Journal: Frontiers in Immunology

Article Title: Premature renal epithelial cell senescence promoted by LXN/Rps3/p53 signaling pathway activation increases calcium oxalate crystal deposition by altering macrophage polarization

doi: 10.3389/fimmu.2025.1658989

Figure Lengend Snippet: LXN knockdown inhibited oxalate induced senescence of HK-2 cells as well as subsequent macrophage polarization. HK-2 cells were treated with 0.5 mM oxalate with/without 10 μM fisetin. (A) LXN, GLS, PTGS1 and CFB mRNA expression were determined via RT-qPCR. The ratio of relative expression of LXN, GLS, PTGS1 and CFB genes induced by oxalate or oxalate+fisetin compared to control group were shown. Relative LXN mRNA levels detected via RT-qPCR (B) and LXN protein expression determined via western blot (C) were significantly increased in oxalate induced HK-2 cells compared to control cells, and further significantly down-regulated by fisetin. LXN knockdown was performed via transfecting specific LXN small interfering RNA (siRNA) into HK-2 cells, and these HK-2 cells were further incubated with/without oxalate for 48h. Analysis of senescence in HK-2 cells were performed via SA-β-gal activity (D) and relative p16 protein expression (E) . Results showed that SA-β-gal activity and p16 protein level were significantly increased by oxalate, and further significantly decreased under LXN knockdown intervention. SASP-associated factors (F) (VEGF, MMP13, MCP-1 and IL-1β) in HK-2 cells and (G) (IL-1β, MMP13, IL-6 and MMP3) in HK-2 cells culture medium demonstrated similar changing trends with p16 protein levels. THP-1 cells were stimulated with PMA for differentiation into macrophages (MΦs). Polarization was performed with LPS & IFN-γ (M1) or IL-4 & IL-13 (M2) for 24 h, and simultaneously, culture medium from fresh medium incubated HK-2 cells for another 48h which cells have been already knocked down by si-LXN with/without oxalate intervention for 48h. THP-1 cells differentiated either to M1 (H, I) or M2 (J, K) macrophages were analyzed by RT-qPCR (H, J) or western blot (I, K) . Based on three independent experiments. Data are presented as means ± SD. * P < 0.05, ** P < 0.01, ** *P < 0.001; CTL, control group. Scr siRNA, Scrambled siRNA control.

Article Snippet: Hematoxylin and eosin (HE) staining, Von Kossa staining and immunohistochemical analysis were performed as described previously ( – ).Immunostaining was performed with specific antibodies against the target proteins LXN (Bioss, bs-1971R), Rps3 (Absin, abs116130), p53 (Bioss, bs-2090R), p16 (Bioss, bs-0740R), iNOS (Bioss, bs-0162R) and CD163 (Bioss, bs-2527R).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Control, Western Blot, Small Interfering RNA, Incubation, Activity Assay

Journal: Frontiers in Immunology

Article Title: Premature renal epithelial cell senescence promoted by LXN/Rps3/p53 signaling pathway activation increases calcium oxalate crystal deposition by altering macrophage polarization

doi: 10.3389/fimmu.2025.1658989

Figure Lengend Snippet: LXN/Rps3/p53 pathway was involved in the oxalate induced senescent cell burden. (A) Laser confocal microscopy was applied to evaluate nuclear localization of Rps3. Nuclear localization of Rps3 were significantly decreased in HK-2 cells under oxalate induction compared to control cells, and these decreased nuclear translocation of Rps3 were significantly up-regulated by fisetin treatment. mRNA (B) and protein (C) expression of Rps3 in HK-2 cells under oxalate with/without fisetin treatment appeared no significant changes compared to control group. Oxalate induction was performed on the basis of LXN knockdown. (D) LXN knockdown has little effect on expression levels of Rps3/p53 mRNA, and Rps3 silencing has little effect on LXN3/p53 mRNA expression. (E) p53 protein expression was significantly down-/up-regulated by LXN/Rps3 knockdown. (F) Nuclear translocation of Rps3 was obviously elevated after LXN silencing. Data are presented as means ± SD. ** P < 0.01, ** *P < 0.001; CTL, control group. Scr siRNA=Scrambled siRNA control.

