RESUMEN
Cisplatin-induced nephrotoxicity restricts its clinical use against solid tumors. The present study elucidated the pharmacological effects of Renogrit, a plant-derived prescription medicine, using cisplatin-induced human renal proximal tubular (HK-2) cells and Caenorhabditis elegans. Quantification of phytochemicals in Renogrit was performed on HPTLC and UHPLC platforms. Renogrit was assessed in vitro in HK-2 cells post-exposure to clinically relevant concentration of cisplatin. It was observed that renoprotective properties of Renogrit against cisplatin-induced injury stem from its ability to regulate renal injury markers (KIM-1, NAG levels; NGAL mRNA expression), redox imbalance (ROS generation; GST levels), and mitochondrial dysfunction (mitochondrial membrane potential; SKN-1, HSP-60 expression). Renogrit was also found to modulate apoptosis (EGL-1 mRNA expression; protein levels of p-ERK, p-JNK, p-p38, c-PARP1), necroptosis (intracellular calcium accumulation; RIPK1, RIPK3, MLKL mRNA expression), mitophagy (lysosome population; mRNA expression of PINK1, PDR1; protein levels of p-PINK1, LC3B), and inflammation (IL-1ß activity; protein levels of LXR-α). More importantly, Renogrit treatment did not hamper normal anti-proliferative effects of cisplatin as observed from cytotoxicity analysis on MCF-7, A549, SiHa, and T24 human cancer cells. Taken together, Renogrit could be a potential clinical candidate to mitigate cisplatin-induced nephrotoxicity without compromising the anti-neoplastic properties of cisplatin.
Asunto(s)
Apoptosis , Caenorhabditis elegans , Cisplatino , Mitofagia , Cisplatino/efectos adversos , Cisplatino/toxicidad , Animales , Humanos , Mitofagia/efectos de los fármacos , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Extractos Vegetales/farmacología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Antineoplásicos/efectos adversos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patologíaRESUMEN
Bisphenol P (BPP) has been detected in human biological samples; however studies on its nephrotoxicity are scarce. Given the susceptibility of kidneys to endocrine-disrupting chemicals, there is an urgent need to investigate the renal toxicity of BPP. This study aimed to evaluate the effects of different concentrations of BPPs on the kidneys of C57BL/6 mice and elucidate the underlying mechanisms of renal damage using a combination of mouse renal transcriptomic data and human renal proximal tubular epithelial cells (HK-2). Mice were exposed to BPP (0, 0.3, 30, 3000 µg/kg bw/d) via gavage for 5 weeks. Renal injury was assessed based on changes in body and kidney weights, serum renal function indices, and histopathological examination. Transcriptomic analysis identified differentially expressed genes and pathways, whereas cellular assays were used to measure cell viability, reactive oxygen species (ROS), apoptosis, and the expression of key genes and proteins. The results show that BPP exposure induces renal injury, as evidenced by increased body weight, abnormal renal function indices, and renal tissue damage. Transcriptomic analysis revealed alterations in genes and pathways related to oxidative stress, p53 signaling, autophagy, and apoptosis. Cellular experiments confirmed that BPP induces oxidative stress and apoptosis. Furthermore, BPP exposure significantly inhibits autophagy, potentially exacerbating apoptosis and contributing to kidney injury. Treatment with a ROS inhibitor (N-Acetylcysteine, NAC) mitigated BPP-induced autophagy inhibition and apoptosis, implicating oxidative stress as a key factor. BPP exposure may lead to renal injury through excessive ROS accumulation, oxidative stress, inflammatory responses, autophagy inhibition, and increased apoptosis. The effects of NAC highlight the role of oxidative stress in BPP-induced nephrotoxicity. These findings enhance our understanding of BPP-induced nephrotoxicity and underscore the need to control BPP exposure to prevent renal disease. This study emphasized the importance of evaluating the safety of new Bisphenol A analogs, including BPP, in environmental toxicology.
