scholarly journals Melatonin Lowers HIF-1α Content in Human Proximal Tubular Cells (HK-2) Due to Preventing Its Deacetylation by Sirtuin 1

2021 ◽  
Vol 11 ◽  
Author(s):  
Aleksandra Owczarek ◽  
Katarzyna B. Gieczewska ◽  
Marta Polanska ◽  
Bohdan Paterczyk ◽  
Andrzej Gruza ◽  
...  
Keyword(s):  
Western Blot ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Melatonin Receptors ◽  
Sirtuin 1 ◽  
Regulatory Subunit ◽  
Proximal Tubular Cells ◽  
Acetylation Level ◽  
Tubular Cells ◽  
Proximal Tubular

Although melatonin is widely known for its nephroprotective properties, there are no reports clearly pointing at its impact on the activity of hypoxia-inducible factor-1 (HIF-1), the main mediator of metabolic responses to hypoxia, in kidneys. The aim of the present study was to elucidate how melatonin affects the expression of the regulatory subunit HIF-1α in renal proximal tubules. HK-2 cells, immortalized human proximal tubular cells, were cultured under hypoxic conditions (1% O2). Melatonin was applied at 100 μM concentration. Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. HIF-1α acetylation level was established by means of immunoprecipitation followed by Western blot. Melatonin receptors MT1 and MT2 localization in HK-2 cells was visualized using immunofluorescence confocal analysis. It was found that melatonin in HK-2 cells (1) lowered HIF-1α protein, but not mRNA, content; (2) attenuated expression of HIF-1 target genes; (3) increased HIF-1α acetylation level; and (4) diminished sirtuin 1 expression (both protein and mRNA). Sirtuin 1 involvement in the regulation of HIF-1α level was confirmed applying cells with silenced Sirt1 gene. Moreover, the presence of membrane MT1 and MT2 receptors was identified in HK-2 cells and their ligand, ramelteon, turned out to mimic melatonin action on both HIF-1α and sirtuin 1 levels. Thus, it is concluded that the mechanism of melatonin-evoked decline in HIF-1α content in renal proximal tubular cells involves increased acetylation of this subunit which results from the attenuated expression of sirtuin 1, an enzyme reported to deacetylate HIF-1α. This observation provides a new insight to the understanding of melatonin action in kidneys.

scholarly journals Intracellular prostaglandin E2 contributes to hypoxia-induced proximal tubular cell death

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Coral García-Pastor ◽  
Selma Benito-Martínez ◽  
Ricardo J. Bosch ◽  
Ana B. Fernández-Martínez ◽  
Francisco J. Lucio-Cazaña
Keyword(s):  
Hypoxia Inducible Factor ◽  
Renal Diseases ◽  
Prostaglandin E ◽  
Relevant Factor ◽  
Tubular Injury ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Cox 2 ◽  
Induced Apoptosis

AbstractProximal tubular cells (PTC) are particularly vulnerable to hypoxia-induced apoptosis, a relevant factor for kidney disease. We hypothesized here that PTC death under hypoxia is mediated by cyclo-oxygenase (COX-2)-dependent production of prostaglandin E2 (PGE2), which was confirmed in human proximal tubular HK-2 cells because hypoxia (1% O2)-induced apoptosis (i) was prevented by a COX-2 inhibitor and by antagonists of prostaglandin (EP) receptors and (ii) was associated to an increase in intracellular PGE2 (iPGE2) due to hypoxia-inducible factor-1α-dependent transcriptional up-regulation of COX-2. Apoptosis was also prevented by inhibitors of the prostaglandin uptake transporter PGT, which indicated that iPGE2 contributes to hypoxia-induced apoptosis (on the contrary, hypoxia/reoxygenation-induced PTC death was exclusively due to extracellular PGE2). Thus, iPGE2 is a new actor in the pathogenesis of hypoxia-induced tubular injury and PGT might be a new therapeutic target for the prevention of hypoxia-dependent lesions in renal diseases.

scholarly journals The exosomes derived from urine of premature infants target MAPK8 via miR-30a-5p to protect cisplatin-induced acute kidney injury in mice

2021 ◽  
Author(s):  
Mingming Ma ◽  
Qiao Luo ◽  
Lijing Fan ◽  
Weilong Li ◽  
Qiang Li ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Premature Infants ◽  
Target Genes ◽  
Kidney Injury ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Renal Tubular ◽  
Hk2 Cells

