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The Journal of International Medical... Dec 2020To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury.
OBJECTIVE
To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury.
METHODS
An model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha () expression were compared.
RESULTS
Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group.
CONCLUSIONS
Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating expression and enhancing autophagy.
Topics: Animals; Apoptosis; Autophagy; Mice; Podocytes; Puromycin; Puromycin Aminonucleoside; Tacrolimus
PubMed: 33322998
DOI: 10.1177/0300060520971422 -
Frontiers in Medicine 2021Podocytes are an integral part of the glomerular filtration barrier. Many genes are already known to be essential for podocyte survival, structure and function, but...
Podocytes are an integral part of the glomerular filtration barrier. Many genes are already known to be essential for podocyte survival, structure and function, but there are more podocyte essential genes to be identified. By single-cell RNA-seq of mouse podocytes, we detected the expression of gene encoding MCC regulator of WNT signaling pathway (MCC) in majority of the podocytes and speculated that MCC is essential for podocytes. We confirmed MCC expression in mouse podocytes and further showed its expression in human podocytes. To experimentally prove the essentiality of MCC for podocytes, we knocked down MCC in cultured podocytes and found marked morphological change of cell shape, cytoskeletal F-actin stress fiber disruption, increased apoptosis, and downregulation of podocyte essential genes, CD2AP and WT1, demonstrating that MCC is essential for podocytes. Since MCC has been implicated in cell cycle and β-catenin signaling, we examined the expression of cell cycle related genes and activity of β-catenin in the MCC knockdown podocytes, but did not find significant changes. To further explore the mechanism underlying the role of MCC in podocytes, we performed RNA-sequencing and bioinformatics analysis of MCC knockdown podocytes and found a significant enrichment of the regulated genes in lamellipodia formation. Consistently, we found that MCC is present in lamellipodia and MCC knockdown resulted in loss of lamellipodia in the cells. Lastly, we found that MCC was downregulated in podocytes treated with puromycin aminonucleosides and in glomeruli of diabetic mice and FSGS patients, implicating MCC is involved in the development of podocytopathy and proteinuria. In conclusion, MCC is potentially essential for podocytes and its downregulation may be involved in podocytopathy.
PubMed: 34926519
DOI: 10.3389/fmed.2021.777563 -
Kidney International Sep 2021Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized...
Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.
Topics: Animals; Apolipoprotein A-I; Endothelial Cells; Kidney; Lymphangiogenesis; Lymphatic Vessels; Mice; Rats; Vascular Endothelial Growth Factor C
PubMed: 34102217
DOI: 10.1016/j.kint.2021.05.028 -
International Journal of Medical... 2021Cathepsin B (CTSB) was well documented in solid tumors, up-regulated of CTSB expression is linked with progression of tumors. However, the study of CTSB in adult... (Observational Study)
Observational Study
Cathepsin B (CTSB) was well documented in solid tumors, up-regulated of CTSB expression is linked with progression of tumors. However, the study of CTSB in adult leukemia has not been reported. Total RNA was isolated from PBMC (peripheral blood mononuclear cell) of AML patients and healthy donors. qRT-PCR was performed to detect the expression of CTSB. The association of CTSB expression with the patients' overall survival (OS) and disease-free survival (DFS) were analyzed. Stable HL-60 CTSB-shRNA cell lines were established by retrovirus infection and puromycin selection. Cell proliferation was detected by CCK-8 analysis. Tumorigenesis ability was analyzed by soft agar and xenograft nude mice model. Western blot was performed to detect the expression of CTSB and the proteins of cell signaling pathway. The mRNA expression level of CTSB was up-regulated in AML patients compared to healthy control (<0.001), and CTSB expression was significantly higher in M1, M2, M4 and M5 AML samples than healthy control. The CTSB expression in AML was associated with WBC count (=0.037). Patients with high CTSB expression had a relatively poor OS (=0.007) and a shorter DFS (=0.018). Moreover, the expression level of CTSB may act as an independent prognostic factor for both OS (=0.011) and DFS (=0.004). Knockdown CTSB expression in HL-60 cells could inhibit the cells' proliferation and tumorigeneses and . Further study showed knockdown CTSB expression in HL-60 cells could inactive the AKT signaling pathway. CTSB mRNA was upregulated in AML patients. CTSB overexpression was correlated with poor prognosis and may serve as an independent prognostic factor for both OS and DFS in AML patients. Knockdown CTSB expression in HL-60 cells could inhibit the cells' proliferation and tumorigenesis. The underlying mechanism may be the inhibition of the AKT signaling pathway.