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Células Epiteliales , Ratones Endogámicos C57BL , Estrés Oxidativo , Fenoles , Animales , Humanos , Ratones , Apoptosis/efectos de los fármacos , Compuestos de Bencidrilo/toxicidad , Disruptores Endocrinos/toxicidad , Células Epiteliales/efectos de los fármacos , Riñón/citología , Riñón/efectos de los fármacos , Riñón/patología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Estrés Oxidativo/efectos de los fármacos , Fenoles/toxicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Dromedary camels can survive and reproduce in desert areas. The unique anatomical structure of the kidney enables the camel to prevent water loss. The present study aimed to investigate the ultrastructure of the peroxisomes in the normal kidney of the adult dromedary camel. Tissue samples were taken from the cortex and outer medulla of the kidney of eight camels. The samples were then processed for histological and ultrastructural investigations. The epithelial cells of the proximal tubules displayed peroxisomes with varying sizes and shapes. The peroxisomes were observed in either dispersed or clustered arrangement. Each peroxisome exhibited a homogenous matrix enveloped by a single membrane. Several peroxisomes exhibited one or more dark marginal plates that were always strongly associated with the smooth endoplasmic reticulum. The intensity of the peroxisomal matrix differed significantly, either within the same cell or across different cells. The intensity was light or dark, with a few peroxisomes presenting a similar intensity to that of the mitochondria. Some peroxisomes contained nucleoids within their matrix. The peroxisomes in the first and second sections of proximal convoluted tubules were scattered and primarily located in the region between the microvilli and the underlying mitochondria. The peroxisomes in the third region were abundant and frequently aggregated in clusters throughout the cytoplasm. In the fourth region, the number of peroxisomes was low. The proximal straight tubule had a limited quantity of peroxisomes. In conclusion, peroxisomes in the proximal tubule in kidney of normal dromedary camel were similar in shape and size to other mammals; however, heterogeneity exists as a result of differences in species-specific peroxisomal proteins. Peroxisomes are suggested to be a major source of metabolic energy and act as hydrogen peroxide (H2O2) scavengers, resulting in the release of water and oxygen.
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Camelus , Riñón , Peroxisomas , Animales , Camelus/anatomía & histología , Camelus/fisiología , Peroxisomas/ultraestructura , Peroxisomas/metabolismo , Riñón/ultraestructura , Riñón/anatomía & histología , Microscopía Electrónica de Transmisión/veterinaria , Masculino , Túbulos Renales Proximales/ultraestructura , Túbulos Renales Proximales/anatomía & histologíaRESUMEN
BACKGROUND/AIM: Angiotensinogen (AGT), a precursor of angiotensin II (AngII), contributes to regulating (patho)physiological conditions, including blood pressure changes, inflammation, and kidney fibrosis. However, the precise role of tissue-specific AGT in kidney fibrosis independent of blood pressure remains to be fully understood. This study investigated the source of intrarenal AGT and its role in kidney injury and fibrosis during obstructive nephropathy. MATERIALS AND METHODS: Proximal tubule- (PT, major source secreting AGT in the kidney; PKO) or liver- (major source of circulating AGT; LKO) AGT knockout (KO) mice were subjected to unilateral ureteral obstruction (UUO), a blood pressure-independent fibrosis model. RESULTS: UUO increased AGT mRNA and protein levels in the kidneys. PKO decreased AGT mRNA, but LKO enhanced it in UUO kidneys compared with the control. In contrast, the intrarenal protein levels of AGT increased in PKO, but not in LKO in UUO kidneys, indicating that the liver is a major source of intrarenal AGT protein. Expression of megalin, a PT receptor involved in the uptake of circulating AGT, was down-regulated in UUO kidneys and was independent of PKO or LKO. However, none of these changes prevented UUO-induced tubular injury and kidney fibrosis. CONCLUSION: Hepatic and proximal tubule AGT play distinct roles in contributing to intrarenal AGT levels during UUO, and their genetic inhibitions fail to prevent kidney injury and fibrosis, suggesting a highly complicated signaling pathway of the renin-angiotensin system and an associated compensatory mechanism in obstructive nephropathy.
