SCM-198 Can Regulate Autophagy Through the Bax/Bcl-2/TLR4 Pathway to Alleviate Renal Ischemia-Reperfusion Injury

SCM-198 (catalog no: HY-N0741, MedChem Express, New Jersey, USA) was dissolved in 0.1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, USA).

Figure 1

In the current research, 40 healthy, 12-week-old, male Wistar albino male rats were weighed (240-260 g) and planned as 4 groups (n=10). Rats were purchased from Ataturk University Medical Experimental Application Research Center (Erzurum, Turkey). All experiments were carried out with permission from Atatürk University Animal Experiments Local Ethics Committee (protocol number: 17.09.2018-174).

Rats were anesthetized via Ketamine 75 mg/ kg (Pfizer, Istanbul, Turkey) and Xylazine 8 mg/ kg (RompunTM, Bayer, Turkey). In the I/R group, the renal pedicles were clamped bilaterally with microvascular clamps by reaching the retroperitoneal region from the back regions in the lateral position, and 60 minutes of ischemia was initiated. Afterward, clamps were removed from the renal pedicles, the incision areas of the animals were closed with 3.0 silk suture (Dogsan, Istanbul, Turkey), and 24-hour reperfusion was initiated (16, 17). In the control group, the renal pedicles were reached but not clamped. In the DMSO group, the kidneys were reached by incision as in the control group, and DMSO was applied. In the SCM-198 group, in addition to the procedures performed in the I/ R group, 30 mg/ kg SCM-198 was administered intraperitoneally (i.p.) 5 minutes before reperfusion. After the I/R process was completed, the rats were sacrificed, renal tissue and blood samples were taken.

The blood collected from the rats was centrifuged at 3,500 rpm for 15 minutes without wasting any time, and serum was obtained. Urea and creatinine (Cre) levels were measured from the serum. Renal tissues were divided into two parts at the point where the renal pedicle enters the kidney. One part was used in the preparation of the kidney homogenate. The kidneys were weighed and homogenized at a ratio of 1:20 in 0.1 M potassium phosphate buffer solution (PBS, pH 7.4). The homogenates were then centrifuged at 5,000 g for 20 minutes at 4°C. Supernatants were stored at -80°C in pre-labeled microcentrifuge tubes until they were used in biochemical analysis. Protein determination; the protein concentration were determined by using the Lowry method using a commercial total protein kit (Sigma Aldrichs, St. Louis, MO) (18). The other kidney piece was fixed in 10% formalin. Then, the samples were embedded in paraffin. Following the paraffinization, the samples were sectioned and stained with hematoxylin and eosin to evaluate the histological changes.

Serum Urea (catalog no: STA-382 Cell Biolabs, Inc San Diego, USA) and Cre (catalog no: E-EL-R0058 Wuhan, China) concentrations were measured using an ELISA reader (BioTEK Powerwave XS Winooski, UK).

Tumor necrosis factor-α (TNF-α) (catalog no: E-EL-R0019, Elabscience, Wuhan, China), interleukin-1β (IL-1β) (catalog no: E-EL-R0012, Elabscience, Wuhan, China), interleukin-18 (IL-18) (catalog no: E-EL-R0567, Elabscience, Wuhan, China), kidney injury molecule-1 (KIM-1) (catalog no: E-EL-R0575, Elabscience, Wuhan, China), neutrophil gelatinase-associated lipocalin (NGAL) (catalog no: E-EL-R0662, Elabscience, Wuhan, China), and podocalyxin (PCX) (catalog no: E-EL-R0764, Elabscience, Wuhan, China) levels were determined via ELISA reader (BioTEK Powerwave XS Winooski, UK) in kidney tissues. All measurements were performed adhering to the kit manufacturer’s protocol.

Renal tissues of the rats were determined in 10% neutral formalin solution. Tissues were taken into paraffin blocks after routine alcohol-xylol follow-up. Sections of 5 μm taken on polylysine slides were exposed to the xylol and alcohol series, and washed with PBS. The samples were kept in 3% H₂O₂ for 10 minutes to inactivate endogenous peroxidase. The antigen was treated with the retrieval solution at 500 watts for 2x5 minutes to reveal the antigen in the tissues. Later, tissues were incubated with LC3B (ABclonal, catalog no: ab15191, 1/200 dilution ratio), TLR4 (Santa Cruz, catalog no: 293072, 1/200 dilution ratio), Bcl-2-associated X protein (Bax) (Bioss, catalog no: bs-0127R, 1/200 dilution ratio), B-cell lymphoma 2 (Bcl‑2) (Bioss, catalog no: bs-0032R, 1/200 dilution rate), and Beclin-1 (Bioss, catalog no: bs-1353R, 1/200 dilution ratio) primary antibodies at the room temperature for 20 minutes. Mouse and Rabbit Specific HRP/ DAB IHC Detection Kit-Micro-polymer kit (Abcam, catalog no: ab236466) was used as the secondary antibody suggested by the manufacturer. DAB (3,3ʹ-Diaminobenzidine) was used as chromogen. After contrast painting with Mayer’s Hematoxylin, it was covered with entellan and examined under a light microscope. Immunopositivity in kidney tissues was evaluated as none (-), mild (+), moderate (++), and severe (+++).