Topics: Carcinogenesis; Case-Control Studies; Cathepsin B; Cell Proliferation; Disease-Free Survival; Female; Follow-Up Studies; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; HL-60 Cells; Healthy Volunteers; Humans; Leukemia, Myeloid, Acute; Leukocyte Count; Male; Middle Aged; Neoplasm Recurrence, Local; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Up-Regulation; Xenograft Model Antitumor Assays
PubMed: 33628106
DOI: 10.7150/ijms.54206 -
ELife Apr 2023Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by...
Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by labeling the nascent proteome with amino acid analogs or isotope-containing amino acids. These methods have been difficult to implement in vivo as they require lengthy amino acid replacement procedures. O-propargyl-puromycin (OPP) is a puromycin analog that incorporates into nascent polypeptide chains. Through its terminal alkyne, OPP can be conjugated to a fluorophore-azide for directly visualizing nascent protein synthesis, or to a biotin-azide for capture and identification of newly-synthesized proteins. To achieve cell type-specific OPP incorporation, we developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog harboring an enzyme-labile blocking group that can be removed by penicillin G acylase (PGA). Here, we show that cell type-specific PGA expression in can be used to achieve OPP labeling of newly-synthesized proteins in targeted cell populations within the brain. Following a brief 2 hr incubation of intact brains with PhAc-OPP, we observe robust imaging and affinity purification of OPP-labeled nascent proteins in PGA-targeted cell populations. We apply this method to show a pronounced age-related decline in neuronal protein synthesis in the fly brain, demonstrating the capability of PhAc-OPP to quantitatively capture in vivo protein synthesis states. This method, which we call POPPi (GA-dependent ncorporation), should be applicable for rapidly visualizing protein synthesis and identifying nascent proteins synthesized under diverse physiological and pathological conditions with cellular specificity in vivo.
Topics: Animals; Proteome; Drosophila; Azides; Amino Acids; Puromycin
PubMed: 37092974
DOI: 10.7554/eLife.83545 -
Journal of Ethnopharmacology Jun 2022Danggui-shaoyao-san (DSS), a representative formula of Traditional Chinese Medicine (TCM) for promoting blood circulation and diuresis (Huo-Xue-Li-Shui) therapy, has...
ETHNOPHARMACOLOGICAL RELEVANCE
Danggui-shaoyao-san (DSS), a representative formula of Traditional Chinese Medicine (TCM) for promoting blood circulation and diuresis (Huo-Xue-Li-Shui) therapy, has been used to clinically nephrotic syndrome (NS) and relieve nephrotic edema.
AIM OF THE STUDY
To explore the effects and mechanisms of DSS in improving sodium retention and to identify the bioactive compounds from DSS.
MATERIALS AND METHODS
DSS prescriptions were disassembled into Yangxue-Huoxue (YXHX) and Jianpi-Lishui (JPLS). A nephrotic rat model was induced with puromycin aminonucleoside (PAN), and the effects on urinary sodium excretion, urinary plasmin(gen) content, and plasmin activity of DSS, YXHX, and JPLS extracts were assessed. The inhibitory effects on urokinase-type plasminogen activator (uPA) and plasmin activity of extracts were evaluated in vitro. Bio-affinity ultrafiltration and high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (BAU-UPLC-Q/TOF-MS) were used to rapidly screen and qualitatively analyze the uPA/plasmin affinity compounds from DSS extract. Additionally, uPA/plasmin inhibition assays and molecular docking were used to verify the activity and affinity mechanisms of the potential bioactive compounds.