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Angiotensinógeno , Modelos Animales de Enfermedad , Fibrosis , Riñón , Ratones Noqueados , Obstrucción Ureteral , Animales , Angiotensinógeno/metabolismo , Angiotensinógeno/genética , Ratones , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/genética , Obstrucción Ureteral/patología , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Enfermedades Renales/metabolismo , Enfermedades Renales/etiología , Enfermedades Renales/patología , Enfermedades Renales/genética , Hígado/metabolismo , Hígado/patología , MasculinoRESUMEN
RATIONALE: Light chain proximal tubulopathy (LCPT) is a rare form of renal impairment associated with multiple myeloma (MM). LCPT is caused by inclusions formed of free light chains that are typically crystalline, but can also be noncrystalline structures. PATIENT CONCERNS: A 62-year-old man was hospitalized for the investigation of abnormal urine test results lasting for 1 year and kidney-function abnormalities persisting for more than 1 month. DIAGNOSES: Noncrystalline LCPT and MM. INTERVENTIONS: The patient was treated with the lenalidomide, bortezomib, and dexamethasone and pomalidomide, bortezomib, and dexamethasone chemotherapy regimens. OUTCOMES: Complete remission of MM was achieved, and the patient's renal function returned to normal. LESSONS: This case report highlights the importance of renal pathology in the diagnosis of patients with unexplained chronic kidney disease and proteinuria.
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Mieloma Múltiple , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/complicaciones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/patología , Cadenas Ligeras de Inmunoglobulina/orina , Túbulos Renales Proximales/patología , Dexametasona/uso terapéutico , Cuerpos de Inclusión/patología , Talidomida/uso terapéutico , Talidomida/análogos & derivados , Lenalidomida/uso terapéutico , Lenalidomida/administración & dosificación , Bortezomib/uso terapéuticoRESUMEN
Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.
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Metabolismo Energético , Inflamación , Estrés Oxidativo , Tramadol , Estrés Oxidativo/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/metabolismo , Tramadol/efectos adversos , Tramadol/farmacología , Metabolismo Energético/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Humanos , Línea Celular , Animales , Analgésicos Opioides/efectos adversos , Analgésicos Opioides/farmacologíaRESUMEN
The inhibition of the autophagolysosomal pathway mediated by transcription factor EB (TFEB) inactivation in proximal tubular epithelial cells (TECs) is a key mechanism of TEC injury in diabetic kidney disease (DKD). Acetylation is a novel mechanism that regulates TFEB activity. However, there are currently no studies on whether the adjustment of the acetylation level of TFEB can reduce the damage of diabetic TECs. In this study, we investigated the effect of Trichostatin A (TSA), a typical deacetylase inhibitor, on TFEB activity and damage to TECs in both in vivo and in vitro models of DKD. Here, we show that TSA treatment can alleviate the pathological damage of glomeruli and renal tubules and delay the DKD progression in db/db mice, which is associated with the increased expression of TFEB and its downstream genes. In vitro studies further confirmed that TSA treatment can upregulate the acetylation level of TFEB, promote its nuclear translocation, and activate the expression of its downstream genes, thereby reducing the apoptosis level of TECs. TFEB deletion or HDAC6 knockdown in TECs can counteract the activation effect of TSA on autophagolysosomal pathway. We also found that TFEB enhances the transcription of Tfeb through binding to its promoter and promotes its own expression. Our results, thus, provide a novel therapeutic mechanism for DKD that the alleviation of TEC damage by activating the autophagic lysosomal pathway through upregulating TFEB acetylation can, thus, delay DKD progression.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Nefropatías Diabéticas , Células Epiteliales , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos , Túbulos Renales Proximales , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Nefropatías Diabéticas/metabolismo , Ratones , Acetilación , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Ácidos Hidroxámicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Masculino , Ratones Endogámicos C57BL , Autofagia/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