The obtained data were analyzed with SPSS v.20 (IBM, Chicago, USA) software. In histopathological evaluations, the difference between the groups was determined by Kruskal Wallis, and the Mann-Whitney U test determined the group that made the difference. In biochemical analyses, One-way ANOVA was used to analyze the differences between groups, and then Duncan’s post hoc test was used for multiple comparisons. All data are expressed as mean ± SE. P<0.05 was accepted statistically significant.

Serum Cre and Urea levels elevated in the I/R group compared to the control and DMSO groups. In the group that received SCM-198 treatment, the levels of these parameters decreased cantly in the control and DMSO groups, Bcl-2 immunopositivity was moderate (Figures 5, 6, 7, 8, 9). While LC3B, TLR4, Bax, Beclin-1 immunopositivity was found to be moderate in the I/ R group, Bcl-2 immunopositivity could not be determined. In the SCM-198 group, LC3B, TLR4, Bax, Bcl-2, and Beclin-1 immunopositivity was mild.

ELISA test was used to measure the inflammatory cytokines. IL-1β, TNF-α, and IL-18 levels were found to be high in the I/R group. TNF-α, IL-18, and IL-1β levels declined significantly in the SCM-198 group (Figures 3A, 3B, and 3C). In the light of all data, inflammation that increased in renal I/ R injury was reduced with SCM-198 administration.

The levels of kidney damage markers (NGAL, KIM-1, and PCX) were significantly higher in the I/R group than in the control and DMSO groups. These levels were significantly lower in the SCM-198 group than in the I/R group (figure 4).

We detected a significant difference among the groups regarding TLR4, Bax, Bcl-2, LC3B, and Beclin-1 immunopositivity in the renal tissues (Figure 10, p<0.05). While LC3B, TLR4, Bax, Beclin-1 immunopositivity could not be determined signifi(21, 22). NGAL is expressed in the intercalated cells of the collecting channel and thick arm of the loop of Henle (23, 24). Approaching normal levels of BUN, KIM-1, Cre, and NGAL indicate reduced renal injury (19, 20, 25). Additionally, PCX is a podocyte-specific marker and membrane protein involved in regulating glomerular filtration (26). We have not previously encountered a renal I/ R study in which PCX levels were investigated. Still, some evidence has been presented showing that changes in PCX levels are associated with renal injury (27, 28). We showed that PCX levels increased after our bilateral I/R model. We think that the probable cause of this increase in PCX levels may be to improve the decreased filtration after bilateral I/ R. We found significant improvements in the levels of parameters indicating renal injury in the SCM- 198 group.

Figure 10

Toll-like receptor (TLR) is a transmembrane protein structurally produced in renal cells. TLRs fall into two categories: those that are localized to the cell membrane (TLR-1, 2, 4, 5, 6, 10, 11, 12, and 13) and those that are located inside the cell (TLR-3, 7, 8, and 9) (29). TLR-4, one of the family members, is structurally expressed in the renal epithelium, and its expression increases in the renal I/R (30, 31). The activation of the

TLRs leads to the transport of NF-κB from the cytoplasm to the nucleus, which increases the inflammatory cytokine levels and proinflammatory responses (30). During AKI induced by I/ R injury, TLR-2 and TLR-4 were found to activate NF-κB and AP-1 signaling pathways in renal tubular epithelial cells. Subsequently, upregulated mediators such as IL-6, IL-1β, IL-18, MCP1, TNF-α, and IFN-γ enhance inflammatory responses that lead to acute tubulointerstitial damage (30, 31, 32). Inhibition of inflammatory cytokine release is one of the main goals in preventing I/ R damage. At this point, we evaluated the effects of SCM-198 on TLR-4 mediated cytokines. Increased TLR-4 immunopositivity and cytokine levels (IL-1β, TNF-α, and IL-18) in I/R were decreased by SCM-198 treatment. Our results are consistent with many studies. SCM-198 has been demonstrated anti-inflammatory effects by suppressing NF-κB activity (11, 33). Besides, it has been reported to downregulate the release of various adhesion molecules and cytokines (34, 35).

The primary pathological mechanism of AKI is apoptosis in tubular cells and accompanying renal dysfunction (25, 36, 37). There are many different findings (changes in caspase‑3 activity, cytochrome c release, and Bcl‑2/Bax ratio) indicating that apoptosis results from I/R (39). From the Bcl-2 protein family, Bcl-2 and Bcl-XL are the best-defined anti-apoptotic factors. Bax, Bad, or Bcl-Xs from other family members supports apoptosis (39, 40, 41). In renal I/R injury, Bax and caspase‑3 levels increase, and Bcl‑2 level decreases, resulting in tubular cell apoptosis (42). The Bcl-2 expression level was increased significantly, and the Bax expression level was reduced considerably in the SCM-198 group compared to the I/R group, and thus the Bcl-2/Bax ratio increased. The high Bcl-2/Bax ratio in the results explains the mechanism of the anti-apoptotic effects of SCM-198. Similar to our results, Liu et al. and Zhu et al. showed in their studies that SCM- 198 has an anti-apoptotic effect by lowering the Bax/Bcl-2 ratio (13, 43).