scholarly journals Transcription Factor ChREBP Mediates High Glucose-Evoked Increase in HIF-1α Content in Epithelial Cells of Renal Proximal Tubules

2021 ◽  
Vol 22 (24) ◽  
pp. 13299
Author(s):  
Aleksandra Owczarek ◽  
Katarzyna B. Gieczewska ◽  
Robert Jarzyna ◽  
Zuzanna Frydzinska ◽  
Katarzyna Winiarska
Keyword(s):  
Transcription Factor ◽  
Epithelial Cells ◽  
Proximal Tubule ◽  
High Glucose ◽  
Target Genes ◽  
Renal Proximal Tubule ◽  
Diabetes Type Ii ◽  
Nadh Ratio ◽  
Proximal Tubular ◽  
Hif1a Gene

Hyperglycemia/diabetes appears to be accompanied by the state of hypoxia, which especially affects kidneys. The aim of the study was to elucidate the mechanism of high glucose action on HIF-1α expression in renal proximal tubule epithelial cells. The research hypotheses included: (1) the participation of transcription factor ChREBP; and (2) the involvement of the effects resulting from pseudohypoxia, i.e., lowered intracellular NAD+/NADH ratio. The experiments were performed on HK-2 cells and primary cells: D-RPTEC (Diseased Human Renal Proximal Tubule Epithelial Cells—Diabetes Type II) and RPTEC (Renal Proximal Tubule Epithelial Cells). Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. ChREBP binding to DNA was detected applying chromatin immunoprecipitation, followed by RT-qPCR. Gene knockdown was performed using siRNA. Sirtuin activity and NAD+/NADH ratio were measured with commercially available kits. It was found that high glucose in HK-2 cells incubated under normoxic conditions: (1) activated transcription of HIF-1 target genes, elevated HIF-1α and ChREBP content, and increased the efficacy of ChREBP binding to promoter region of HIF1A gene; and (2), although it lowered NAD+/NADH ratio, it affected neither sirtuin activity nor HIF-1α acetylation level. The stimulatory effect of high glucose on HIF-1α expression was not observed upon the knockdown of ChREBP encoding gene. Experiments on RPTEC and D-RPTEC cells demonstrated that HIF-1α content in diabetic proximal tubular cells was lower than that in normal ones but remained high glucose-sensitive, and the latter phenomenon was mediated by ChREBP. Thus, it is concluded that the mechanism of high glucose-evoked increase in HIF-1α content in renal proximal tubule endothelial cells involves activation of ChREBP, indirectly capable of HIF1A gene up-regulation.

scholarly journals Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vidya Chandrasekaran ◽  
Giada Carta ◽  
Daniel da Costa Pereira ◽  
Rajinder Gupta ◽  
Cormac Murphy ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Retinoic Acid Receptor ◽  
Renal Proximal Tubule ◽  
Whole Body ◽  
Proper Function ◽  
Genome Wide ◽  
Glomerular Filtrate ◽  
Proximal Tubular ◽  
Induced Pluripotent

AbstractThe renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

Abstract P415: ACE N-domain Regulates High-glucose Mediated Interleukin-1 beta Production by Renal Epithelial Cells Independently From Angiotensin II Generation

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Jorge F Giani ◽  
Ellen A Bernstein ◽  
Masahiro Eriguchi ◽  
Romer A Gonzalez-Villalobos ◽  
Kenneth E Bernstein
Keyword(s):  
Diabetic Nephropathy ◽  
Angiotensin Ii ◽  
Epithelial Cells ◽  
High Glucose ◽  
Interleukin 1 ◽  
Kidney Injury ◽  
Fold Increase ◽  
Renal Epithelial Cells ◽  
Proximal Tubular ◽  
Sense And Respond