Adult polycystic kidney disease (APKD) is a genetic disorder leading to premature renal dysfunction and failure. The prevalence of malignant renal tumors occurring in the APKD setting has been rarely reported. OBJECTIVE: To better characterize malignant renal tumors in nephrectomy specimens of APKD and apply modern pathologic evaluation. METHODS: We reviewed our database of APKD specimens over the past 11 years (from 2012 to 2023) for primary malignant tumors within the kidneys of APKD. RESULTS: Of 48 nephrectomy specimens with APKD evaluated, 10 malignant renal tumors were identified, indicating a prevalence of 20.8 % (10/48). These included three clear cell (cc) renal cell carcinomas (RCC) (ranging from 1 mm to 6.7 cm), three papillary RCCs (2.5, 3.5, and 14 cm with lymph node metastasis), two cases of clear cell papillary (CCP) RCC, one acquired cystic disease (ACD) with associated RCC (4 mm), and one urothelial adenocarcinoma. The urothelial adenocarcinoma was found near a tubulovillous adenoma in a collecting duct and stained positively for GATA3 and Uroplakin-2 but was negative for PAX8 & CDX2. The tumor showed extensive invasion into perirenal fatty tissue and the rectum. Next generating sequencing (NGS) analysis of the tumor showed mutations in TERT, RB1, TP53, ERBB2, and TET1 genes, further supporting its urothelial origin. CONCLUSIONS: We found a prevalence of 20.8%, which was higher than in previous reports of malignant renal tumors in patients who underwent resections for APKD. Renal tumors were mostly from damaged proximal tubular origins (clear cell or papillary RCC), but less commonly were from distal tubular or urothelial cells as well (clear cell papillary RCC and urothelial adenocarcinoma).
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Neoplasias Renales , Enfermedades Renales Poliquísticas , Humanos , Neoplasias Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/epidemiología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Enfermedades Renales Poliquísticas/patología , Enfermedades Renales Poliquísticas/genética , Adulto , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/epidemiología , Túbulos Renales Proximales/patología , NefrectomíaRESUMEN
Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.
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Técnicas de Cultivo Tridimensional de Células , Humanos , Técnicas de Cultivo Tridimensional de Células/métodos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Técnicas de Cultivo de Célula/métodos , Antineoplásicos/farmacología , Automatización , Automatización de Laboratorios/métodos , Neoplasias/patología , Neoplasias/tratamiento farmacológicoRESUMEN
The complex pathogenesis of kidney disease is closely related to the diversity of kidney intrinsic cells. In this study, single-cell transcriptome sequencing technology was used to sequence and analyze blood and kidney tissue cells in normal control rats and rats with chronic kidney disease (CKD), focusing on key cell populations and functional enrichment to explore the pathogenesis of CKD. Oil red O staining and enzyme-linked immunosorbent assay (ELISA) were used to detect lipid droplets and free fatty acid (FFA). Quantitative real-time polymerase chain reaction (RT-PCR), western blot (WB) were used to verify the differential gene hydroxyacid oxidase 2 (HAO2) and fatty acid metabolic process in tissue to ensure the reliability of single-cell sequencing results. We successfully established a single-cell transcriptome atlas of blood and kidney tissue in rats with CKD, which were annotated into 14 cell subsets (MPCs, PT, Tc, DCT, B-IC, A-IC, CNT, ALOH, BC, Neu, Endo, Pla, NKT, Baso) according to marker gene, and the integrated single-cell atlas of rats showed a significant increase and decrease of MPCs and PTs in the CKD group, respectively. Functional analysis found extensive enrichment of metabolic-related pathways in PT cells, includes fatty acid metabolic process, cellular amino acid metabolic process and generation of precursor metabolites and energy. Immunohistochemical experiments determined that the differential gene HAO2 was localized in the renal tubules, and its expression was significantly reduced in CKD group compared with control, and oil red O staining showed that lipid droplets increased in the CKD group, after overexpression of HAO2 the lipid droplets was inhibited. ELISA assay showed that ATP content decreased in the CKD group and FFA increased in the CKD group. Moreover, the mitochondrial membrane potential of the cells in the OE-HAO2 group was significantly increased compared with OE-NC. The acyl-CoA oxidase 1(ACOX1), peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were decreased in the CKD group, while genes and proteins were increased after overexpression of HAO2, and the AMP-activated protein kinase (AMPK) phosphorylated proteins were increased, the acetyl-CoA carboxylase (ACC) phosphorylated proteins were decreased, reversely. Therefore, HAO2 may be an important regulator of fatty acid metabolic processes in CKD, and overexpression of HAO2 can enhance fatty acid metabolism by promoting fatty acid oxidation (FAO) pathway.
