Mouse Kidney Cells Research Articles

MTORC1 and mTORC2 Co-Protect against Cadmium-Induced Renal Tubular Epithelial Cell Apoptosis and Acute Kidney Injury by Regulating Protein Kinase B.

The potential threat of cadmium (Cd)-induced acute kidney injury (AKI) is increasing. In this study, our primary goal was to investigate the individual roles played by mTOR complexes, specifically mTORC1 and mTORC2, in Cd-induced apoptosis in mouse kidney cells. We constructed a mouse model with specific deletion of Raptor/Rictor renal cells. Inhibitors and activators of mTORC1 or mTORC2 were also applied. The effects of protein kinase B (AKT) activation and autophagy were studied. Both mTORC1 and mTORC2 were found to mediate the antiapoptotic mechanism of renal cells by regulating the AKT activity. Inhibition of mTORC1 or mTORC2 exacerbated Cd-induced kidney cell apoptosis, suggesting that both proteins exert antiapoptotic effects under Cd exposure. We further found that the AKT activation plays a key role in mTORC1/TORC2-mediated antiapoptosis, protecting Cd-exposed kidney cells from apoptosis. We also found that mTOR activators inhibited excessive autophagy, alleviated apoptosis, and promoted cell survival. These findings provide new insights into the regulatory mechanisms of mTOR in renal diseases and provide a theoretical basis for the development of novel therapeutic strategies to treat renal injury.

#2023 The role of neutrophil extracellular traps in cisplatin-induced acute kidney injury

Abstract Background and Aims Acute kidney injury (AKI) is a public health problem that seriously endangers human health. The exact pathogenesis of AKI has not been fully elucidated. Previous studies have shown that the immune system plays a key role in the inflammatory response of AKI in the acute phase, especially innate immunity. Recent discoveries have highlighted that neutrophils, in particular the neutrophil extracellular traps (NETs), have central roles in the occurrence and development of AKI. However, the specific role and mechanism of NETs in AKI of different etiologies are remain unclear. This study aimed to investigate the role of NETs in the development of cisplatin-induced AKI and provide potential therapeutic targets for AKI treatment. Method Wild-type (WT) and NETs-deficient (PAD4-/-) mouse models of AKI were constructed by intraperitoneal injection of cisplatin. Mice were weighed at 24, 48, and 72 hours after modeling, and sacrificed at 72 hours. Serum and kidney specimens were collected for renal function analysis, pathological, immunohistochemical staining, and molecular biology experiments. Mouse bone marrow neutrophils were isolated from healthy mouse. NETs were induced in vitro by adding phorbol 12 myristate 13-acetate (PMA). Primary renal tubular epithelial cells (TECs) of mouse kidney were extracted, and the cells were collected after 24 h of cisplatin and cisplatin combined with NETs intervention, respectively. The inflammatory indicators such as TNF-α, IL-1β, MCP-1, and renal injury indicators such as KIM-1 and Ngal were detected by RT-PCR. Results Compared with the WT-cisplatin group, the PAD4-/- -cisplatin group showed a reduced degree of weight loss after modeling, lower serum creatinine and urea nitrogen levels, and significantly improved renal function. The PAD4-/- -cisplatin group showed a significant decrease in the inflammatory indicators TNF-α, IL-1β, IL-6, and renal injury indicators KIM-1 and Ngal. Cisplatin intervention for 6 hours could induce the generation of NETs. Compared with the cisplatin intervention group, the expression of inflammatory indicators TNF-α, IL-1β, and MCP-1 were significantly higher in the cisplatin combined with NETs intervention TECs group. Conclusion Cisplatin directly induces neutrophils to generate NETs, which can exacerbate cisplatin-induced injury to TECs in vitro. The inhibition of NETs by PAD4-/- improves cisplatin-induced AKI. The inhibition of NETs may be a novel target for the treatment of cisplatin AKI.

