scholarly journals A linear relationship between the ex-vivo sodium mediated expression of two sodium regulatory pathways as a surrogate marker of salt sensitivity of blood pressure in exfoliated human renal proximal tubule cells: The virtual renal biopsy

2013 ◽  
Vol 421 ◽  
pp. 236-242 ◽  
Author(s):  
John J. Gildea ◽  
Dylan T. Lahiff ◽  
Robert E. Van Sciver ◽  
Ryan S. Weiss ◽  
Neema Shah ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Proximal Tubule ◽  
Renal Biopsy ◽  
Linear Relationship ◽  
Ex Vivo ◽  
Surrogate Marker ◽  
Proximal Tubule Cells ◽  
Regulatory Pathways ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Abstract 79: Caveolin-1 Knockout Mice Have Salt-Sensitive Hypertension and Dopamine-1 Receptor Defects in Renal Cortex and Isolated Renal Proximal Tubule Cells

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
John J Gildea ◽  
Nancy L Howell ◽  
Robert E Van Sciver ◽  
Brandon A Kemp ◽  
Robert M Carey ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Proximal Tubule ◽  
Kinase Activity ◽  
Renal Cortex ◽  
Renal Proximal Tubule ◽  
Normal Blood Pressure ◽  
Proximal Tubule Cells ◽  
Caveolin 1 ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Dopamine-1 receptors (D1R) are necessary for kidney proximal tubule-dependent natriuresis and maintenance of normal blood pressure, especially under high salt conditions. G-protein coupled receptor kinase 4 (GRK4) is a negative regulator of D1R function and single nucleotide polymorphisms in GRK4 have been associated with both hypertension and salt sensitivity in humans. Caveolin-1 (CAV1) directly binds to GRK4 and decreases kinase activity. We hypothesized that CAV1 knockout mice (CAV1KO) would have increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the D1R. Mean arterial blood pressure (MAP, in mmHg ±SEM) measured over 5 days was not significantly different for Wild-type mice (WT, 128.9±4.2 mmHg, n=4) vs CAV1KO (129.5±3.5 mmHg, n=4) on normal chow (0.3% sodium). However, on a 4% high sodium diet, the MAP of CAV1KO mice increased in just 2 days by 20.1±4.2 mmHg (p<0.05 vs either Day 0 CAV1KO or Day 2 WT, n=4). The CAV1KO MAP increased by 25.9±6.6 mmHg by day 7 (p<0.05 vs either Day 0 CAV1KO or Day 7 WT, n=4). Hyperphosphorylation and inactivation of the D1R in renal cortex was examined by looking at phospho-serine D1R by immuno-precipitation and Western dot blotting. A 92.5% ± 18.8 SEM increase in phospho-D1R was found in the CAV1KO renal cortex (n=4, p<0.01 vs WT; 14,574/7570 RFU). Cortical slices were made and incubated for 30 minutes with fenoldopam (FEN, 10 μM) with or without LE300 (D1R-like antagonist, 10 μM) or vehicle (VEH). Cyclic AMP was measured by TR-FRET (Lance, Perkin Elmer). FEN significantly increased cAMP 5.6 fold ± 1.2 SEM (n=4, p<0.01 vs VEH; 7.84/1.4 pmole/mg protein) in WT but not in CAV1KO slices, and this effect was completely blocked by LE300. Primary mouse CAV1KO and WT renal proximal tubule cell lines were established and monensin (sodium ionophore, 5 μM, 30 minutes)-induced plasma membrane D1R recruitment increased as measured by confocal microscopy in WT (30.4% ± 7.4 SEM, n=11, p<0.01 vs VEH; 8990/6894 RFU) but not in CAV1KO proximal tubule cells. In summary, CAV1 is necessary in high salt conditions for maintaining normal blood pressure in mice and for preserving normal D1R function in kidney cortex and in mouse renal proximal tubule cells.

Abstract MP61: Sex Difference In The Response Of Human Renal Proximal Tubule Cells To A Nephrotoxic Agent.