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Ácidos Grasos , Túbulos Renales Proximales , Insuficiencia Renal Crónica , Animales , Ratas , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/genética , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Ácidos Grasos/metabolismo , Ratas Sprague-Dawley , Transcriptoma , Modelos Animales de EnfermedadRESUMEN
Epigenetic mechanisms are considered to contribute to diabetic nephropathy by maintaining memory of poor glycemic control during the early stages of diabetes. However, DNA methylation changes in the human kidney are poorly characterized, because of the lack of cell type-specific analysis. We examined DNA methylation in proximal tubules (PTs) purified from patients with diabetic nephropathy and identified differentially methylated CpG sites, given the critical role of proximal tubules in the kidney injury. Hypermethylation was observed at CpG sites annotated to genes responsible for proximal tubule functions, including gluconeogenesis, nicotinamide adenine dinucleotide synthesis, transporters of glucose, water, phosphate, and drugs, in diabetic kidneys, whereas genes involved in oxidative stress and the cytoskeleton exhibited demethylation. Methylation levels of CpG sites annotated to ACTN1, BCAR1, MYH9, UBE4B, AFMID, TRAF2, TXNIP, FOXO3, and HNF4A were correlated with the estimated glomerular filtration rate, whereas methylation of the CpG site in RUNX1 was associated with interstitial fibrosis and tubular atrophy. Hypermethylation of G6PC and HNF4A was accompanied by decreased expression in diabetic kidneys. Proximal tubule-specific hypomethylation of metabolic genes related to HNF4A observed in control kidneys was compromised in diabetic kidneys, suggesting a role for aberrant DNA methylation in the dedifferentiation process. Multiple genes with aberrant DNA methylation in diabetes overlapped genes with altered expressions in maladaptive proximal tubule cells, including transcription factors PPARA and RREB1. In conclusion, DNA methylation derangement in the proximal tubules of patients with diabetes may drive phenotypic changes, characterized by inflammatory and fibrotic features, along with impaired function in metabolism and transport.NEW & NOTEWORTHY Cell type-specific DNA methylation patterns in the human kidney are not known. We examined DNA methylation in proximal tubules of patients with diabetic nephropathy and revealed that oxidative stress, cytoskeleton, and metabolism genes were aberrantly methylated. The results indicate that aberrant DNA methylation in proximal tubules underlies kidney dysfunction in diabetic nephropathy. Aberrant methylation could be a target for reversing memory of poor glycemic control.
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Islas de CpG , Metilación de ADN , Nefropatías Diabéticas , Epigénesis Genética , Túbulos Renales Proximales , Fenotipo , Humanos , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/fisiopatología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Estudios de Casos y Controles , Tasa de Filtración GlomerularRESUMEN
It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10-7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.
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Albúminas , Angiotensina II , Endocitosis , Células Epiteliales , Glucosa , Túbulos Renales Proximales , Receptor de Angiotensina Tipo 1 , Endocitosis/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/efectos de los fármacos , Angiotensina II/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Albúminas/metabolismo , Porcinos , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Línea Celular , Losartán/farmacologíaRESUMEN
ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivo IR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.
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Adenosina Trifosfato , Cisplatino , Glucólisis , Túbulos Renales Proximales , Fosforilación Oxidativa , Animales , Adenosina Trifosfato/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Podocitos/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Modelos Animales de Enfermedad , Cimetidina/farmacología , Masculino , Túbulos Renales Distales/metabolismo , Técnicas de Cultivo de Órganos , Ratones Transgénicos , Oligomicinas/farmacología , Floretina/farmacología , Ratones Endogámicos C57BLRESUMEN
Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl-/H+exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at exploring therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease.
