The role of Testis-Specific Protein Y-encoded-Like 2 in kidney injury

Abstract

Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI). Recent findings suggest that Testis-Specific Protein Y-encoded-Like 2 (TSPYL2) plays a fibrogenic role in diabetes-associated renal injury. However, the role of TSPYL2 in IRI-induced kidney damage is not entirely clear. In this study, we found that the expression of TSPYL2 was upregulated in a mouse model of AKI and in the hypoxia/reoxygenation (H/R) cell model. Knockdown of TSPYL2 attenuated kidney injury after IRI. More specifically, the knockdown of TSPYL2 or aminocarboxymuconate-semialdehyde decarboxylase (ACMSD) alleviated renal IRI-induced mitochondrial dysfunction and oxidative stress in vitro and in vivo. Further investigation showed that TSPYL2 regulated SREBP-2 acetylation by inhibiting SIRT1 and promoting p300 activity, thereby promoting the transcriptional activity of ACMSD. In conclusion, TSPYL2 was identified as a pivotal regulator of IRI-induced kidney damage by activating ACMSD, which may lead to NAD+ content and the damaging response in the kidney.

Keywords: Cancer; Cancer systems biology; Genomic analysis; Genomics.

Graphical abstract

Figure 1

Expression of TSPYL2 is induced in the animal model of acute kidney injury (AKI) and in an H/R cell model (A) Renal TSPYL2 mRNA levels were measured by quantitative real-time RT-PCR in sham-treated mice and ischemic mice exposed to reperfusion after 6h, 12h, or 24 h. (B) TSPYL2 protein levels in the kidneys were detected by Western blot analysis in sham-treated and ischemic mice after different reperfusion times. Levels of GADH were used as loading control. A representative blot is shown and a bar graph of multiple experiments. (C and D) Kidney sections of sham-treated or ischemic mice with reperfusion after 6h, 12h, or 24h were stained immunohistochemically with specific antibodies against TSPYL2 and DAPI. (C) Representative images indicate TSPYL2 protein expression in red and DAPI in blue. Bar = 50 μm. (D) Bar graph shows the average of several experiments of the percentage TSPYL2-positive cells relative to the sham group. (E) TSPYL2 protein levels in HK-2 cells were detected by Western blot analysis in control cells and in hypoxia-treated cells with reoxygenation after 2h, 4h, or 6h. GAPDH was used as a loading control. A representative blot is shown and a bar graph of TSPYL2 expression relative to sham group from three independent experiments. Data are represented as mean ± SD (n = 3–6). p value calculated was determined by a two-tailed unpaired Student’s t test. ∗p < 0.05, ∗∗p < 0.01 versus sham mice or control cells.

Figure 2

Knockdown of TSPYL2 attenuated kidney injury after ischemic-reperfusion injury (IRI) (A) Experimental design. Injection of a short hairpin RNA plasmid in mice was done two days before exposure to IRI. After 24h, blood and urine were collected, and tissue was isolated. (B) A representative Western blot of renal TSPYL2 protein expression in mice groups as indicated, with GAPDH as a loading control. The two lanes in the same group represent two samples from two different mice. In addition, a bar graph of several experiments showing TSPYL2 expression relative to the sham-shControl group (n = 6). (C) Representative images show renal TSPYL2 expression in red and DAPI in blue in mice. Quantitative analyses of several experiments of the TSPYL2-positive area for four mice groups as indicated. At least five randomly selected fields were evaluated per mice. Bar = 50 μm. (D) The level of Scr was determined for the four mice groups as indicated. (E) BUN levels were measured in mice as indicated. (F) Injury in kidney proximal tubular cells in mice was evaluated by measuring the concentration of urinary Kim-1. (G) Kidney sections of mice were subjected to H&E staining. A bar graph with quantitative analyses of injured tubules assessed by kidney injury score is shown for the four groups as indicated. (H) Representative images of H&E stainings of mice. Images in the right upper corner are enlarged and presented in the larger images. Upper panels, bar = 700 μm; lower panels, bar = 50 μm. Data are represented as mean ± SD (n = 4–6). p value calculated was determined by a two-tailed unpaired Student’s t test. ∗p < 0.05, ∗∗p < 0.01 versus sham-shControl mice; #p < 0.05, ##p < 0.01 versus IRI-shControl mice.

