Abstract MP11: Decreased Expression Of Ace2 Is Associated Urine Derived Human Renal Proximal Tubule Cells From Inverse Salt Sensitivity Participants Cultured In Low Salt Conditions

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
John J Gildea ◽  
Peng Xu ◽  
Katie Schiermeyer ◽  
Wei Yue ◽  
Robin A Felder
Keyword(s):  
Proximal Tubule ◽  
Salt Sensitivity ◽  
Renal Proximal Tubule ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Low Sodium Diet ◽  
Low Sodium ◽  
Low Salt ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. Inverse salt sensitivity (ISS) is the paradoxical increase in blood pressure of individuals to a low sodium diet. Our group previously reported decreased expression of dopamine type 2 receptor (D 2 R), increased expression Aminopeptidase N, and increased Ang II dependent sodium transport in human urine derived renal proximal tubule cells isolated from ISS participants. In an attempt to understand the increased Ang II sensitivity demonstrated in ISS cells, we examined angiotensin converting enzyme 2 (ACE2), a membrane associated enzyme involved in the metabolism of Ang II. Urine derived renal proximal tubule cells grown and immortalized from ISS participants were compared to cells from salt resistant (SR) participants cultured in iso-osmotic media with low salt (LS, 90 mM NaCl) normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl). Cells were incubated in LS, NS, and HS media with and without losartan (LOS,1 μM) overnight (18 hours) and ACE2 expression levels determined by in-cell western blot. A monoclonal antibody specific to an extracellular epitope of ACE2 was used as the primary antibody and an Alexa-647 anti-mouse secondary antibody. ACE2 expression was only reduced in ISS cells in LS condition (28.7±2.1 % reduction, ISS LS vs SR LS, N=4 per group, p<0.05). Addition of losartan completely blocked the decrease in ACE2 expression in low salt conditions in ISS in urine derived human renal proximal tubule cells. No other changes in ACE2 expression were found between ISS and SR in either NS or HS conditions and with or without losartan. In conclusion, a decreased expression of ACE2 in ISS urine proximal tubule cells could explain the previously reported increased sensitivity of ISS cells to Ang II by increasing the half-life of Ang II under low salt conditions.

Abstract MP29: A Cloned Anti-aminopeptidase N Scfv Antibody Blocks Enzymatic Activity And Rescues A Low Sodium Phenotype In Proximal Tubule Cells Isolated From Inverse Salt Sensitive Participants

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
John J Gildea ◽  
Peng Xu ◽  
Wei Yue ◽  
Robin A Felder
Keyword(s):  
Enzymatic Activity ◽  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Aminopeptidase N ◽  
Proximal Tubule Cells ◽  
Sodium Diet ◽  
Low Sodium Diet ◽  
Low Sodium ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Inverse Salt Sensitivity (ISS), defined as the paradoxical increase in blood pressure of individuals on a low sodium diet compared to a high sodium diet, may be associated with the increase in mortality and morbidity found in individuals on a low sodium diet. Our group has previously found that urine derived human renal proximal tubule cells (RPTC) isolated from ISS participants express higher Aminopeptidase N (APN) protein than cells isolated from salt resistant (SR) participants. An anti-Aminopeptidase N (APN) hybridoma was used construct a single chain variable fragment (scFv) into a bacterial expression system. The purified anti-APN scFv bound to live human renal proximal tubule cells, detected using an Alexa594 directly labelled anti-myc monoclonal antibody by immunofluorescent confocal microscopy. The anti-APN scFv was then tested to determine if it blocks APN enzymatic activity. We have previously shown that under low sodium conditions the amount of reduced-glutathione as determined by live cell fluorescence staining with the redox sensitive dye, monochlorobimane (mBCl), is decreased only in ISS cells under low sodium conditions at the two-hour time point. We hypothesized that the decreased mBCl signal may be due to the increased APN expression and activity altering the Ang II/Ang III ratio and reducing the Ang III activation of the AT 2 R. Low sodium reduced the mBCl signal in ISS (-29.2±4.3%, ISS vs SR, N=3 per group, p<0.05) and the addition of the anti-APN scFv at 1 ug/ml completely blocked the mBCl signal back to levels found in SR in normal salt conditions. In order to verify that the full rescue of this ISS specific response is due to enhanced Ang III – AT 2 R signaling, we next tried to block the effect of the anti-APN scFv by the addition of the AT 2 R antagonist, PD123319 (PD, 1 uM). Addition of PD alone to the ISS LS RPTCs did not significantly alter the mBCl signal, but when PD and anti-APN scFv are added to the LS treated ISS cells, there is complete reversal of the effect of anti-APN scFv alone (-38.1±3.9%, ISS anti-APN scFv + PD vs ISS anti-APN scFv, N=3 per group, p<0.05). The anti-APN scFv has potential therapeutic value by reducing APN enzymatic activity in ISS individuals and inducing the protective AT 2 R arm of the renin angiotensin system in low sodium conditions.