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Canales de Cloruro , Modelos Animales de Enfermedad , Ratones Transgénicos , Animales , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Ratones , Enfermedad de Dent/genética , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Mutación Missense , Humanos , Lipocalina 2/genética , Lipocalina 2/metabolismo , Autofagia/genética , Apoptosis/genética , Enfermedades Genéticas Ligadas al Cromosoma X , NefrolitiasisRESUMEN
BACKGROUND: Autosomal recessive renal tubular dysgenesis is a rare, usually fatal inherited disorder of the renin-angiotensis system (RAS). Herein, we report an adolescent individual experiencing an unknown chronic kidney disease and aim to provide novel insights into disease mechanisms. METHODS: Exome sequencing for a gene panel associated with renal disease was performed. The RAS was assessed by comprehensive biochemical analysis in blood. Renin expression was determined in primary tubular cells by quantitative polymerase chain reaction and in situ hybridization on kidney biopsy samples. Allele frequencies of heterozygous and biallelic deleterious variants were determined by analysis of the Genomics England 100,000 Genomes Project. RESULTS: The patient was delivered prematurely after oligohydramnios was detected during pregnancy. Postnatally, he recovered from third-degree acute kidney injury but developed chronic kidney disease stage G3b over time. Exome sequencing revealed a previously reported pathogenic homozygous missense variant, p.(Arg375Gln), in the AGT (angiotensinogen) gene. Blood AGT concentrations were low, but plasma renin concentration and gene expression in kidney biopsy, vascular, and tubular cells revealed strong upregulation of renin. Angiotensin II and aldosterone in blood were not abnormally elevated. CONCLUSIONS: Renal tubular dysgenesis may present as chronic kidney disease with a variable phenotype, necessitating broad genetic analysis for diagnosis. Functional analysis of the RAS in a patient with AGT mutation revealed novel insights regarding compensatory upregulation of renin in vascular and tubular cells of the kidney and in plasma in response to depletion of AGT substrate as a source of Ang II (similarly observed with hepatic AGT silencing for the treatment of hypertension).
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Angiotensinógeno , Humanos , Angiotensinógeno/genética , Masculino , Adolescente , Sistema Renina-Angiotensina/genética , Sistema Renina-Angiotensina/fisiología , Progresión de la Enfermedad , Renina/genética , Renina/sangre , Renina/metabolismo , Mutación Missense/genética , Secuenciación del Exoma/métodos , Femenino , Túbulos Renales Proximales/anomalías , Anomalías UrogenitalesRESUMEN
BACKGROUND: The protection of the diabetic kidney by Empagliflozin (EMPA) is attributed to its interaction with the sodium glucose cotransporter 2 located on proximal tubular epithelial cells (PTECs). Estrogen-related receptor α (ESRRA), known for its high expression in PTECs and association with mitochondrial biogenesis, plays a crucial role in this process. This study aimed to explore the impact of ESRRA on mitochondrial mass in diabetic tubular injury and elucidate the mechanism underlying the protective effects of EMPA. METHODS: Mitochondrial changes in PTECs of 16-week-old diabetic mice were assessed using transmission electron microscopy (TEM) and RNA-sequences. In vivo, EMPA administration was carried out in db/db mice for 8 weeks, while in vitro experiments involved modifying ESRRA expression in HK2 cells using pcDNA-ESRRA or EMPA. RESULTS: Evaluation through TEM revealed reduced mitochondrial mass and swollen mitochondria in PTECs, whereas no significant changes were observed under light microscopy. Analysis of RNA-sequences identified 110 downregulated genes, including Esrra, associated with mitochondrial function. Notably, ESRRA overexpression rescued the loss of mitochondrial mass induced by high glucose (HG) in HK2 cells. EMPA treatment ameliorated the ultrastructural alterations and mitigated the downregulation of ESRRA both in db/db mice and HG-treated HK2 cells. CONCLUSION: The diminished expression of ESRRA is implicated in the decline of mitochondrial mass in PTECs during the early stages of diabetes, highlighting it as a key target of EMPA for preventing the progression of diabetic kidney injury.