Figure 3

Knockdown of TSPYL2 inhibited IR-induced apoptosis and oxidative stress in the kidney in vivo (A) Representative images show TUNEL-positive cells in green in the kidneys of mice and the bar graph depicts the percentage of TUNEL-positive cells. Bar = 50 μm. (B) Representative images show cleaved caspase 3 staining in red and DAPI in blue in the kidneys of mice as indicated. The bar graph shows the percentage of caspase 3-positive cells. Bar = 50μm. (C) Representative Western blots show the renal expression of Bax, Bcl-2, and cleaved caspase 3 in mice as indicated. GADPH was used as a loading control. The two lanes in same group represent two samples from two different mice. Quantitative analyses of Western blots of several experiments for the renal expression of Bax, Bcl-2, and cleaved caspase 3 in mice (n = 6). (D) Representative flow cytometry plots of ROS staining in renal tissues of mice and quantitative analysis of several experiments of renal ROS levels in each group relative to the sham-shControl group. (E–G) ELISA assays were used to measure the concentration of MDA, GSH and SOD in the kidney of mice. Data are represented as mean ± SD (n = 4–6). p value calculated was determined by a two-tailed unpaired Student’s t test. ∗p < 0.05, ∗∗p < 0.01 versus sham-shControl mice; #p < 0.05 versus IRI-shControl mice.

Figure 4

Knockdown of TSPYL2 attenuates NAD+ loss and decreases ACMSD expression after IRI (A) Renal NAD+ levels were measured in mice. (B) NADH measurements were performed in the kidneys of mice. (C) The ratio of NAD⁺/NADH was analyzed and expressed relative to that of the sham-shControl group. (D) Representative images show renal ACMSD staining in blue in mice as indicated. Bar = 50 μm. The bar graph shows the average of quantitative analyses of several experiments of the percentage ACMSD-positive area in the kidneys for the four mice groups as indicated. (E) A representative Western blot of renal ACMSD protein expression is shown for mice as indicated. The same mice are tested in Western blot of ACMSD and TSPYL2 protein. The bar graph shows renal ACMSD protein expression relative to the sham-shControl group. (F) ACMSD mRNA levels in the kidneys of mice were determined for the four groups. Data are represented as mean ± SD (n = 4–6). p value calculated was determined by a two-tailed unpaired Student’s t test. ∗p < 0.05, ∗∗p < 0.01 versus sham-shControl group; #p < 0.05, ##p < 0.01 versus IRI-shControl group, “ns” means not statistically different.