Macrophages communicate with mesangial cells through the CXCL12/DPP4 axis in lupus nephritis pathogenesis

Lupus nephritis (LN) occurs in 50% of cases of systemic lupus erythematosus (SLE) and is one of the most serious complications that can occur during lupus progression. Mesangial cells (MCs) are intrinsic cells in the kidney that can regulate capillary blood flow, phagocytose apoptotic cells, and secrete vasoactive substances and growth factors. Previous studies have shown that various types of inflammatory cells can activate MCs for hyperproliferation, leading to disruption of the filtration barrier and impairment of renal function in LN. Here, we characterized the heterogeneity of kidney cells of LN mice by single-nucleus RNA sequencing (snRNA-seq) and revealed the interaction between macrophages and MCs through the CXC motif chemokine ligand 12 (CXCL12)/dipeptidyl peptidase 4 (DPP4) axis. In culture, macrophages modulated the proliferation and migration of MCs through this ligand–receptor interaction. In LN mice, treatment with linagliptin, a DPP4 inhibitor, effectively inhibited MC proliferation and reduced urinary protein levels. Together, our findings indicated that targeting the CXCL12/DPP4 axis with linagliptin treatment may serve as a novel strategy for the treatment of LN via the CXCL12/DPP4 axis.

Macula densa mTORC1 is an important regulator of cellular and aging-dependent kidney functions

Macula densa (MD) cells are major regulators of nephron, kidney, and whole body functions, including the control of renal blood flow, glomerular filtration rate (GFR), renin release and thus the maintenance of body fluid and electrolyte balance. Our recent studies identified the high rate of MD protein synthesis regulated by the mTORC1 complex of the mechanistic target of rapamycin (mTOR) as a novel component of MD cell and glomerular hemodynamic regulatory functions. Raptor is an essential player in mTORC1’s key roles to regulate cell energy homeostasis, growth, protein synthesis, mitochondrial function, apoptosis, and autophagy via phosphorylation of substrates including ribosomal protein S6 kinase (S6K) and eukaryotic translation-initiation factor 4E-binding proteins (4E-BPs). mTORC1 signaling controls the aging process, and deficient mTORC1 activity extends health and lifespan. Therefore, we hypothesized that MD cell-specific mTOR loss of function (lof) prevents aging-related kidney function decline. The present study aimed to demonstrate the key importance of MD cell mTOR activity in renal physiology and aging by characterizing the phenotype of a newly generated genetic MD-mTOR lof mouse model. MD-mTOR lof mice were generated by crossing Nos1-CreERT2 and Raptor floxed mice. Tamoxifen induction (at 3 weeks of age) of Nos1-CreERT2+/- Raptor−/− mice resulted in conditional and inducible Raptor deletion in cells of the Nos1 lineage, which in the kidney is entirely specific to MD cells. Kidney structure/function phenotyping was performed using intravital multiphoton microscopy (MPM), the Medibeacon transdermal GFR measurement system, and tissue harvest, immunohistochemistry, and the OPP incorporation assay of protein synthesis. High and MD-specific Raptor, pS6K, and p4EBP1 immunofluorescence labeling was observed in WT kidney sections. The immunolabeling of these mTOR component and targets were absent specifically in MD cells in MD-mTOR lof mouse kidneys, validating the MD cell-specific genetic approach. The intensity of incorporated OPP labeling was highest in MD cells among all renal cell types in WT. In contrast, MD OPP labeling was significantly reduced in MD-mTOR lof mice indicating diminished, mTORC1-dependent global MD cell protein synthesis. Importantly, in 15-month old aged mice the GFR was significantly higher (preserved) in MD-mTOR lof (1794±197 uL/min/100g BW) compared to WT (1117±61) and MD-mTOR gof (929±61) mice (n=6-18 mice in each group). Altogether, these studies identified mTORC1 as an essential regulator of MD cell biology, and the importance of MD cells in aging-related kidney function decline. MD cells may be important new targets in future anti-aging therapies. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Upregulation of PD-1 and its ligands and expansion of T peripheral helper cells in the nephritic kidneys of lupus-prone BXSB-Yaa mice.