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Shaun Moore ◽  
Megha Kumar ◽  
Daniel Yaqub ◽  
John J Gildea ◽  
Robin Felder ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Proximal Tubule ◽  
Dopamine D2 Receptor ◽  
Renal Proximal Tubule ◽  
Ki 67 ◽  
Proximal Tubule Cells ◽  
Renal Inflammation ◽  
Males And Females ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Our previous work indicated that the renal dopamine D2 receptor (D2R) has a significant role in regulating renal inflammation and injury, as well as in blood pressure control. In mice, D2R has protective effects in the kidney by limiting the inflammatory and fibrotic reaction; impaired D2R function results in renal inflammation and damage. Some common single nucleotide polymorphisms (SNPs; rs 6276 and 6277) in the human DRD2 gene are associated with decreased D 2 R expression and function and high blood pressure. To determine the effects of the presence of SNPs in the response to the nephrotoxic aristolochic acid (AA, 5μg/ml, 24 h), we studied immortalized human renal proximal tubule cells isolated from normal tissue of nephrectomies and genotyped for DRD2 SNPs and DRD2 wild-type (WT). We also determined whether this response is sex dependent. D2R protein was higher in male than in female WT (135±5 vs 100±4%; n=3/group; P<0.04) and lower in males with SNPs (43±2%, P<0.05) and females with SNPs (23±2%,P<0.05), compared with their respective WT counterparts. In both male groups (WT and SNPs), AA increased D2R protein by 80-100% but had no effect in WT females and increased ~50% in females with SNPs. The TNFα mRNA was higher in males with WT and SNPs which was increased by AA 9-10-fold in WT males and females but only 2-3-fold in those with SNPs. The TGFβ mRNA was similar in WT males and females and increased to the same extent in both groups with SNPs and was not affected by AA in all groups. Col1a1 mRNA was higher (30%) in WT males and females than those with SNPs; AA decreased Col1a1 mRNA in all groups. FN1 mRNA was higher (30-40%) in males and females with SNPs than WT; AA increased FN1 mRNA only in males and females with SNPs. The mRNA expression of the cell proliferation marker Ki-67 was higher in WT females than WT males (1.5-2-fold) and higher with SNPs than WT in both groups; AA increased Ki-67 mRNA in both groups and to a greater extent in males than in females. Taken together our data indicate that the presence of DRD2 SNPs affects the baseline expression of inflammatory and fibrotic factors and the response to AA is dependent on both sex and the presence of DRD2 SNPs. These data may have potential clinical translation since rs6276/6277 is commonly expressed (42%/23%) in the human population.

Abstract P009: Differential Redox Signaling In Urine Derived Renal Proximal Tubule Cells Isolated From Inverse Salt Sensitive Vs Salt Resistant Clinical Study Participants

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
John J Gildea ◽  
Michael G Daley ◽  
Peng Xu ◽  
Katherine Schiermeyer ◽  
Wei Yue ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Proximal Tubule ◽  
Redox Signaling ◽  
T Test ◽  
Wild Type ◽  
Proximal Tubule Cells ◽  
Low Sodium ◽  
Insulator Function ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. We demonstrated that single nucleotide polymorphisms (SNPs) in the dopamine type 2 receptor (D2R) as well as lower expression of cellular and plasma membrane D2R are associated with inverse salt sensitivity (ISS, a paradoxical increase in blood pressure while on the low sodium diet). Urine derived human renal proximal tubule cells (RPTC) were cultured from 3 separate ISS and 3 salt resistant (SR, resists blood pressure change on both low and high sodium diets) participants. D2R reduces cellular redox signaling so we hypothesized that an aberrant redox state occurs in ISS and SR RPTC exposed to different NaCl concentrations. RPTC incubated in 90 mM NaCl (low salt - LS) for 2 hours in both ISS and SR RPTCs had a significant reduction of monochlorobimane (mBCl - reduced glutathione indicator) compared to 140 mM NaCl (normal salt -NS) (SR -7.1± 2.1% and ISS -9.7 ± 1.6% vs NS, n=12, p<0.05, t-test). Only ISS RPTC had a significant increase in mBCl fluorescence in 190 mM NaCl (ISS +7.0 ± 2.5% vs NS, n=12, p<0.05, t-test) and mBCl fluorescence was not affected by incubation with losartan (angiotensin type 1 receptor inhibitor). 3-Aminobenzamide (3AB, 1 mM) inhibits Parp1 (poly[ADP-ribose] synthase), a protein necessary for the transcriptional repressor CTCF insulator function. 3AB reversed the lower mBCl fluorescence only in the SR RPTC, suggesting that reducing CTCF insulator function only affects D2R wild type cells. mBCl fluorescence in ISS RPTC showed a greater reduction by LOS vs SR RPTC (ISS -49.3 ± 1.5% vs SR -37.3 ± 4.8%, n=12, p<0.05, t-test), which was blocked by nemonapride (NEM, 1 uM, D2R antagonist) and further reduced the mBCl fluorescence only in the SR RPTC. Whether these differences in salt sensitivities are due to differential effects of CTCF insulator function at the homozygous variant or wild type D2R warrants further studies.