RESULTS
In vivo, DSS, YXHX, and JPLS prevented sodium retention in nephrotic rats. DSS and YXHX treatment decreased urinary plasmin activity but did not alter urinary plasmin(ogen) concentration, and their extracts showed strong uPA and plasmin inhibitory activity in vitro. These results suggested that uPA and plasmin are direct targets of DSS and YXHX in intervening NS sodium retention. Using BAU-UPLC-Q/TOF-MS, gallic acids, methyl gallate, albiflorin, and 1,2,3,4,6-O-pentagalloylglucose (PGG) were screened as uPA or plasmin affinity compounds. Among them, PGG was found to be a uPA and plasmin dual inhibitor, with an IC of 6.861 μM against uPA and an IC of 149.0 μM against plasmin. The molecular docking results of PGG with uPA and plasmin were consistent with the verification results.
CONCLUSION
Intervening in sodium retention by inhibiting uPA-mediated plasmin generation and plasmin activity in the kidneys could be possible mechanisms for DSS, as indicated by the results in PAN-induced nephrotic rats. We conclude that PGG is a potential bioactive compound responsible for the effect of DSS on natriuresis.
Topics: Animals; Drugs, Chinese Herbal; Female; Fibrinolysin; Humans; Male; Molecular Docking Simulation; Nephrotic Syndrome; Rats; Sodium; Ultrafiltration; Urokinase-Type Plasminogen Activator
PubMed: 35259444
DOI: 10.1016/j.jep.2022.115171 -
American Journal of Physiology. Renal... Dec 2021Macula densa (MD) cells, a chief sensory cell type in the nephron, are endowed with unique microanatomic features including a high density of protein synthetic...
Macula densa (MD) cells, a chief sensory cell type in the nephron, are endowed with unique microanatomic features including a high density of protein synthetic organelles and secretory vesicles in basal cell processes ("maculapodia") that suggest a so far unknown high rate of MD protein synthesis. This study aimed to explore the rate and regulation of MD protein synthesis and their effects on glomerular function using novel transgenic mouse models, newly established fluorescence cell biology techniques, and intravital microscopy. Sox2-tdTomato kidney tissue sections and an -propargyl puromycin incorporation-based fluorescence imaging assay showed that MD cells have the highest level of protein synthesis within the kidney cortex followed by intercalated cells and podocytes. Genetic gain of function of mammalian target of rapamycin (mTOR) signaling specifically in MD cells (in MD-mTOR mice) or their physiological activation by low-salt diet resulted in further significant increases in the synthesis of MD proteins. Specifically, these included both classic and recently identified MD-specific proteins such as cyclooxygenase 2, microsomal prostaglandin E synthase 1, and pappalysin 2. Intravital imaging of the kidney using multiphoton microscopy showed significant increases in afferent and efferent arteriole and glomerular capillary diameters and blood flow in MD-mTOR mice coupled with an elevated glomerular filtration rate. The presently identified high rate of MD protein synthesis that is regulated by mTOR signaling is a novel component of the physiological activation and glomerular hemodynamic regulatory functions of MD cells that remains to be fully characterized. This study discovered the high rate of protein synthesis in macula densa (MD) cells by applying direct imaging techniques with single cell resolution. Physiological activation and mammalian target of rapamycin signaling played important regulatory roles in this process. This new feature is a novel component of the tubuloglomerular cross talk and glomerular hemodynamic regulatory functions of MD cells. Future work is needed to elucidate the nature and (patho)physiological role of the specific proteins synthesized by MD cells.
Topics: Animals; Autocrine Communication; Diet, Sodium-Restricted; Glomerular Filtration Rate; Green Fluorescent Proteins; Humans; Intravital Microscopy; Juxtaglomerular Apparatus; Luminescent Proteins; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; Nitric Oxide Synthase Type I; Paracrine Communication; Protein Biosynthesis; Renin; Signal Transduction; Sodium, Dietary; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Red Fluorescent Protein; Mice
PubMed: 34693742
DOI: 10.1152/ajprenal.00222.2021 -
Bioscience Reports May 2021Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts...
Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts its effects remain unclear. In the present study, we investigated the effects of Shensu IV on glomerular podocyte injury in nephrotic rats and puromycin-induced injury in cultured podocytes, and assessed the associated molecular mechanisms. Liquid chromatography-mass spectrometry (LC-MS) results showed that the main components of Shensu IV were l-Carnitine, P-lysoPC (LPC) 16:0, Coumaroyl tyramine, Tetramethylpyrazine, LPC 18:1, Choline, (S,S)-Butane-2,3-diol, and Scopoletin. We further found that nephrotic rats displayed pathological alterations in kidney tissues and ultrastructural changes in glomerular podocytes; however, these effects were reversed with Shensu IV treatment. Compared with the control, the numbers of autophagosomes were markedly reduced in the model group, but not in the Shensu IV treatment group. Furthermore, the expression of p62 was significantly higher in the model group than in the controls, whereas the LC3-II/I ratio was significantly lower; however, these changes were not observed when Shensu IV was administered. The protective effects of Shensu IV were further confirmed in podocytes displaying puromycin-induced injury. Compared with control group, the expression of long non-coding RNA (lncRNA) H19, mTOR, p-mTOR, and p62 was significantly increased in the puromycin group, whereas that of distinct subgroup of the RAS family member 3 (DIRAS3) was significantly decreased, as was the LC3-II/I ratio. The opposite results were obtained for both shH19- and Shensu IV-treated cells. Collectively, our data demonstrated that Shensu IV can prevent glomerular podocyte injury in nephrotic rats and puromycin-treated podocytes, likely via promoting lncRNA H19/DIRAS3-regulated autophagy.
Topics: Animals; Autophagy; Cells, Cultured; Drugs, Chinese Herbal; Male; Microtubule-Associated Proteins; Nephrosis; Podocytes; Puromycin; RNA, Long Noncoding; Rats; Rats, Sprague-Dawley; TOR Serine-Threonine Kinases; rho GTP-Binding Proteins
PubMed: 33881140
DOI: 10.1042/BSR20203362 -
Stem Cell Research & Therapy Jul 2022Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X...
BACKGROUND
Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B.
METHODS
First, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system.
RESULTS
The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system.
CONCLUSIONS
The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level.
Topics: Animals; CRISPR-Cas Systems; Codon, Nonsense; Factor IX; Hemophilia A; Hemophilia B; Humans; Mice; Mouse Embryonic Stem Cells; Mutation; Technology
PubMed: 35883203
DOI: 10.1186/s13287-022-03036-2 -
International Journal of Reproductive... Oct 2022The relationship between the biochemical characteristics of follicular fluid (FF), oocyte quality and embryonic development has not yet been elucidated. We compared...
BACKGROUND
The relationship between the biochemical characteristics of follicular fluid (FF), oocyte quality and embryonic development has not yet been elucidated. We compared samples of FF with a normal metabolic profile against samples with metabolic abnormalities to identify potential predictive biomarkers of reproductive success.
OBJECTIVE
To analyze peptide activity in the FF of women undergoing in vitro fertilization using 3 samples of FF per individual.
MATERIALS AND METHODS
FF samples were obtained by ovum pick-up. Pathological samples were defined as samples of FF obtained from women with a gynecological condition or with infertility. A total of 30 women participated in this study. 3 samples of FF were obtained per individual (90 samples), but 8 samples were excluded because they were hemolyzed. The samples (n = 82 FF) included controls (n = 36, donors without fertility problems), women with endometriosis (n = 15), unexplained infertility (n = 19), and aged 39 (n = 12). We assessed local encephalinergics: aminopeptidase-N (puromycin sensitive aminopeptidase and neutral endopeptidase; and components of the angiotensin system of the reproductive tract: prolyl-endopeptidase, APN, aspartate-aminopeptidase, and basic-aminopeptidase.
RESULTS
No differences were observed in peptide metabolism based on the presence or absence of oocytes in the FF. Women with endometriosis and aged 39 yr showed alterations in puromycin sensitive aminopeptidase (p = 0.01), aminopeptidase-B (p = 0.01), aspartate-aminopeptidase (p 0.001) and neutral endopeptidase (p 0.001).
CONCLUSION
This study reveals alterations in the metabolism of enkephalin and angiotensin in pathological FF, which points to these components as potential diagnostic biomarkers.
PubMed: 36381359
DOI: 10.18502/ijrm.v20i10.12269