Research studies demonstrated that interleukin (IL)-1β contributes to the development of diabetic nephropathy and hypertension. However, the origin and regulation of IL-1β synthesis during diabetic kidney injury are still unknown. Here, we hypothesize that renal epithelial cells produce IL-1β in response to a high glucose stress and that angiotensin converting enzyme (ACE) plays a key role in this process. To study this, we isolated proximal tubular (PT) epithelial cells from wild-type (WT) and mice lacking either the ACE N-domain (NKO) or the C-domain (CKO) catalytic activity. These cells were exposed to normal (5 mM) or high (30 mM) glucose for 24 hours. IL-1β produced by PT cells were assessed by ELISA and RT-PCR. High glucose induced WT PT cells to release significant amounts of IL-1β (from 5±1 to 70±6 pg/ml, p<0.001; n=6). When WT PT cells were exposed to a high glucose media in the presence of an ACE inhibitor (lisinopril, 10 mM), IL-1β levels were significantly reduced (from 70±6 to 38±6 pg/ml, p<0.01). In contrast, AT1 receptor blockade by losartan did not change the amount of IL-1β produced by WT PT cells. To determine which ACE domain is associated with IL-1β production, NKO and CKO PT cells were exposed to high glucose. Strikingly, NKO PT cells released lower amounts of IL-1β when exposed to high glucose compared to WT (NKO: 15±7 vs. WT: 79±9 pg/ml, p<0.01, n=4). No differences were observed between WT and CKO PT cells. Since the ACE N-domain degrades the anti-inflammatory tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), we tested whether the lower IL-1β production in NKO PT cells was due to an accumulation of AcSDKP. For this, we pre-treated NKO PT cells with a prolyl endopeptidase inhibitor (S17092, 50μM) to prevent the production of AcSDKP. Notably, this treatment increased the IL-1β response to high glucose in NKO PT cells (2.1±0.3-fold increase, p<0.01, n=4). Our data indicate that: 1) PT cells can sense and respond to high glucose by secreting IL-1β and 2) the absence of the ACE N-domain blunts the production of IL-1β through a mechanism that involves AcSDKP accumulation. In conclusion, ACE might contribute to the inflammatory response that underlays diabetic nephropathy independently from angiotensin II generation.

QS394. The Genomic Analysis of the Heterogeneity Hypoxia Responses in Renal Proximal Tubule Epithelial Cells After Knock-Down of HIF-1α and HIF-2α

2008 ◽  
Vol 144 (2) ◽  
pp. 424
Author(s):  
Zhen Wang ◽  
Edwin H. Rodriguez ◽  
Michael T. Longaker ◽  
Patrick O. Brown ◽  
Jen-Tsan A. Chi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proximal Tubule ◽  
Genomic Analysis ◽  
Renal Proximal Tubule ◽  
Knock Down

scholarly journals Therapy with antisense TGF-β1 oligodeoxynucleotides reduces kidney weight and matrix mRNAs in diabetic mice

2000 ◽  
Vol 278 (4) ◽  
pp. F628-F634 ◽  
Author(s):  
Dong Cheol Han ◽  
Brenda B. Hoffman ◽  
Soon Won Hong ◽  
Jia Guo ◽  
Fuad N. Ziyadeh
Keyword(s):  
Proximal Tubule ◽  
High Glucose ◽  
Leucine Incorporation ◽  
Diabetic Mice ◽  
Renal Hypertrophy ◽  
Kidney Weight ◽  
Cell Hypertrophy ◽  
Proximal Tubular ◽  
Tgf Β1

Inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) relies on their ability to bind complementary mRNA sequences and prevent translation. The proximal tubule is a suitable target for ODN therapy in vivo because circulating ODNs accumulate in the proximal tubule in high concentrations. Because increased proximal tubular transforming growth factor- β1 (TGF-β1) expression may mediate diabetic renal hypertrophy, we investigated the effects of antisense TGF-β1 ODN on the high-glucose-induced proximal tubular epithelial cell hypertrophy in tissue culture and on diabetic renal hypertrophy in vivo. Mouse proximal tubular cells grown in 25 mM d-glucose and exposed to sense ODN as control (1 μM) exhibited increased3[H]leucine incorporation by 120% and total TGF-β1 protein by 50% vs. culture in 5.5 mM d-glucose. Antisense ODN significantly decreased the high-glucose-stimulated TGF-β1 secretion and leucine incorporation. Continuous infusion for 10 days of ODN (100 μg/day) was achieved via osmotic minipumps in diabetic and nondiabetic mice. Sense ODN-treated streptozotocin-diabetic mice had 15.3% increase in kidney weight, 70% increase in α1(IV) collagen and 46% increase in fibronectin mRNA levels compared with nondiabetic mice. Treatment of diabetic mice with antisense ODN partially but significantly decreased kidney TGF-β1 protein levels and attenuated the increase in kidney weight and the α1(IV) collagen and fibronectin mRNAs. In conclusion, therapy with antisense TGF-β1 ODN decreases TGF-β1 production and attenuates high-glucose-induced proximal tubular cell hypertrophy in vitro and partially prevents the increase in kidney weight and extracellular matrix expression in diabetic mice.

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