This study aimed to investigate the role of the programmed cell death protein 1 (PD-1) pathway and T peripheral helper (Tph) cells in the pathogenesis of lupus nephritis using lupus-prone BXSB-Yaa mice. Male BXSB-Yaa mice and age-matched male C57BL/6 mice were used. The expression of PD-1 and its ligands (programmed cell death 1 ligand-1, PD-L1 and programmed cell death 1 ligand-2, PD-L2) and the phenotypes of kidney-derived cells and splenocytes expressing these molecules were analyzed by immunofluorescence and flow cytometry. Nephritis spontaneously developed in 16-week-old but not in 8-week-old BXSB-Yaa or C57BL/6 mice. PD-1 was expressed on CD4+ mononuclear cells (MNCs) that infiltrated the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of CD4+PD-1+CXCR5-ICOS+ kidney-derived Tph cells was higher in 16-week-old than in 8-week-old BXSB-Yaa and C57BL/6 mice, whereas the frequency of CD4+PD-1+CXCR5+ICOS+ kidney-derived T follicular helper (Tfh) cells was not significantly different between the mice. PD-L1 was constitutively expressed in the renal tubules. PD-L2 was expressed in the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of PD-L1highCD11c+CD3-CD19- and PD-L2+CD11c+CD3-CD19- kidney-derived MNCs in 16-week-old BXSB-Yaa mice was significantly higher than that of the control mice. The percentage of kidney-derived Tph cells but not Tfh cells was correlated with the urinary protein levels in the nephritic mice. The results of this study suggest that kidney-infiltrating PD-1+ Tph cells expanded concomitantly with the upregulation of PD-L1 and PD-L2 in the kidneys and the progression of lupus nephritis.

Co-exposure of arsenic and polystyrene-nanoplastics induced kidney injury by disrupting mitochondrial homeostasis and mtROS-mediated ferritinophagy and ferroptosis

Arsenic (As) and polystyrene nanoplastics (PSNPs) co-exposure induced biotoxicity and ecological risks have attracted wide attention. However, the combined effects of As and PSNPs on the kidney and their underlying mechanisms of toxicities remain to be explored. Here, we investigated the effects of As and PSNPs co-exposure on structure and function in mice kidney, and further explored the possible mechanisms. In this study, we identified that co-exposure to As and PSNPs exhibited conspicuous renal structural damage and pathological changes, accompanied by renal tissue fibrosis (increased protein expression of Collagen I and α-SMA and deposition of collagen fibers), whereas alone exposure to As or PSNPs does not exhibit nephrotoxicity. Subsequently, our results further showed that combined action of As and PSNPs induced mitochondrial oxidative damage and impaired mitochondrial dynamic balance. Furthermore, co-treatment with As and PSNPs activated NCOA4-mediated ferritinophagy and ferroptosis in mice kidney and TCMK-1 cells, which was confirmed by the changes in the expression of ferritinophagy and ferroptosis related indicators (NCOA4, LC3, ATG5, ATG7, FTH1, FTL, GPX4, SLC7A11, FSP1, ACSL4 and PTGS2). Meaningfully, pretreatment with the mtROS-targeted scavenger Mito-TEMPO significantly attenuated As and PSNPs co-exposure induced mitochondrial damage, ferritinophagy and ferroptosis. In conclusion, these findings demonstrated that mtROS-dependent ferritinophagy and ferroptosis are important factors in As and PSNPs co-exposure induced kidney injury and fibrosis. This study provides a new insight into the study of combined toxicity of nanoplastics and heavy metal pollutants.

Alternating projection combined with fast gradient projection (FGP-AP) method for intensity-only measurement optical diffraction tomography in LED array microscopy.

Optical diffraction tomography (ODT) is a powerful label-free measurement tool that can quantitatively image the three-dimensional (3D) refractive index (RI) distribution of samples. However, the inherent "missing cone problem," limited illumination angles, and dependence on intensity-only measurements in a simplified imaging setup can all lead to insufficient information mapping in the Fourier domain, affecting 3D reconstruction results. In this paper, we propose the alternating projection combined with the fast gradient projection (FGP-AP) method to compensate for the above problem, which effectively reconstructs the 3D RI distribution of samples using intensity-only images captured from LED array microscopy. The FGP-AP method employs the alternating projection (AP) algorithm for gradient descent and the fast gradient projection (FGP) algorithm for regularization constraints. This approach is equivalent to incorporating prior knowledge of sample non-negativity and smoothness into the 3D reconstruction process. Simulations demonstrate that the FGP-AP method improves reconstruction quality compared to the original AP method, particularly in the presence of noise. Experimental results, obtained from mouse kidney cells and label-free blood cells, further affirm the superior 3D imaging efficacy of the FGP-AP method.

