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

García-Pastor, Coral; Benito-Martínez, Selma; Moreno‐Manzano, Victoria; Fernández‐Martínez, Ana B.; Lucio-Cazaña, Javier

doi:10.1038/s41598-019-52310-6

Cited by 29 publications

(30 citation statements)

References 62 publications

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“…Furthermore, impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabeticlike 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 hyperglycemic, hypoxic diabetic-like milieu [18]. Finally, neither HG alone nor hypoxia alone affected the migration of HK-2 cells [18] and the release of microparticles under hyperglycemia was greatly diminished in the absence of hypoxia (unpublished results).…”

Section: Introductionmentioning

confidence: 84%

“…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 hyperglycemic, hypoxic diabetic-like milieu [18]. Finally, neither HG alone nor hypoxia alone affected the migration of HK-2 cells [18] and the release of microparticles under hyperglycemia was greatly diminished in the absence of hypoxia (unpublished results). In summary, HK-2 cells exposed simultaneously to hyperglycemia and hypoxia (i.e.…”

Section: Introductionmentioning

confidence: 84%

“…In these conditions, HIF-1α fails to stabilize and accumulate in HK-2 cells (i.e. the same defective response found in the hypoxic diabetic kidney) [18]. Furthermore, impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabeticlike milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair).…”

Section: Introductionmentioning

confidence: 94%

“…For the proteomic study, and after incubation, the growth medium from three culture plates was removed by aspiration, washed with PBS, and proteins were directly extracted from attached cells. An additional plate per time and treatment condition was used to check by Western blot analysis the interaction between HG and hypoxia resulting in specific loss of hypoxia-induced accumulation of HIF-1α in HK-2 cells [18].…”

Section: Cell Culture Conditionsmentioning

confidence: 99%

“…For this study, equal number of cells (5 × 10 5 per mL) were plated at 90% confluence in p35 cultured dishes and when completely attached, they were treated for 5, 24 and 48 h with HG (25 mM) medium in hypoxic conditions (1% O 2 ), or with NG medium (5.5 mM) in normoxic conditions (21% O 2 ). The interaction between HG and hypoxia resulting in specific loss of hypoxia-induced accumulation of HIF-1α in HK-2 cells, which has important pathological implications in the context of proximal tubular disfunction in DKD [18], was routinely checked (results are not shown).…”

Section: Cell Viability and Protein Contentmentioning

confidence: 99%

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Time-series proteomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu

et al. 2020

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During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O 2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent to in vitro studies, propose new mechanisms and targets to be considered in the management of DKD.

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Section: Introductionmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 94%

Section: Cell Culture Conditionsmentioning

confidence: 99%

Section: Cell Viability and Protein Contentmentioning

confidence: 99%

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Time-series proteomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu

et al. 2020

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RACK1 mediates the advanced glycation end product‐induced degradation of HIF‐1α in nucleus pulposus cells via competing with HSP90 for HIF‐1α binding

Yang

et al. 2021

Cell Biology International

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Hyperglycemia can drive advanced glycation end product (AGE) accumulation and associated nucleus pulposus cell (NPC) dysfunction, but the basis for this activity has not been elucidated. Hypoxia‐inducible factor‐1α (HIF‐1α) is subject to cell‐type‐specific AGE‐mediated regulation. In the current study, we assessed the mechanistic relationship between AGE accumulation and HIF‐1α degradation in NPCs. Immunohistochemical staining of degenerated nucleus pulposus (NP) samples was used to assess AGE levels. AGE impact on NPC survival and glycolysis‐related gene expression was assessed via 3‐(4,5)‐dimethylthiazol(‐z‐y1)‐3,5‐di‐phenyltetrazolium bromide assay and quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), while HIF‐1α expression in NPCs following AGE treatment was monitored via Western blot analysis and qRT‐PCR. Additionally, a luciferase reporter assay was used to monitor HIF‐1α transcriptional activity. The importance of the receptor for activated C‐kinase 1 (RACK1) as a mediator of HIF‐1α degradation was evaluated through gain‐ and loss‐of‐function experiments. Competitive binding of RACK1 and HSP90 to HIF‐1α was evaluated via immunoprecipitation. Increased AGE accumulation was evident in NP samples from diabetic patients, and AGE treatment resulted in reduced HIF‐1α protein levels in NPCs that coincided with reduced HIF‐1α transcriptional activity. AGE treatment impaired the stability of HIF‐1α, leading to its RACK1‐mediated proteasomal degradation in a manner independent of the canonical PHD‐mediated degradation pathway. Additionally, RACK1 competed with HSP90 for HIF‐1α binding following AGE treatment. AGE treatment of NPCs leads to HIF‐1α protein degradation. RACK1 competes with HSP90 for HIF‐1α binding following AGE treatment, resulting in posttranslational HIF‐1α degradation. These results suggest that AGE is an intervertebral disc degeneration risk factor, and highlight potential avenues for the treatment or prevention of this disease.

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TRIM7 suppresses cell invasion and migration through inhibiting HIF‐1α accumulation in clear cell renal cell carcinoma

Yuan

Liu

Jia

et al. 2021

Cell Biology International

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Clear cell renal cell carcinoma (ccRCC) is one frequent form of urologic malignancy characterized by deregulated hypoxia-inducible factor signaling, genetic and epigenetic alterations. Metastasis is the leading cause of mortality from ccRCC, and understanding the underlying mechanism of this event will provide better strategies for its management. Here, we identify tripartite motif containing 7 (TRIM7) as a tumor suppressor in ccRCC cells, which negatively regulates hypoxia-inducible factor 1α (HIF-1α) signaling through targeting the proto-oncogene Src. We observed the downregulated expression of TRIM7 in clinical ccRCC tissues and its correlation with the poor prognosis. In Caki-1 cells, depletion of TRIM7 increased cell migration and invasion under normoxic and hypoxic conditions. TRIM7 markedly reduced the abundance of Src protein via the ubiquitin-proteasome pathway. Further study showed that TRIM7 affected HIF-1α accumulation through targeting either the Srctriggered PI3K/AKT/mTOR signaling pathway or reactive oxygen species production. Overall, our findings highlight a novel mechanism for negative regulation of HIF-1 signaling pathway by TRIM7 and define a promising therapeutic strategy for ccRCC by modulating TRIM7.

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

Cited by 29 publications

References 62 publications

Time-series proteomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu

Time-series proteomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu

RACK1 mediates the advanced glycation end product‐induced degradation of HIF‐1α in nucleus pulposus cells via competing with HSP90 for HIF‐1α binding

TRIM7 suppresses cell invasion and migration through inhibiting HIF‐1α accumulation in clear cell renal cell carcinoma

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