The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells

2002 ◽  
Vol 282 (2) ◽  
pp. F228-F237 ◽  
Author(s):  
Soo Hyun Park ◽  
Ho Jae Han
Keyword(s):  
Proximal Tubule ◽  
High Glucose ◽  
Renal Proximal Tubule ◽  
Channel Blockers ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Pkc Inhibitors ◽  
Renal Proximal Tubule Cells ◽  
Tgf Β1

The renin-angiotensin system plays an important role in the development of diabetic nephropathy. However, the mechanism of ANG II receptor regulation in the renal proximal tubule in the diabetic condition has not been elucidated. Thus we investigated the signal pathways involved in high-glucose-induced downregulation of ANG II binding in primary cultured renal proximal tubule cells. Twenty-five millimolar glucose, but not mannitol andl-glucose, induced downregulation of the AT1receptor (AT1R) because of a significant decline in maximal binding with no significant change in the affinity constant. Twenty-five millimolar glucose also decreased AT1R mRNA and protein levels. The 25 mM glucose-induced increase in the formation of lipid peroxides was prevented by antioxidants, protein kinase C (PKC) inhibitors, or L-type calcium channel blockers. These agents also blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. In addition, 25 mM glucose increased transforming growth factor (TGF)-β1 secretion, and anti-TGF-β antibody significantly blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. Furthermore, the 25 mM glucose-induced increase in TGF-β1 secretion was inhibited by PKC inhibitors, L-type calcium channel blockers, or antioxidants. In conclusion, high glucose may induce downregulation of 125I-ANG II binding via a PKC-oxidative stress-TGF-β signal cascade in primary cultured rabbit renal proximal tubule cells.

Signaling cascade of ANG II-induced inhibition of α-MG uptake in renal proximal tubule cells

2004 ◽  
Vol 286 (4) ◽  
pp. F634-F642 ◽  
Author(s):  
Ho Jae Han ◽  
Soo Hyun Park ◽  
Yun Jung Lee
Keyword(s):  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Receptor Blocker ◽  
Dependent Manner ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Tk Inhibitors ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Mapk Inhibitor

ANG II and Na+-glucose cotransporter have been reported to be associated with the onset of diverse renal diseases. However, the effect of ANG II on Na+-glucose cotransporter activity was not elucidated. The effects of ANG II on α-methyl-d-[14C]glucopyranoside (α-MG) uptake and its related signal pathways were examined in the primary cultured rabbit renal proximal tubule cells (PTCs). ANG II (>2 h; >10-9 M) inhibited α-MG uptake in a time- and concentration-dependent manner and decreased the protein level of Na+-glucose cotransporters, the expression of which was abrogated by both actinomycin D and cycloheximide exposure. ANG II-induced inhibition of α-MG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD-123319, an ANG II type 2 receptor blocker. ANG II-induced inhibition of α-MG uptake was blocked by genistein, herbimycin A [tyrosine kinase (TK) inhibitors], mepacrine, and AACOCF3 (phospholipase A2 inhibitors), suggesting the role of TK phosphorylation and arachidonic acid (AA). Indeed, ANG II increased AA release, which was blocked by losartan or TK inhibitors. The effects of ANG II on AA release and α-MG uptake also were abolished by staurosporine and bisindolylmaleimide I (protein kinase C inhibitors) or PD-98059 (p44/42 MAPK inhibitor), but not SB-203580 (p38 MAPK inhibitor), respectively. Indeed, ANG II increased p44/42 MAPK activity. ANG II-induced activation of p44/42 MAPK was blocked by staurosporine. In conclusion, ANG II inhibited α-MG uptake via PKC-MAPK-cPLA2 signal cascade through the AT1 receptor in the PTCs.

Effects of TCDD and estradiol-17β on the proliferation and Na+/glucose cotransporter in renal proximal tubule cells

2005 ◽  
Vol 19 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Ho Jae Han ◽  
Min Jin Lim ◽  
Yun Jung Lee ◽  
Eun Jung Kim ◽  
Young Jin Jeon ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Estradiol 17Β

Abstract P238: Regulation Of G Protein-coupled Receptor Kinase 4 Expression By Serum In Breast Cancer And Renal Proximal Tubule Cells

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Wei Yue ◽  
Peng Xu ◽  
John J Gildea ◽  
Robin A Felder
Keyword(s):  
Breast Cancer ◽  
Protein Synthesis ◽  
Proximal Tubule ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
G Protein Coupled

G protein-coupled receptor kinase 4 (GRK4) is a member of the GRK family which play critical role in regulation of the function of G protein-coupled receptors. Our previous studies have shown that GRK4 not only plays a role in regulating sodium excretion in renal proximal tubule cells but also acts as a stimulator on proliferation of breast cancer cells. Uncontrolled proliferation is a characteristics of cancer cells and GRK4 is upregulated in breast cancer cells. We hypothesized that expression of GRK4 may be regulated differently in cancer and non-cancer cells. To test this hypothesis, expression of GRK4 in response to serum was compared in breast cancer cells and renal proximal tubule cells by Western analysis. In three breast cancer cell lines serum withdrawal caused rapid reduction in the levels of GRK4 which occurred as early as 15 min. GRK4 levels correlated with the concentrations of serum added to the culture media. To determine if growth factors were a critical element for maintaining GRK4 levels in the cells, EGF (10-20 ng/ml) was added to serum free medium for 24 h. There was no increase in GRK4 levels in the cells treated with EGF compared with the serum starvation control. Similarly, serum withdrawal (16 h) led to 40-80% decrease of GRK4 levels in renal proximal tubule cells even in the presence of EFG supplement. Serum feeding for 30 min after starvation dramatically increased the levels of GRK4 in both breast cancer cells and RPTC which exceeded the steady state levels. This rapid recovery of GRK4 protein do not need de novo protein synthesis because pretreatment of the cells with protein synthesis inhibitor, cycloheximide (10 μg/ml, 24 h), did not prevent this event. Expression of GRK2, another member of the GRK family, was not affected by serum starvation. Our results have shown that GRK4 is very sensitive to serum concentration in breast cancer cells as well as in RPTC. Preliminary studies suggest that rapid protein degradation rather than shutting down the protein synthesis plays a major role in this kind of GRK4 regulation. The biological significance of serum regulation of GRK4 in cancer and non-cancerous cells needs further investigation.

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