Histone deacetylase-mediated silencing of PSTPIP2 expression contributes to aristolochic acid nephropathy-induced PANoptosis.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by using herbal medicines. Currently, no therapies are available to treat or prevent aristolochic acid nephropathy. Histone deacetylase (HDAC) plays a crucial role in the development and progression of renal disease. We tested whether HDAC inhibitors could prevent aristolochic acid nephropathy and determined the underlying mechanism. HDACs expression in the aristolochic acid nephropathy model was examined. The activation of PANoptosis of mouse kidney and renal tubular epithelial cell were assessed after exposure to HDAC1 and HDAC2 blockade. Kidney-specific knock-in of proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) mice were used to investigate whether PSTPIP2 affected the production of PANoptosome. Aristolochic acid upregulated the expression of HDAC1 and HDAC2 in the kidneys. Notably, the HDAC1 and HDAC2 specific inhibitor, romidepsin (FK228, depsipeptide), suppressed aristolochic acid-induced kidney injury, epithelial cell pyroptosis, apoptosis and necroptosis (PANoptosis). Moreover, romidepsin upregulated PSTPIP2 in renal tubular epithelial cells, which was enhanced by aristolochic acid treatment. Conditional knock-in of PSTPIP2 in the kidney protected against aristolochic acid nephropathy. In contrast, the knockdown of PSTPIP2 expression in PSTPIP2-knock-in mice restored kidney damage and PANoptosis. PSTPIP2 function was determined in vitro using PSTPIP2 knockdown or overexpression in mouse renal tubular epithelial cells (mTECs). Additionally, PSTPIP2 was found to regulate caspase 8 in aristolochic acid nephropathy. HDAC-mediated silencing of PSTPIP2 may contribute to aristolochic acid nephropathy. Hence, HDAC1 and HDAC2 specific inhibitors or PSTPIP2 could be valuable therapeutic agents for preventing aristolochic acid nephropathy.

Lack of mitochondrial Cyp2E1 drives acetaminophen-induced ER stress-mediated apoptosis in mouse and human kidneys: Inhibition by 4-methylpyrazole but not N-acetylcysteine

Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney injury.

Farnesoid X receptor activation protects against renal fibrosis via modulation of β-catenin signaling

ObjectiveActivation of farnesoid X receptor (FXR), a bile acid nuclear receptor, may be implicated in the pathophysiology of diabetic nephropathy. We explored a possible role for FXR activation in preventing renal fibrosis in high fat diet (HFD)-fed mice. MethodsWe investigated the effects of HFD on mouse kidney and renal tubular epithelial cells both in vivo and in vitro, and observed the changes of FXR and β-catenin pathway. FXR agonist was also used to alleviate this HFD-induced effect, and the interaction between FXR and β-catenin was further verified. ResultsMice were fed by a 60% kcal fat diet for 20 weeks developed the typical traits of metabolic syndrome with subsequent renal lipid accumulation and renal injury. Treatment with the FXR agonist CDCA or GW4064 decreased body weight, renal lipid accumulation, as well as renal injury. Moreover, renal β-catenin signaling was activated and improved with FXR-agonist treatment in HFD-fed mice. To examine whether FXR affected β-catenin signaling, and was involved in tubulo-interstitial fibrosis, we explored the FXR expression and function in ox-LDL induced-renal tubular injury. In rat proximal tubular epithelial cells (NRK-52E) stimulated by ox-LDL, FXR protein was decreased compared to control group, and phosphorylated (Ser675) β-catenin was activated by ox-LDL in a dose- and time-dependent manner. Ox-LDL enhanced α-SMA and fibronectin expressions and reduced E-cadherin levels, whereas FXR agonism or FXR overexpression inhibited fibronectin and α-SMA expressions and restored E-cadherin. Moreover, FXR agonist treatment also decreased phosphorylated (Ser675) β-catenin, nuclear translocation and β-catenin-mediated transcription induced by ox-LDL in NRK-52E cells. We showed that FXR could bind with β-catenin via the AF1 domain, and disrupt the assembly of the core β-catenin/TCF4 complex. ConclusionThese experimental data suggest that FXR activation, via modulating β-catenin signaling, may contribute to attenuating the development of lipid-mediated tubulo-interstitial fibrosis.

