200).

Discussion

It is well known that the PI3K/Akt pathway is closely associated with the onset and progression of a variety of cancers (21). PI3Ks can phosphorylate phosphatidylinositol (4,5)-bisphosphate (PIP2) to produce phosphatidylinositol 3, 4, 5 triphosphate (PIP3), and the produced PIP3 can active Akt to phosphorylate a variety of target proteins associated with the regulation of cell growth, survival, proliferation and other cellular processes (22). PTEN protein, which is a tumor suppressor, serves essential roles in the regulation of the PI3K/Akt pathway. PTEN can convert PIP3 to the inactive form of PIP2 through hydrolyzation, so as to inhibit signaling transduction of PI3k/Akt pathway (23). PTEN-Long, which is derived from PTEN, is the permeable form of PTEN that can be secreted by one cell to enter neighboring cells. Similar to the functions of PTEN, PTEN-Long can also dephosphorylate PIP3 to reduce the signaling transduction of the PI3K-Akt pathway, which in turn induces cell death and inhibit tumor growth (24). Previous studies have demonstrated that PTEN-Long serves pivotal roles in the development and progression of various cancers (13,25,26). A previous study on the treatment of pancreatic cancer have demonstrated that PTEN-Long targeted therapy combined with chemotherapy may significantly extend overall survival in mice model (15). In the clinical study of ccRCC, expression levels of PTEN-Long protein were observed to be significantly reduced in patients with ccRCC, and the reduced level of PTEN-Long protein caused the increased phosphorylation levels of Akt (16). In contrast, PTEN-Long overexpression in the ccRCC cell line 786-0 decreased the phosphorylation level of Akt, which in turn inhibit the proliferation, migration and invasion of cancer cells or may induce cells death (16). Therefore, expression level of PTEN-Long protein was negatively associated with the development of ccRCC. In the present study, reduced serum level of PTEN-Long protein was observed in patients with RCC compared with healthy controls. In addition, serum levels of PTEN-Long protein in patients with nephritis were lower than that of healthy controls but higher than that of patients with RCC. These data suggest that PTEN-Long is also associated with the development of nephritis and its expression level is negatively associated with the severity of kidney diseases.

The development of nephritis is a complex process with a variety of associated factors, particularly inflammatory factors. Expression levels of TNF-α, IL-6, IL-12 and IL-18, which are proinflammatory cytokines, may be used to sensitively reflect the status of inflammation (27,28). It has previously been demonstrated that expression levels of IL-1β, IL-18 and IL-6 were significantly increased in patients with acute renal failure (29). TNF-α can mediate the expression of chemokines and cytokines to interact with inflammatory responses caused by renal injury (30). In the present study, serum levels of TNF-α, IL-6, IL-12 and IL-18 were significantly increased in mice with nephritis than in healthy control mice. In addition, expression levels of TNF-α, IL-6, IL-12 and IL-18 mRNA in renal tissue were also increased in nephritis mice compared with controls, indicating the inflammation response caused by nephritis. Furthermore, PTEN knock-in significantly decreased the serum levels of TNF-α, IL-6, IL-12 and IL-18 and their expression in retinal tissue. In contrast, PTEN knock-out served an opposite role. The development of nephritis is accompanied by histological changes in renal tissue that can significantly reduce renal function (31). In the present study, PTEN knock-in was demonstrated to improve the histological changes caused by nephritis, but PTEN knock-out promoted those changes. These data suggest that PTEN-Long can protect kidney from nephritis injuries by inhibiting the expression of inflammatory factors.

Based on these findings, PTEN-Long expression was significantly reduced due to kidney diseases, and the reduced level of PTEN-Long expression was positively correlated with the severity of disease. Therefore, PTEN-Long protein can potentially serve as a biomarker for the diagnosis of nephritis. The protein levels of PTEN-Long in serum and renal tissue of mice in different groups were evaluated, and protein levels of PTEN-Long were observed to be significantly lower in serum than in retinal tissue. Therefore, serum level of PTEN-Long may more sensitively reflect the status of kidney diseases.

In conclusion, serum level of PTEN-long was negatively associated with the severity of kidney disease, and PTEN-long may protect retinal tissue from the damage caused by nephritis. Therefore, PTEN-long may potentially be a target for the diagnosis and treatment of nephritis.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

HW and QY conceived and designed the experiments. HW, QY, LW, YLi, MX, YLu and YC performed the experiments and prepared the manuscript. HW, QY, LW, YLi, MX, YLu and YC analyzed the data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of The Affiliated Yantai Yuhuangding Hospital of Qingdao University, and all participants provided written informed consent.

Patient consent for publication

All participants provided written informed consent.

Competing interests

The authors declare that they have no competing interests.

References