Figure 5

Knockdown of TSPYL2 or ACSMD ameliorated mitochondrial dysfunction, oxidative stress, and apoptosis induced by hypoxia-reoxygenation (H/R) injury in HK-2 cells (A) HK-2 cells were transfected with siTSPYL2 or siACSMD or a negative control (siControl) 24h before exposure to H/R or no exposure (Control). Representative Western blots for the expression of TSPYL2 and ACSMD are shown. GAPDH was used as a loading control. The bar graphs show the quantification of Western blots for TSPYL2 and ACMSD of three independent experiments. (B) Cell viability of HK-2 cells was detected using the CCK8 assay in the indicated groups. (C and D) Apoptosis of HK-2 cells was measured by TUNEL assay. Bar = 50 μm. The bar graph shows the percentage of TUNEL apoptotic cells. (E) Representative Western blots of staining for Bax, Bcl-2, and cleaved caspase-3 in HK-2 cells are shown. GADPH was used as a loading control. Bar graphs show the results of Bax, Bcl-2, and cleaved caspase-3 levels in HK-2 cells from three independent experiments. (F) NAD⁺ levels in HK-2 cells were measured. (G) mtDNA copy number in HK-2 cells was determined by analysis of the ND1 segment-18S segment ratio using qPCR. (H) Cellular ATP production was measured in HK-2 cells. (I) Staining of cytoplasmic ROS (cyto-ROS) in green and mitochondrial ROS (mito-ROS) in red in HK-2 cells was performed using CellROX green reagent and MitoSOX red reagent, respectively. Bar = 20 μm. (J and K) Bar graphs showing relative fluorescence levels of cyto-ROS (J) and mito-ROS (K) are shown. (L) Representative images of mitochondrial divisions in HK-2 cells are shown by the immunohistochemical staining of mitochondria in green. Bar = 5 μm. (M) Mitochondrial fission was quantified in HK-2 cells by counting the percentage of cells with fragmented mitochondria. Data are represented as mean ± SD (n = 3). p value calculated was determined by a two-tailed unpaired Student’s t test. ∗p < 0.05, ∗∗p < 0.01 versus Control-shControl group; #p < 0.05, ##p < 0.01 versus H/R-shControl group.

Figure 6

ACMSD overexpression aggravated mitochondrial dysfunction, oxidative stress and apoptosis induced by H/R injury in TSPYL2-knockdown HK-2 cells (A) HK-2 cells were transfected with siTSPYL2 or siControl and a plasmid carrying the human ACMSD or the mock plasmid without ACMSD 24 h before exposure to H/R or no exposure (Control). Representative Western blots show the protein expression of TSPYL2 and ACMSD in HK-2 cells and the bar graph shows the quantification of blots from three independent experiments. (B) Cell viability was measured using the CCK8 kit in HK-2 cells in the indicated groups. (C) Apoptosis in HK-2 cells was detected by TUNEL assay. Bar = 50 μm. The bar graph shows the percentage of TUNEL apoptotic cells. (D) Representative Western blots of the protein expression of Bax, Bcl-2, and cleaved caspase-3 in HK-2 cells are shown. GAPDH was a loading control. Bar graphs show the results of protein levels of Bax, Bcl-2, and cleaved caspase-3 in HK-2 cells from three independent experiments. (E) NAD⁺ levels in HK-2 cells were measured. (F) mtDNA copy number in HK-2 cells was determined by analysis of the ND1 segment-18S segment ratio using qPCR. (G) Cellular ATP production was measured in HK-2 cells. (H–J) Staining of cytoplasmic ROS (cyto-ROS) in green and mitochondrial ROS (mito-ROS) in red in HK-2 cells was performed using CellROX green reagent and MitoSOX red reagent, respectively. Bar = 20 μm. Bar graphs showing relative fluorescence levels of cyto-ROS (I) and mito-ROS (J) are shown. (K) Representative images of mitochondrial divisions in HK-2 cells are shown by the immunohistochemical staining of mitochondria in green. Bar = 5 μm. (L) Mitochondrial fission was quantified in HK-2 cells by counting the percentage of cells with fragmented mitochondria. Data are represented as mean ± SD. p value calculated was determined by a two-tailed unpaired Student’s t test. ∗, #, ★ p < 0.05, ∗∗, ##, ★★ p < 0.01; ∗ versus Control-siControl+MOCK group; # versus the H/R-siControl+MOCK group.

Figure 7

TSPYL2 promotes ACMSD transcriptional activation by regulating SREBP-2 acetylation (A) HK-2 cells were transfected with siTSPYL2 or siControl for 24 h before exposure to H/R or no exposure (Control). The mRNA level of ACMSD was determined for the four groups. ∗∗p < 0.01 versus Control-siControl group; ##p < 0.01 versus the H/R-siControl group. (B) A predicted binding sites of SREBP-2 to the ACMSD promoter was found when searching the JASPAR database. Base pair (bp) numbers indicate positions relative to the ACMSD transcription start site (TSS). The blue box indicates the SREBP-2-binding motif. (C) HK-2 cells were transfected with siTSPYL2 or siControl and Control vector or a plasmid carrying SREBP-2 (p-SREBP-2). Luciferase reporter analysis was performed 24h later based on the wild-type and mutant ACMSD promoter regions expressed in HK-2 cells. ∗∗p < 0.01 versus ACMSD WT-siControl+Control vector group. (D) HK-2 cells were transfected with siTSPYL2 or siControl and exposed to H/R after 24h or not exposed (Control). Thereafter, cells were collected for ChIP assays to detect the binding of SREBP-2 at the ACMSD promoter. Pull-down was performed with anti-IgG or anti-SREBP-2 antibodies. Levels of ACMSD promoter were measured in samples and normalized to the levels of the IgG pull-down assay in the Control-siControl group. ∗p < 0.05, ∗∗p < 0.01 versus anti-IgG-Control-siControl group. (E) The protein expression of SREBP-2, SIRT1, and p300 in the kidneys of sham-treated mice and IRI-exposed mice earlier injected with shControl or shTSPYL2 was analyzed by Western blotting. Representative blots are shown and bar graphs of the quantitative analyses of several experiments. ∗∗p < 0.01 versus Sham-shControl group; #p < 0.05 versus IRI-shControl group. (F) HK-2 cells were transfected with siTSPYL2 or siControl. Cells were exposed to H/R after 24h or not exposed (Control). Protein levels of SREBP-2, SIRT1, and p300 were detected by Western blotting. Representative blots are shown and bar graphs of the quantitative analyses of several experiments. ∗∗p < 0.01 versus Control-siControl group; #p < 0.05 versus the H/R-siControl group. (G) Endogenous SREBP-2 was immunoprecipitated from mouse kidney tissues of sham-treated mice or IRI-exposed mice which were earlier injected with shControl or shTSPYL2. In immunoprecipitates, acetylated SREBP-2 was detected by Western blotting. Staining for SREBP-2 was done to check for the immunoprecipitation of SREBP-2. Quantification of band of acetylated and total SREBP-2 were performed and acetylated SREBP-1 levels relative to total levels were calculated and normalized to the value of sham-shControl mice. ∗p < 0.05 versus Sham-shControl group; #p < 0.05 versus IRI-shControl group. (H) Endogenous SREBP-2 was immunoprecipitated from HK-2 cells exposed to H/R or not exposed (Control) which were earlier transfected with siTSPYL2 or siControl. Acetylated SREBP-2 was detected by Western blotting in immunoprecipitates. SREBP-2 acetylation was detected by Western blotting by incubating with acetyl-Lys antibody. Staining for SREBP-2 was done to check for the immunoprecipitation of SREBP-2. Quantification of band of acetylated and total SREBP-2 were performed and acetylated SREBP-1 levels relative to total levels were calculated and normalized to the value of Control-cells. ∗∗p < 0.01 versus Control-siControl group; ##p < 0.01 versus the H/R-siControl group. (I) HK-2 cells were transfected with siTSPYL2 in combination with a plasmid carrying p300 or siSIRT1. After 24h, cells were exposed to H/R or not exposed (Control). Protein levels of SREBP-2, SIRT1, and p300 were detected by Western blotting. Representative blots are shown and bar graphs of the quantitative analyses of three experiments. ∗p < 0.05, ∗∗p < 0.01. (J) HK-2 cells were transfected with siTSPYL2 in combination with a plasmid carrying p300 or siSIRT1. After 24h, cells were exposed to H/R or not exposed (Control). Endogenous SREBP-2 was immunoprecipitated and acetylated SREBP-2 was detected by Western blotting. A bar graph shows the results of the SREBP-2 acetylation/total SREBP-2 ratio in HK-2 cells which is normalized to the value of Control cells. ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SD. p value calculated was determined by a two-tailed unpaired Student’s t test.