Alpha-linolenic acid protects against methotrexate-induced nephrotoxicity in mouse kidney cells.

Methotrexate (MTX) is a folic acid antagonist used in chronic inflammatory diseases and various cancer treatments. Although the main mechanism of the toxic effect of MTX is not known, it is stated that it causes oxidative stress and inflammation. Alpha-linolenic acid (ALA) protects against oxidative stress, apoptosis, and inflammation. For this reason, we aimed to find out the useful effect of ALA on MTX-induced nephrotoxicity MATERIALS AND METHODS: The mice were divided into 4 groups randomly. The control group was treated with physiological saline solution; the ALA group was treated with ALA (200 mg/kg) by gavage; MTX-treated group received 20 mg/kg i.p. (intraperitoneal) MTX; and MTX+ALA treated group received 20 mg/kg i.p. MTX and ALA 200 mg/kg by gavage. All of the drugs were performed once a day for 9 days. Alpha-linolenic acid significantly decreased oxidative stress parameters and MTX-induced inflammatory and apoptotic mediators. Furthermore, histopathological examination showed that MTX induced significant edematous damage, and ALA treatment attenuated this damage in renal tissue. Our results revealed that ALA may be helpful against MTX-induced nephrotoxicity in mice via its antioxidant and anti-inflammatory properties.

The SGLT2 inhibitor dapagliflozin improves kidney function in glycogen storage disease XI.

Glycogen storage disease XI, also known as Fanconi-Bickel syndrome (FBS), is a rare autosomal recessive disorder caused by mutations in the SLC2A2 gene that encodes the glucose-facilitated transporter type 2 (GLUT2). Patients develop a life-threatening renal proximal tubule dysfunction for which no treatment is available apart from electrolyte replacement. To investigate the renal pathogenesis of FBS, SLC2A2 expression was ablated in mouse kidney and HK-2 proximal tubule cells. GLUT2Pax8Cre+ mice developed time-dependent glycogen accumulation in proximal tubule cells and recapitulated the renal Fanconi phenotype seen in patients. In vitro suppression of GLUT2 impaired lysosomal autophagy as shown by transcriptomic and biochemical analysis. However, this effect was reversed by exposure to a low glucose concentration, suggesting that GLUT2 facilitates the homeostasis of key cellular pathways in proximal tubule cells by preventing glucose toxicity. To investigate whether targeting proximal tubule glucose influx can limit glycogen accumulation and correct symptoms in vivo, we treated mice with the selective SGLT2 inhibitor dapagliflozin. Dapagliflozin reduced glycogen accumulation and improved metabolic acidosis and phosphaturia in the animals by normalizing the expression of Napi2a and NHE3 transporters. In addition, in a patient with FBS, dapagliflozin was safe, improved serum potassium and phosphate concentrations, and reduced glycogen content in urinary shed cells. Overall, this study provides proof of concept for dapagliflozin as a potentially suitable therapy for FBS.

3-MCPD Induced Mitochondrial Damage of Renal Cells Via the Rhythmic Protein BMAL1 Targeting SIRT3/SOD2.

Biorhythm regulates a variety of physiological functions and enables organisms to adapt to changing environments. 3-Monochloro-1,2-propanediol (3-MCPD) is a common food thermal processing contaminant, and the kidney is its toxic target organ. However, the nephrotoxicity mechanism of 3-MCPD has not been fully elucidated. In the study, we found that 3-MCPD caused mitochondrial damage in renal cells by inhibiting the SIRT3/SOD2 pathway. Further, we found that 3-MCPD could interfere with rhythm protein BMAL1 expression at protein and mRNA levels in mice kidney and NRK-52E cells. Simultaneously, the balance of the daily oscillation of SIRT3/SOD2 pathway proteins was impeded under 3-MCPD treatment. To determine the role of BAML1 in mitochondrial damage, we overexpressed the BMAL1 protein. The data showed that BMAL1 overexpression upregulated SIRT3 and SOD2 expression and attenuated mitochondrial damage caused by 3-MCPD. These results indicated that 3-MCPD inhibited the SIRT3/SOD2 pathway by affecting the expression of the rhythm protein BMAL1, thereby inducing mitochondrial damage in renal cells. Taken together, our work reveals that 3-MCPD may possess a toxic effect via circadian clock mechanisms.

