MMP-9-mediated regulation of hypoxia-reperfusion injury-related neutrophil inflammation in an in vitro proximal tubular cell model

Figures & data

Table 1. The primer sequences used for RT-PCR.

Primer name	Sequence (5′–3′)
MMP-9	F: 5′ -GCTACGTGACCTATGACATCCTG-3′
	R: 5′ -AGAAACACTCCAACAAAAAACAAAG-3′
MMP-2	F: 5′ -GTGGATGATGCCTTTGCTCG-3′
	R: 5′ -GGAGTCCGTCCTTACCGTCAA-3′
TIMP-1	F: 5′ -TCCTGTTGTTGCTGTGGCTGA-3′
	R: 5′ -GGTTGTGGGACCTGTGGAAGTAT-3′
CD11b	F: 5′ -GGATAGTGACATTGCCTTCTTGAT-3′
	R: 5′ -GGTTAGGGTTGTTCTGGAACTCTT-3′
CD18	F: 5′ -AGGTTCTTCCCCAGTGTGAGTG-3′
	R: 5′ -GTTTTTCCCAATGTAGCCAGTGTC-3′
β-actin	F: 5′ -CCTTCCTGGGCATGGAGTC-3′
	R: 5′ -TGATCTTCATTGTGCTGGGTG-3′

MMP-9: matrix metalloproteinase 9; MMP-2: matrix metalloproteinase 2; TIMP-1: tissue inhibitor of metalloproteinases 1; CD11b: integrin subunit alpha M; CD18: integrin subunit beta 2; F: forward; R: reverse

Figure 1. Changes in the viability of HK-2 cells were assessed after the HR treatment and BB-94 intervention using CCK-8 kits. All experiments were repeated three times, and the mean values are presented and compared (*p < .05).

Figure 2. Relative fluorescence intensity and ratio of ROS + HK-2 cells. (A) Fluorescence images of HK-2 cells captured using a fluorescence microscope. (B) Quantitative analysis of the relative fluorescence intensity. (C) Flow cytometry plots of HK-2 cells. (D) Quantitative flow cytometry data. All experiments were repeated three times, and the mean values are presented and compared (**p < .01 and ***p < .005).

Figure 3. Levels of the MMP-2, MMP-9, and TIMP-1 mRNAs in HK-2 cells. (A) Real-time PCR analysis of levels of the MMP-2 mRNA in HK-2 cells. (B) Levels of the MMP-9 mRNA in HK-2 cells. (C) Levels of the TIMP-1 mRNA in HK-2 cells. All experiments were repeated three times, and the mean values are presented and compared (*p < .05; **p < .005; and ****p < .001).

Figure 4. Levels of the MMP-9 and MMP-2 proteins in HK-2 cells. (A) Western blot analysis of MMP-2 protein levels. (B) Quantitation of the Western blot analysis of MMP-2 protein levels. (C) Western blot analysis of MMP-9 protein levels. (D) Quantitation of the Western blot analysis of MMP-9 protein levels. All experiments were repeated three times, and the mean values are presented and compared (*p < .05).

Figure 5. Levels of the CD-11b, CD-18, and MMP-9 mRNAs in PMNs. (A) Real-time PCR analysis of levels of the CD-11b mRNA in PMNs. (B) Levels of the CD-18 mRNA in PMNs. (C) Levels of the MMP-9 mRNA in PMNs. All experiments were repeated three times, and the mean values are presented and compared (*p < .05 and **p < .01).

Figure 6. Levels of the MMP-9 protein in PMNs. (A) Western blot analysis of MMP-9 protein levels. (B) Quantitation of the Western blot analysis of MMP-9 protein levels. All experiments were repeated three times, and the mean values are presented and compared (*p < .05).

Figure 7. Effects of HK-2 cell supernatants on PMN MPO activity. All experiments were repeated three times, and the mean values are presented and compared (****p < .001).

Data availability statement

The data used to support the findings of this study are included in the article.