Article Snippet: Hematoxylin and eosin (HE) staining, Von Kossa staining and immunohistochemical analysis were performed as described previously ( – ).Immunostaining was performed with specific antibodies against the target proteins LXN (Bioss, bs-1971R), Rps3 (Absin, abs116130), p53 (Bioss, bs-2090R), p16 (Bioss, bs-0740R), iNOS (Bioss, bs-0162R) and CD163 (Bioss, bs-2527R).

Techniques: Confocal Microscopy, Control, Translocation Assay, Expressing, Knockdown

Journal: Frontiers in Immunology

Article Title: Premature renal epithelial cell senescence promoted by LXN/Rps3/p53 signaling pathway activation increases calcium oxalate crystal deposition by altering macrophage polarization

doi: 10.3389/fimmu.2025.1658989

Figure Lengend Snippet: Cellular senescence-targeting therapy could ameliorate the renal impairment and crystal depositions in the animal model. (A) Process diagrams are demonstrated. (B, C) The levels of Scr (B) and BUN (C) in kidney stone model rats were measured. Compared with those in the normal group, Scr and BUN levels in the oxalate group were increased notably on Day 30, and fisetin markedly ameliorated the renal functional damage caused by oxalate crystals. The elevated levels of Scr and BUN in oxalate group could be significantly relieved by injection of AAV-shLXN. (D) Renal histological analyses of H&E staining showed that fisetin lowered the grade of tubules dilatation along with decreased levels of mononuclear cell infiltrates in the interstitium compared to that in oxalate group. AAV-shLXN showed a more significant inhibitory effect on tubules dilatation and mononuclear cell infiltrates compared to fisetin. (E) Von Kossa staining revealed similar changing trends of crystal formation in kidney tissue as histological H&E staining analysis. (F) Immunohistochemistry analysis demonstrated that immunostaining intensity of LXN and p53 were significantly stronger in the oxalate group compared with normal group, and the staining intensity of LXN and p53 were significantly decreased under fisetin or AAV-shLXN administration, even more significantly in AAV-shLXN group. There was no significant change of total Rps3 staining among different groups. Data are presented as means ± SD. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. “ns” indicates no significant difference.

Article Snippet: Hematoxylin and eosin (HE) staining, Von Kossa staining and immunohistochemical analysis were performed as described previously ( – ).Immunostaining was performed with specific antibodies against the target proteins LXN (Bioss, bs-1971R), Rps3 (Absin, abs116130), p53 (Bioss, bs-2090R), p16 (Bioss, bs-0740R), iNOS (Bioss, bs-0162R) and CD163 (Bioss, bs-2527R).

Techniques: Animal Model, Functional Assay, Injection, Staining, Immunohistochemistry, Immunostaining

Journal: Frontiers in Immunology

Article Title: Premature renal epithelial cell senescence promoted by LXN/Rps3/p53 signaling pathway activation increases calcium oxalate crystal deposition by altering macrophage polarization

doi: 10.3389/fimmu.2025.1658989

Figure Lengend Snippet: Schematic illustration of the mechanism of LXN/Rps3/p53 pathway on cellular senescence in CaOx stone formation. Activation of LXN gene in renal tubular epithelial cells (RTECs) is evoked by hyperoxaluria and oxalate. Since ribosomal protein subunit 3 (Rps3) is a LXN binding protein, the nuclear translocation of Rps3 were significantly down-regulated due to the more combination conducted by LXN protein. And then p53 which might be a downstream target of Rps3 is up-regulated, with a consequence of triggering RTECs’ senescence. Senescence-Associated Secretory Phenotype (SASP) factors are then released, skewing macrophage polarization towards pro-inflammatory M1 phenotype, not anti-inflammatory M2 phenotype, demonstrating a low crystals phagocytic ability. Therefore LXN gene targeting silencing could effectively inhibit cellular senescence, thus reduce macrophage polarization towards M1 phenotype and ultimately behave a protective effect on kidney stone formation.

Article Snippet: Hematoxylin and eosin (HE) staining, Von Kossa staining and immunohistochemical analysis were performed as described previously ( – ).Immunostaining was performed with specific antibodies against the target proteins LXN (Bioss, bs-1971R), Rps3 (Absin, abs116130), p53 (Bioss, bs-2090R), p16 (Bioss, bs-0740R), iNOS (Bioss, bs-0162R) and CD163 (Bioss, bs-2527R).

Techniques: Activation Assay, Binding Assay, Translocation Assay