Abstract P169: Antenatal Betamethasone Attenuates the Angiotensin-(1-7)/Nitric Oxide Axis in Adult Male but not Female Renal Proximal Tubule Cells

Our studies have revealed a sex-specific effect of fetal programming on sodium (Na+) excretion in adult sheep whereby the males exhibit reduced Na+ excretion and an attenuated natriuretic response to Ang-(1-7) as compared to the females. We hypothesize that the renal proximal tubules are a key target for the early programming effects of glucocorticoids exposure to regulate Na+ handling in the adult males. Therefore, we isolated and cultured cortical proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to betamethasone (Beta) or vehicle. Na+ uptake and nitric oxide (NO) were assessed with Sodium Green and DAF fluorescence prior to and following a low dose of Ang-(1-7) (1x10-11 M) in isolated RPTC from sheep at ~1.5 years of age. Data are expressed as % of basal uptake or area under the curve (AUC) for Na+ or % of control for NO. Male Beta RPTC exhibit greater Na+ uptake than male vehicle cells (427±32%, n=13, vs. 315±28%, n=14, p<0.05; however, Beta had no effect on Na+ uptake in the female cells (242±18%, n=9, vs. 250±15%, n=10, p>0.05). Ang-(1-7) inhibited Na+ uptake in RPTC from vehicle male (255±40%) and from both vehicle (191±14%) and Beta (209±11%) females (Figure 1B), but failed to attenuate Na+ uptake in Beta male cells (Figure 1A). Beta exposure also abolished NO stimulation by Ang-(1-7) in male but not female RPTC (Figure 1C). We conclude that an Ang-(1-7)-NO-dependent pathway contributes to the sex-dependent consequences of programming on Na+ regulation in the proximal tubules of the kidney. Moreover, the RPTC retain both the sex and Beta-induced phenotype of the adult and may reflect an appropriate cell model of fetal programming.

Download Full-text

Antenatal betamethasone attenuates the angiotensin-(1–7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00593.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. F1056-F1062 ◽

Cited By ~ 6

Author(s):

Yixin Su ◽

Jianli Bi ◽

Victor M. Pulgar ◽

Mark C. Chappell ◽

James C. Rose

Keyword(s):

Nitric Oxide ◽

Proximal Tubule ◽

Cell Model ◽

Primary Cultures ◽

Specific Effect ◽

Renal Tubules ◽

Proximal Tubule Cells ◽

Mas Receptor ◽

Tubule Cells

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1–7) [Ang-(1–7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1–7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1–7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1–7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1–7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1–7). We conclude that the tubular Ang-(1–7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

Download Full-text

Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner

AJP Renal Physiology ◽

10.1152/ajprenal.00047.2015 ◽

2015 ◽

Vol 308 (11) ◽

pp. F1268-F1275 ◽

Cited By ~ 13

Author(s):

Yixin Su ◽

Jianli Bi ◽

Victor M. Pulgar ◽

Jorge Figueroa ◽

Mark Chappell ◽

...

Keyword(s):

Proximal Tubule ◽

Fetal Programming ◽

Specific Effect ◽

Ang Ii ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Renal Proximal Tubule Cells ◽

Female Sheep ◽

Male Sheep ◽

In Cells

We have shown a sex-specific effect of fetal programming on Na+ excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na+ uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na+ uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na+/H+ exchanger 3. Basal Na+ uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na+ uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na+ uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na+ uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells.

Download Full-text

Sex specific effect of antenatal glucocorticoids on nitric oxide production stimulated by angiotensin peptides in renal proximal tubule cells (1085.6)

The FASEB Journal ◽

10.1096/fasebj.28.1_supplement.1085.6 ◽

2014 ◽

Vol 28 (S1) ◽

Author(s):

Jianli Bi ◽

Kai Chen ◽

Yixin Su ◽

Mark Chappell ◽

James Rose

Keyword(s):

Nitric Oxide ◽

Proximal Tubule ◽

Specific Effect ◽

Renal Proximal Tubule ◽

Nitric Oxide Production ◽

Proximal Tubule Cells ◽

Oxide Production ◽

Angiotensin Peptides ◽

Tubule Cells ◽

Renal Proximal Tubule Cells

Download Full-text

Immunolocalization of NHE8 in rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00229.2004 ◽

2005 ◽

Vol 288 (3) ◽

pp. F530-F538 ◽

Cited By ~ 65

Author(s):

Sunita Goyal ◽

SueAnn Mentone ◽

Peter S. Aronson

Keyword(s):