Melatonin attenuates cellular senescence and apoptosis in diabetic nephropathy by regulating STAT3 phosphorylation

AimsMelatonin is an endogenous hormone related to the regulation of biorhythm. Previous researchers have found that melatonin can ameliorate diabetic nephropathy (DN), but the mechanism remains to be elucidated. To discover the possible mechanism by which melatonin prevents DN, we investigated the potential effects of melatonin on signal transducer and activator of transcription 3 (STAT3) on the progression of cellular senescence and apoptosis. Main methodsCellular senescence, apoptosis and the underlying mechanism of melatonin were investigated both in vivo and in vitro. C57BL/6 mice were intraperitoneally injected with streptozotocin (STZ) to establish DN. For an in vitro model of DN, human renal cortex proximal epithelial tubule (HK-2) cells were exposed to high glucose conditions. Key findingsMelatonin inhibited the phosphorylation of STAT3, decreased the expression of senescence proteins p53, p21 and p16INK4A. Melatonin also downregulated the expression of apoptotic proteins, including cleaved PARP1, cleaved caspase-9 and -3. Melatonin treatment decreased the positive area of senescence-associated galactosidase (SA-β-gal) staining and the number of TUNEL-positive cells in kidneys of DN mice. In vitro, melatonin inhibited STAT3 phosphorylation and lowered cellular senescence and apoptosis markers, in a manner similar to the STAT3 inhibitor S3I-201. In addition, the inhibition effect of melatonin on cellular senescence and apoptosis in HK-2 cells was reversed by the usage of recombinant IL-6 (rIL-6), which can induce STAT3 phosphorylation. SignificanceWe, for the first time, demonstrate that melatonin inhibits STAT3 phosphorylation, which is involved in alleviating the cellular senescence and apoptosis in DN.

Biotin-modified cyclometalated iridium-based photosensitizers as mitochondria-targeted theranostic agents for tumor photodynamic therapy in vitro and in vivo

Photodynamic therapy (PDT) is a promising non-invasive treatment technique for various types of cancer. Photoluminescent cyclometalated Ir(III) complexes, a distinctive genre of photosensitizers (PSS), have garnered significant interest due to their exceptional selective toxicity towards cancerous cells and minimal cytotoxicity towards normal cells. This study aimed to investigate the application of three newly designed biotin-modified cyclometalated Ir(III) complexes (Ir1-Ir3) for the treatment of cancer. The three complexes were found to have good photocatalytic activity, with singlet oxygen (1O2) yields ranging from 0.24 to 0.65 in aqueous solutions. The results suggest that these complexes have the potential as phototherapeutic and photodiagnostic agents for cancer treatment. Additionally, the biotin fragments in the Ir1-Ir3 structures enable their selective targeting of tumor cells. Notably, Ir3 displayed negligible dark toxicity towards A549 human lung cancer cells (IC50 > 100 μM), but its value decreased to 0.25 μM after 15 min of 425 nm (40 mW/cm2) light exposure. Furthermore, these complexes exhibited low phototoxicity towards non-cancerous BHK mouse kidney cells. Further investigation demonstrated that Ir3 had potent targeting abilities for mitochondria, and could trigger cell apoptosis through the activation of ROS-mediated mitochondrial signaling pathways under 425 nm light irradiation. In vivo studies revealed that Ir3 effectively suppressed tumor growth in A549 xenograft-bearing mice when exposed to 425 nm light. These results indicate that Ir3 has the potential to be a valuable photosensitizer drug for the photodynamic treatment of tumors.

Abstract A006: Identification of a potential lymphatic progenitor cell population in Vhlh conditional knockout mouse and in ccRCC tissue

Abstract Introduction. We aim to elucidate the role of lymphatic cells in the progression of clear-cell renal cell carcinoma (ccRCC). Objective. Inactivation of tumor suppressor gene von Hippel–Lindau (VHL) is the main cause of ccRCC, whose progression is extensively influenced by the tumor microenvironment (TME). It has been shown that tissue inflammation, with extensive infiltration of lymphocytes, is a feature of ccRCC tissue. Here, the role of the lymphatic cells in the inflammatory microenvironment was analyzed in the kidney with or without VHL inactivation. Methods. The conditional Vhlh knockout mouse (Hoxb7-Cre-GFP; Vhlh loxP/loxP) was established as a ccRCC model. Single-cell RNA sequencing (scRNA-seq) of podoplanin-positive (Pdpn+) cells revealed heterogeneity of lymphatic cells in mouse kidney. Immunostaining of 3-dimensional tissue was used to visualize lymphatic outgrowth from lymphatic vessels. Protein marker expression in mouse kidney and human ccRCC sections was detected by immunofluorescence. Relevant molecular mechanisms were quantified by qPCR, Western blot, flow cytometry, and ELISA. Results. The lymphatic vasculature is responsible for draining interstitial fluid, mediating lymphocyte recruitment, and participating in immune responses, and is a major route for metastasis. Immunofluorescence of the classical lymphatic markers such as Lyve1, Prox1, CD31, and VEGFR3 was observed in the lymphatic vessels including collecting lymphatics, lymphatic valves, and lymphatic capillaries. Moreover, immunostaining of 3-dimensional tissue showed sprouting Pdpn+ cells from lymphatic vessels, more numerous in Vhlh mutants. In Vhlh-deficient kidney tissue, the Pdpn+ cells form linearly arrayed clusters with no or small lumen and exhibit cuboidal cell shape instead of the flattened endothelial shape. Similar PDPN+ clusters of cells were also present in ccRCC tissues, compared with normal adjacent tissues. Interestingly, scRNA-seq identified a subpopulation of the progenitor marker CD133+ cells within the Pdpn+ population, providing evidence for the differentiation of novel lymphatic endothelial progenitor cells (LEPCs) in postnatal tissues in the kidney. Especially, there was a concomitant threefold increase in CD45+ lymphocyte populations in Vhlh conditional knockout mice compared to wild-type. Interestingly, the chemokine CXCl13 was highly expressed in LEPCs and the receptor CXCR5 was expressed in Vhlh-deficient tubule cells and in infiltrating immune cells. These results suggest a crosstalk between LEPCs and Vhlh-deficient cells and immune cells to modulate metastasis and immune responses. Double knockout (Hoxb7-Cre-GFP; VhlhloxP/loxP; Cxcl13-/-) will be designed for further experiments to investigate the role of CXCL13/CXCR5 axis in the progression of ccRCC. Conclusion. These investigations provide novel insights into CXCL13/CXCR5 axis that may orchestrate LEPCs and Vhlh-deficient tubule cells interactions that promote lymphocyte infiltration and potential metastasis within the tumor microenvironment. Citation Format: Tuong-Vi Nguyen, Hieu-Huy Nguyen-Tran, Thi-Ngoc Nguyen, Sheng-Chieh Lin, Tien Hsu. Identification of a potential lymphatic progenitor cell population in Vhlh conditional knockout mouse and in ccRCC tissue [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A006.

Cinnamyl Chalcone Based AIE Fluorescent Probes for Sensitive Detection of Hydrazine and its Application in Living Cells.

Widely utilized in the chemical industry and agriculture, hydrazine is easily absorbed by living things and can cause physical harm when in touch for an extended period of time. As a result, a novel cinnamaldehyde chalcone C5 was produced by Friedel Crafts process and aldol condensation reaction. Triphenylamine was used as the raw material for hydrazine determination in both reactions. Chalcone C5 exhibits significant AIE behavior in a mixed mixture of ethanol and water in addition to having great selectivity and a low detection limit (0.119nm) for hydrazine. The solvent effect test revealed a linear relationship between the Stokes shift of C5 in the solvent and the rise in solvent orientation polarization. It is important to note that C5 is not harmful to MCF-7 cells, mouse kidney cells, or pig kidney cells. Furthermore, research on cell imaging has demonstrated that probe C5 may be utilized to image the fluorescence of hydrazine in active MCF-7 cells.

Pharmacological and Pathological Effects of Mulberry Leaf Extract on the Treatment of Type 1 Diabetes Mellitus Mice.

This study investigated the pharmacological and pathological effects of aqueous mulberry leaf extract on type 1 diabetes mellitus mice induced with an intraperitoneal injection of streptozotocin (STZ). Diabetic mice were randomized into six groups: control (normal group), model, metformin-treated mice, and high-dose, medium-dose, and low-dose mulberry. The mulberry-treated mice were divided into high-, medium-, and low-dose groups based on the various doses of aqueous mulberry leaf extract during gavage. The efficacy of the six-week intervention was evaluated by measuring levels of fasting plasma glucose, alkaline phosphatase, alanine aminotransferase, aspartate transaminase, blood urea nitrogen, gamma-glutamyl transferase, glucose, high-density lipoprotein cholesterol, lactate dehydrogenase, and low-density lipoprotein cholesterol and recording body weight. Results revealed that mulberry leaf extract exhibited an ideal hypoglycemic effect, and the high-dose group was the most affected. Histology analysis, glycogen staining and apoptosis detection were used to study the extract's effects on the liver, kidney, and pancreatic cells of diabetic mice, enabling the assessment of its effectiveness and complications on a clinical and theoretical basis. It was shown that a certain concentration of aqueous mulberry leaf extract repaired the islet cells of type 1 diabetes mellitus mice, promoting normal insulin secretion. Herein, it was confirmed that mulberry leaf could be used to develop new hypoglycemic drugs or functional health food with broad applicability.

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Haploinsufficiency of progranulin causes cell type specific impairments in PINK1/Parkin mitophagy

AbstractBackgroundGRN mutations that result in progranulin haploinsufficiency cause frontotemporal dementia (FTD). Mitophagy, the selective autophagy of damaged mitochondria, is impaired in several neurodegenerative diseases. A number of genes linked to FTD (e.g. OPTN, SQSTM, VCP and TBK1) are also known to play a role in mitophagy. Xenophagy, the selective autophagy of non‐host pathogens, relies on some of the same proteins as mitophagy (Parkin and TBK1) and is reduced in GRN knockout mice. Progranulin has also been found to play a role in mitophagy in mouse kidney cells. We therefore hypothesised that loss of progranulin could lead to defective mitophagy in neurons, astrocytes and microglia.MethodsWe investigated PINK1/Parkin mitophagy in astrocytic‐like H4 cells and neuron‐like Parkin overexpressing SHSY5Y cells (PoE‐5Ys) +/‐ siRNA against GRN. We also examined induced pluripotent stem cells (iPSCs) from four controls, three patients with FTD‐associated GRN mutations (R493X and C31fs) and a CRISPR series from the human iPSC Neurodegenerative Disease Initiative1,2,3 (iNDI) with an isogenic control, heterozygous and homozygous R493X mutation lines. The iPSCs were differentiated to cortical neurons, astrocytes and microglia. PINK1/Parkin mitophagy was induced using Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb), other mitophagy markers as well as proteins hypothesised to play a role in the mechanism were examined using western blotting and immunofluorescence (ICC).ResultsLower levels of pUb were detected in PoE‐5Ys treated with oligomycin/antimycin following GRN knockdown. There was a significant reduction in mitophagy in GRN siRNA treated H4 cells, detected by ICC and western blotting of mitophagy markers. Initial results from iPSC neurons found no significant difference in mitophagy between control and patient lines. Work is ongoing in a larger set of iPSC neurons, astrocytes and microglia. Preliminary results suggest that reducing progranulin affects mitophagy in astrocytes but has a limited effect on mitophagy in neurons.ConclusionsThe results suggest that progranulin has a cell specific role in mitophagy with progranulin regulating stability and/or activity of PINK1. Current work aims to understand the mechanisms of this process and to dissect cell‐type specific contributions of progranulin to mitophagy.