Abstract 88: Single Nucleotide Polymorphisms of the Dopamine D2 Receptor Increase Inflammation and Fibrosis in Human Renal Proximal Tubule Cells

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Xiaoliang Jiang ◽  
Prasad Konkalmatt ◽  
John Gildea ◽  
John Jones ◽  
Robin A Felder ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Single Nucleotide Polymorphisms ◽  
Proximal Tubule ◽  
Cell Lines ◽  
High Blood Pressure ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

We have reported that lack or downregulation of dopamine D(2) receptor (D 2 R) function in mice increases the vulnerability to renal inflammation independent of blood pressure and that the D 2 R negatively regulates inflammation and prevents injury in mouse renal proximal tubule cells (RPTCs). Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277 and 1800497) in the human DRD2 gene are associated with decreased D 2 R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs expressing these SNPs have increased expression of inflammatory markers and injury. We studied immortalized human RPTCs isolated from normal tissue of nephrectomies which were genotyped for the D 2 R SNPs; 10 cell lines were selected: wild type (WT) n=5; heterozygous for both rs6276 and 6277 n=3; heterozygous for both rs6276 and 1800497 n=2. All 5 cell lines expressing SNPs (hRPTC-SNP) had decreased D 2 R mRNA (0.52±0.03 vs. 1±0.02 (WT) fold; P<0.02 ) and protein (62±4 vs. 100±6%; P<0.04) and were pooled together for analysis. D 2 R function was also decreased as determined by the decreased ability of a D 2 R agonist to increase PMA-induced cAMP accumulation (hRPTC-SNP: 201±16; hRPTC-WT 270 ±5 %; n=4; P<0.05). The expression of the pro-inflammatory cytokines TNFα (2.5 ± 0.3 fold; P<0.04) and CD40L (2.3 ± 0.5 fold; P<0.05), as well as the inflammation marker CRP (1.5 ± 0.2 fold; P<0.04) was increased in hRPTC-SNP. The expression of the pro-fibrotic TGFβ1 (1.8 ± 0.4 fold; P<0.04) and its signaling targets PAI-1 (6.3± 0.5 fold; P<0.01), SMAD3 (2.8± 0.4 fold; P<0.02) and VEGFa (2.3± 0.6 fold; P<0.04) were also increased. Furthermore hRPTC-SNP showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins as shown by increased fibronectin -1 (3.1± 0.7 fold; P<0.03), col 1a (1.6± 0.2 fold; P<0.05), col 3a (2.2± 0.5 fold; P<0.05), MMP3 (5.6± 0.8 fold; P<0.04), and MMP9 (3.5± 0.5 fold; P<0.04). These data support the hypothesis that D 2 R function has protective effects in human RPTCs and suggest that carriers of these SNPs may be prone to chronic renal disease and high blood pressure.

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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