Aim: Acute kidney injury (AKI), a global public health issue, not only causes millions of deaths every year, but is also a susceptible factor for chronic kidney disease (CKD). Nephrotoxic drugs are an important cause of AKI. There is still a lack of effective and satisfactory prevention method in clinical practice. This study investigated the protective effect of the exosomes derived from urine of premature infants on cisplatin-induced acute kidney injury. Methods: Isolation of exosomes from fresh urine of premature infants: The characteristics of exosomes were determined by flow cytometry, transmission electron microscopy and Western blotting. A C57BL/6 mice model of cisplatin-induced acute kidney injury was established. The mice in the experimental group were given 100ug exosomes dissolved in 200ul solution. The mice in the control group were given normal saline (200ul). These treatments were performed 24 hours after AKI was induced by intraperitoneal injection of cisplatin. To evaluate renal function, blood was drawn 24 hours after AKI model was established and serum creatinine (sCr) was measured. The mice were euthanized 72 hours after exosome treatment. The kidneys were collected for pathological examination, RNA and protein extraction, and the evaluation of renal tubular damage and apoptosis. In the in-vitro experiment, human renal cortex/proximal tubular cells (HK2) was induced by cisplatin to assess the protective ability of the exosomes derived from urine of premature infants. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blotting were used to evaluate the effect of exosomes treatment on the apoptosis of HK2 cells induced by cisplatin. Exosome microRNA sequencing technology and bioinformatics analysis method were applied to investigate the miRNAs enriched in exosomes and their target genes. The dual luciferase gene reporter system was used to detect the interaction of target genes. Results: Treatment of exosomes derived from urine of premature infants could decrease the level of serum creatinine and the apoptosis of renal tubular cell, inhibit the infiltration of inflammatory cell, protect mice from acute kidney injury induced by cisplatin and reduce mortality. In addition, miR-30a-5p was the most abundant miRNA in the exosomes derived from urine of premature infants. It protected HK2 cells from cisplatin-induced apoptosis by targeting and down-regulating the 3'UTR of mitogen-activated protein kinases (MAPK8) mRNA. Conclusions: According to our results, the exosomes derived from urine of premature infants alleviated cisplatin-induced acute kidney injury in mice and inhibited the apoptosis of human proximal tubular cells (HK2) induced by cisplatin in vitro. MiR-30a-5p in exosomes inhibited cisplatin-induced MAPK activation, ameliorated apoptosis, and protected renal function. The exosomes derived from urine of premature infants provided a promising acellular therapy for AKI.

scholarly journals Advanced Glycated End-Products Affect HIF-Transcriptional Activity in Renal Cells

2013 ◽  
Vol 27 (11) ◽  
pp. 1918-1933 ◽  
Author(s):  
Tzvetanka Bondeva ◽  
Juliane Heinzig ◽  
Carola Ruhe ◽  
Gunter Wolf
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Mesangial Cells ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
End Products ◽  
Proximal Tubular

Advanced glycated end-products (AGEs) are ligands of the receptor for AGEs and increase in diabetic disease. MAPK organizer 1 (Morg1) via its binding partner prolyl-hydroxylase domain (PHD)-3 presumably plays a role in the regulation of hypoxia-inducible factor (HIF)-1α and HIF-2α transcriptional activation. The purpose of this study was to analyze the influence of AGEs on Morg1 expression and its correlation to PHD3 activity and HIF-transcriptional activity in various renal cell types. The addition of glycated BSA (AGE-BSA) significantly up-regulated Morg1 mRNA levels in murine mesangial cells and down-regulated it in murine proximal tubular cells and differentiated podocytes. These effects were reversible when the cells were preincubated with a receptor for α-AGE antibody. AGE-BSA treatment induced a relocalization of the Morg1 cellular distribution compared with nonglycated control-BSA. Analysis of PHD3 activity demonstrated an elevated PHD3 enzymatic activity in murine mesangial cells but an inhibition in murine proximal tubular cells and podocytes after the addition of AGE-BSA. HIF-transcriptional activity was also affected by AGE-BSA treatment. Reporter gene assays and EMSAs showed that AGEs regulate HIF- transcriptional activity under nonhypoxic conditions in a cell type-specific manner. In proximal tubular cells, AGE-BSA stimulation elevated mainly HIF-1α transcriptional activity and to a lesser extent HIF-2α. We also detected an increased expression of the HIF-1α and the HIF-2α proteins in kidneys from Morg1 heterozygous (HZ) placebo mice compared with the Morg1 wild-type (WT) placebo-treated mice, and the HIF-1α protein expression in the Morg1 HZ streptozotocin-treated mice was significantly higher than the WT streptozotocin-treated mice. Analysis of isolated mesangial cells from Morg1 HZ (±) and WT mice showed an inhibited PHD3 activity and an increased HIF-transcriptional activity in cells with only one Morg1 allele. These findings are important for a better understanding of the molecular mechanisms of diabetic nephropathy.