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Compuestos de Bencidrilo , Diabetes Mellitus Experimental , Nefropatías Diabéticas , Glucósidos , Mitocondrias , Animales , Glucósidos/farmacología , Glucósidos/uso terapéutico , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Compuestos de Bencidrilo/farmacología , Ratones , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/metabolismo , Masculino , Receptor Relacionado con Estrógeno ERRalfa , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/ultraestructura , Ratones Endogámicos C57BL , Línea Celular , Receptores de Estrógenos/metabolismoRESUMEN
This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson's staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1's 3'-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.
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Apoptosis , Proliferación Celular , Células Epiteliales , Glucosa , Factor I del Crecimiento Similar a la Insulina , Túbulos Renales , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Glucosa/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Línea Celular , Túbulos Renales/metabolismo , Túbulos Renales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 3/genéticaRESUMEN
Megalin (Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. Studies in our lab have shown that knockout (KO) of Lrp2 in opossum PT cells leads to a dramatic reduction in sodium-glucose co-transporter 2 (SGLT2) transcript and protein levels, as well as differential expression of genes involved in mitochondrial and metabolic function. SGLT2 transcript levels are reduced more modestly in Lrp2 KO mice. Here, we investigated the effects of Lrp2 KO on kidney function and health in mice fed regular chow (RC) or a Western-style diet (WD) high in fat and refined sugar. Despite a modest reduction in SGLT2 expression, Lrp2 KO mice on either diet showed increased glucose tolerance compared to control mice. Moreover, Lrp2 KO mice were protected against WD-induced fat gain. Surprisingly, renal function in male Lrp2 KO mice on WD was compromised, and the mice exhibited significant kidney injury compared with control mice on WD. Female Lrp2 KO mice were less susceptible to WD-induced kidney injury than male Lrp2 KO. Together, our findings reveal both positive and negative contributions of megalin expression to metabolic health, and highlight a megalin-mediated sex-dependent response to injury following WD.
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Dieta Occidental , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones Noqueados , Transportador 2 de Sodio-Glucosa , Animales , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Dieta Occidental/efectos adversos , Masculino , Ratones , Femenino , Transportador 2 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Ratones Endogámicos C57BL , Riñón/metabolismo , Riñón/patologíaRESUMEN
A detailed knowledge of the lipid composition of components of nephrons is crucial for understanding physiological processes and the development of kidney diseases. However, the lipidomic composition of kidney tubular segments is unknown. We manually isolated the proximal convoluted tubule (PCT), the cortical thick ascending limb of Henle's loop, and the cortical collecting duct from 5 lean and obese mice and subjected the samples to shotgun lipidomics analysis by high-resolution mass spectrometry acquisition. Across all samples, more than 500 lipid species were identified, quantified, and compared. We observed significant compositional differences among the 3 tubular segments, which serve as true signatures. These intrinsic lipidomic features are associated with a distinct proteomic program that regulates highly specific physiological functions. The distinctive lipidomic features of each of the 3 segments are mostly based on the relative composition of neutral lipids, long-chain polyunsaturated fatty acids, sphingolipids, and ether phospholipids. These features support the hypothesis of a lipotype assigned to specific tubular segments. Obesity profoundly impacts the lipotype of PCT. In conclusion, we present a comprehensive lipidomic analysis of 3 cortical segments of mouse kidney tubules. This valuable resource provides unparalleled detail that enhances our understanding of tubular physiology and the potential impact of pathological conditions.
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Lipidómica , Animales , Ratones , Ratones Endogámicos C57BL , Masculino , Obesidad/metabolismo , Túbulos Renales Proximales/metabolismo , Corteza Renal/metabolismo , Corteza Renal/química , Lípidos/análisis , Metabolismo de los Lípidos/fisiología , Esfingolípidos/metabolismoRESUMEN
The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.