Proximal Tubule ◽

Brush Border ◽

Brush Border Membrane ◽

Rat Kidney ◽

Surface Membrane ◽

Proximal Tubules ◽

Intense Staining ◽

Proximal Tubule Cells ◽

Nephron Segment ◽

Tubule Cells

In situ hybridization studies demonstrated that Na+/H+ exchanger NHE8 is expressed in kidney proximal tubules. Although membrane fractionation studies suggested apical brush-border localization, precise membrane localization could not be definitively established. The goal of the present study was to develop isoform-specific NHE8 antibodies as a tool to directly establish the localization of NHE8 protein in the kidney by immunocytochemistry. Toward this goal, two sets of antibodies that label different NHE8 epitopes were developed. Monoclonal antibody 7A11 and polyclonal antibody Rab65 both specifically labeled NHE8 by Western blotting as well as by immunofluorescence microscopy. The immunolocalization pattern in the kidney seen with both antibodies was the same, thereby validating NHE8 specificity. In particular, NHE8 expression was observed on the apical brush-border membrane of all proximal tubules from S1 to S3. The most intense staining was evident in proximal tubules in the deeper cortex and medulla with a significant but somewhat weaker staining in superficial proximal tubules. Colocalization studies with γ-glutamyltranspeptidase and megalin indicated expression of NHE8 on both the microvillar surface membrane and the coated-pit region of proximal tubule cells, suggesting that NHE8 may be subject to endocytic retrieval and recycling. Although colocalizing in the proximal tubule with NHE3, no significant alteration in NHE8 protein expression was evident in NHE3-null mice. We conclude that NHE8 is expressed on the apical brush-border membrane of proximal tubule cells, where it may play a role in mediating or regulating ion transport in this nephron segment.

Download Full-text

Angiotensin II AT2 receptor decreases AT1 receptor expression and function via nitric oxide/cGMP/Sp1 in renal proximal tubule cells from Wistar–Kyoto rats

Journal of Hypertension ◽

10.1097/hjh.0b013e3283532099 ◽

2012 ◽

Vol 30 (6) ◽

pp. 1176-1184 ◽

Cited By ~ 40

Author(s):

Jian Yang ◽

Caiyu Chen ◽

Hongmei Ren ◽

Yu Han ◽

Duofen He ◽

...

Keyword(s):

Nitric Oxide ◽

Angiotensin Ii ◽

Proximal Tubule ◽

Receptor Expression ◽

At2 Receptor ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Wistar Kyoto ◽

Renal Proximal Tubule Cells ◽

And Function

Download Full-text

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

AJP Cell Physiology ◽

10.1152/ajpcell.00006.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. C517-C526 ◽

Cited By ~ 101

Author(s):

Ruben M. Sandoval ◽

Michael D. Kennedy ◽

Philip S. Low ◽

Bruce A. Molitoris

Keyword(s):

Folic Acid ◽

Proximal Tubule ◽

Rat Kidney ◽

Proximal Tubules ◽

Apical Surface ◽

Proximal Tubule Cells ◽

Two Photon Microscopy ◽

Two Photon ◽

Tubule Cells ◽

Folate Conjugate

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments.

Download Full-text

Tandem Requirement for Full Renal D1R and D5R Activity

10.1101/736611 ◽

2019 ◽

Author(s):

Selim Rozyyev ◽

Annabelle P. Crusan ◽

Andrew C. Tiu ◽

Julie A. Jurgens ◽

Justin Michael B. Quion ◽

...

Keyword(s):

Proximal Tubule ◽

Structural Features ◽

Renal Proximal Tubule ◽

Proximal Tubules ◽

Proximal Tubule Cells ◽

Renal Proximal Tubules ◽

Genetic Ablation ◽

R And D ◽

Tubule Cells ◽

Renal Proximal Tubule Cells

ABSTRACTThe peripheral dopaminergic system promotes the maintenance of blood pressure homeostasis by engendering natriuresis, mainly through the renal D1R and D5R receptors. This effect is most apparent under conditions of moderate body sodium excess. Human and rodent renal proximal tubules express both receptors, which share common structural features and pharmacological profiles. Genetic ablation of either receptor in the kidney results in hypertension in mice. In this study, we demonstrated that in renal proximal tubules, these two receptors colocalized, co-immunoprecipitated, co-segregated in lipid rafts, and heterodimerized with one another, which was enhanced by treatment with the D1R/D5R agonist fenoldopam (1 μM, 30 min). Gene silencing via antisense oligonucleotides in renal proximal tubule cells abrogated cAMP production and sodium transport in response to fenoldopam. Our results highlight the cooperation and co-dependence of these two receptors through heterodimerization in renal proximal tubule cells.

Download Full-text

Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells

PLoS ONE ◽

10.1371/journal.pone.0250996 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0250996

Author(s):

Yasaman Ramazani ◽

Noël Knops ◽

Sante Princiero Berlingerio ◽

Oyindamola Christiana Adebayo ◽

Celien Lismont ◽

...

Keyword(s):

Proximal Tubule ◽

Cell Model ◽

Calcineurin Inhibitors ◽

Redox Balance ