Fig 2: TP53RK was upregulated in kidneys of chronic kidney disease (CKD) patients, kidney fibrosis murine models, and transforming growth factor ß1 (TGF-ß1) induced kidney cells. A) RNA sequencing analysis of human fibrotic kidneys identifies an upregulation of TP53RK expression. Expression of genes was measured using fragments per kilobase million (FPKM). N = 8 in normal group and n = 53 in CKD group. Data were extracted from https://www.ncbi.nlm.nih.gov/geo, accession: GSE66494. B) Representative immunofluorescence detection of TP53RK expression in biopsy samples from normal individual and CKD patients hospitalized in Department of Nephrology, Children's Hospital of Nanjing Medical University. Scale bar: 10 µm. C) Quantitative analysis of TP53RK expression in hospitalized CKD patients, with mild (n = 5), moderate (n = 7), or severe (n = 6) fibrosis compared to the normal group (n = 5) based on immunohistochemical staining. The interstitial fibrosis score was evaluated by a blinded pathologist by estimating the extent of tubular atrophy, infiltration of inflammatory cells, and interstitial fibrosis (0, None; 1, =25%; 2, 25–50%; 3, =50% for each aspect). Individuals with an interstitial fibrosis score of 1–3 were classified as mild, 4–6 as moderate, and those between 7 and 9 as severe fibrosis. D) Pearson correlation analysis of TP53RK levels and renal interstitial fibrosis scores, blood urea nitrogen (BUN) levels, serum creatinine (sCr) levels and estimated glomerular filtration rates (eGFR) of 18 hospitalized CKD patients. E) Representative immunohistochemical staining of TP53RK in kidneys of unilateral ureteral obstruction (UUO) and sham operated mice (7 days after surgery). Scale bar: 20 µm. F) Immunoblot and semiquantification of TP53RK in kidneys of UUO and sham operated mice at day 7 (n = 6). G) Representative immunohistochemical staining of TP53RK in kidneys of unilateral ischemia reperfusion (UIR) and sham operated mice (3 weeks after surgery). Scale bar: 20 µm. H) Representative immunoblotting and semiquantification of TP53RK in day 21 kidneys of UIR and sham operated mice (n = 7). I) Immunofluorescence co-staining of TP53RK and tubular specific marker lotus tetragonolobus lectin (LTL) in UUO and UIR murine kidneys. Red, TP53RK; Green, LTL; Scale bar: 10 µm. J) Immunofluorescence co-staining of TP53RK and fibroblast specific marker S100A4 in UUO and UIR kidneys. Red, TP53RK; Green, S100A4, Scale bar: 10 µm. K) Protein expression of TP53RK in mouse proximal tubular epithelial cells (mPTCs) treated with TGF-ß1 (2.5, 5, and 10 ng mL-1) or vehicle (0 ng mL-1) for 24 h. L) Protein expression of TP53RK in NRK-49Fs treated with TGF-ß1 (1, 2.5, and 5 ng mL-1) or vehicle (0 ng mL-1) for 24 h. Data are presented as mean ± SEM; two-tailed unpaired t-test was used to determine statistical significance of (A), (F) and (H); one-way ANOVA followed by Dunnett's multiple comparisons test was used to determine statistical significance of (C), (K) and (L); *p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the normal/sham or vehicle group.

Fig 3: Genetic and pharmaceutic inhibition of TP53RK alleviated transforming growth factor ß1 (TGF-ß1) induced renal interstitial fibroblasts activation and attenuated unilateral ureteral obstruction (UUO) induced renal fibrosis. A) TP53RK was efficiently deleted in renal interstitial fibroblasts of conditional TP53RK knockout mice (TP53RKfl/fl; S100A4-Cre). B) TP53RKfl/fl and TP53RKfl/fl; S100A4-Cre mice of were subjected to sham or UUO operation and euthanized 7 days after establishment of the model in Figure B–F. Masson's trichrome staining of kidney tissues of mice in corresponding groups. Scale bar: 20 µm. C) Quantification analysis of fibrotic area based on Masson's trichrome staining (n = 5–8). D,E) Western blot and semiquantification of a-SMA, POSTN, VIM, and FN expression (n = 5–8). F) mRNA levels of Acta2, Postn, Vim, Fn, and Col1a1 in kidney of corresponding groups (n = 5–7). K) Control (vector) and TP53RK knockdown (sgTP53RK) NRK-49Fs were treated with vehicle or TGF-ß1 (5 ng mL-1) for 24 h. Immunoblots of TP53RK, POSTN, and FN are shown. Protein semiquantification is presented in Figure S3B (Supporting Information) (n = 3). L) Control (vector) and TP53RK KD NRK-49F cells (sgTP53RK) were treated as in (K). Cells were stained for EdU and Hoechst 33342. The EdU incorporation rate was quantified by normalizing the number of EdU positive cells against total cell nucleus number (n = 4). Representative images are shown in Figure S3D (Supporting Information). M) NRK-49Fs were pretreated with vehicle or FA (5 × 10-6, 10 × 10-6, or 20 × 10-6 m) 2 h before TGF-ß1 (5 ng mL-1) treatment for another 24 h. Representative Western blot analyses of TP53RK, POSTN, and FN in each group are shown. Protein semiquantification is presented in Figure S3E (Supporting Information) (n = 3). Data are presented as mean ± SEM; two-way ANOVA followed by Tukey's multiple comparisons test was used to determine statistical significance of (C), (E), (F), and (L); *** p < 0.001 compared with the TP53RKfl/fl + Sham or vector + vehicle group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the TP53RKfl/fl + UUO or vector + TGF-ß1 group.

Fig 4: Inhibition of TP53RK alleviated transforming growth factor ß1 (TGF-ß1) induced partial epithelial–mesenchymal transition (p-EMT) and attenuated unilateral ureteral obstruction (UUO) induced renal fibrosis. A) CRISPR/Cas9 genome engineering was employed to generate floxed TP53RK mice (TP53RKfl/fl). B) TP53RK was efficiently deleted in renal tubular of tubular conditional TP53RK knockout mice (TP53RKfl/fl; Kap-Cre). C) TP53RKfl/fl and TP53RKfl/fl; Kap-Cre mice were subjected to sham or UUO operation and euthanized at day 7 in Figure (C–G). Masson's trichrome staining of kidney tissues of corresponding groups. Scale bar: 20 µm. D) Quantification analysis of fibrotic area based on Masson's trichrome staining (n = 7–8). E,F) Immunoblot and semiquantification of a-SMA, POSTN, and FN expression (n = 6–8). G) Quantitative reverse transcription PCR (qRT-PCR) analysis of the mRNA level of Acta2, Vim, Postn, Col1a1, and Col3a1 in kidney of corresponding groups (n = 7 in each group). H) Control and TP53RK knockdown (KD) mouse proximal tubular epithelial cells (mPTCs) were treated with TGF-ß1 (10 ng mL-1) for 24 h and harvested for Western blot analysis of TP53RK, POSTN, and FN. Protein semiquantification is presented in Figure S2B (Supporting Information). I) mPTCs were pretreated with vehicle or fusidic acid (FA) (5 × 10-6, 10 × 10-6, or 20 × 10-6 m) 2 h before TGF-ß1 (10 ng mL-1) treatment for another 24 h. Representative Western blot analyses of TP53RK, POSTN, and FN in each group are shown. Protein semiquantification is presented in Figure S2C (Supporting Information). Data are presented as mean ± SEM; two-way ANOVA followed by Tukey's multiple comparisons test was used to determine statistical significance of (D), (F), and (G); ** p < 0.01, and *** p < 0.001 compared with the TP53RKfl/fl + Sham group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the TP53RKfl/fl + UUO group.

Fig 5: Schematic diagram illustrating the profibrotic role of TP53RK in kidney. In response to damage factors such as transforming growth factor ß1 (TGF-ß1), expression of TP53RK in renal tubular epithelial cells and interstitial fibroblasts are both upregulated. Strengthened expression of TP53RK phosphorylates and stabilizes downstream Birc5 and facilitates its nuclear translocation. Enhanced Birc5 mediates partial epithelial–mesenchymal transition (p-EMT) of renal tubular epithelial cells, proliferation, and activation of renal interstitial fibroblasts, and finally drives chronic kidney disease (CKD) progression.