Abstract 385: TRPA1 Mediates Angiotensinogen Augmentation in Renal Proximal Tubular Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Andrea Zsombok ◽  
Andrei V Derbenev
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
P38 Mapk ◽  
Rt Pcr ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular ◽  
Stimulation Of

Overactivity of the intrarenal renin-angiotensin system contributes to the development of hypertension and renal injury. In this process, regulation of angiotensinogen (AGT) in renal proximal tubular cells (RPTC) is a key factor. Oxidative stress stimulates AGT expression accompanied by activation of p38 MAPK in RPTC. Transient receptor potential A1 (TRPA1) is known to be a sensor activated by a variety of noxious stimuli including hydrogen peroxide. Activated TRPA1 induces calcium influx across the plasma membrane leading to activation of AGT-inducible signal transducers. However, the presence and function of TRPA1 in the kidney have not been delineated. Therefore, this study was performed to demonstrate expression of intrarenal TRPA1 and its role in AGT augmentation in RPTC. Expression of TRPA1 in mouse kidney and cultured mouse RPTC were determined by RT-PCR, in situ hybridization, western blot analysis and immunocytochemistry. The RPTC were treated with 100 μM H2O2 for 1 hr with or without 10μM HC030031, a TRPA1 specific antagonist. EGTA was used to the culture medium for depletion of extracellular calcium. Thereafter, AGT expression levels were evaluated by real-time RT-PCR, and TRPA1 expression levels and p38 MAPK activity were determined by western blot analyses. TRPA1 expression was observed in tubules of renal cortex, and the cultured PRTC expressed TRPA1 mRNA and protein. H2O2 increased AGT expression (1.54 ± 0.08, ratio to control) in RPTC. The AGT augmentation was suppressed by HC030031 (1.19 ± 0.05, ratio to control). Calcium depletion also resulted in attenuation of the AGT augmentation induced by H2O2. Although H2O2 induced phosphorylation of p38 MAPK, HC030031 did not inhibit the p38 MAPK activation. TRPA1 expression was increased by H2O2 under the experimental condition (1.90 ± 0.06, ratio to control). These results suggest that TRPA1 is expressed in RPTC, which contributes to H2O2-induced AGT augmentation via calcium-dependent but p38 MAPK-independent pathways. Furthermore, increase in TRPA1 expression by H2O2 likely facilitates the stimulation of AGT expression. Therefore, TRPA1 may play an important role in the progression of oxidative stress-associated renal injury and hypertension via the stimulation of AGT expression in RPTC.

scholarly journals Mechanism and Consequences of The Impaired Hif-1α Response to Hypoxia in Human Proximal Tubular HK-2 Cells Exposed to High Glucose

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Coral García-Pastor ◽  
Selma Benito-Martínez ◽  
Victoria Moreno-Manzano ◽  
Ana B. Fernández-Martínez ◽  
Francisco Javier Lucio-Cazaña
Keyword(s):  
Hypoxia Inducible Factor ◽  
Vascular Endothelial ◽  
Adaptive Responses ◽  
Negative Effects ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Endothelial Growth Factor ◽  
Different Levels

Abstract Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.

Low-affinity transport of pyroglutamyl-histidine in renal brush-border membrane vesicles

1989 ◽  
Vol 257 (5) ◽  
pp. C971-C975 ◽  
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
G. Flouret ◽  
D. R. Peterson
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Luminal Membrane ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Brush Border

These studies were performed to determine if a low-affinity carrier is present in the luminal membrane of proximal tubular cells for the transport of the dipeptide, pyroglutamyl-histidine (pGlu-His). We have previously described the existence of a specific, high-affinity, low-capacity [transport constant (Kt) = 9.3 X 10(-8) M, Vmax = 6.1 X 10(-12) mol.mg-1.min-1] carrier for pGlu-His in renal brush-border membrane vesicles. In the present study, we sought to demonstrate that multiple carriers exist for the transport of a single dipeptide by determining whether a low-affinity carrier also exists for the uptake of pGlu-His. Transport of pGlu-His into brush-border membrane vesicles was saturable over the concentration range of 10(-5)-10(-3) M, yielding a Kt of 6.3 X 10(-5) M and a Vmax of 2.2 X 10(-10) mol.mg-1.min-1. Uptake was inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and carnosine but not by the tripeptide pyroglutamyl-histidyl-prolinamide. We conclude that 1) pGlu-His is transported across the luminal membrane of the proximal tubule by multiple carriers and 2) the lower affinity carrier, unlike the higher affinity carrier, is nonspecific with respect to other dipeptides.

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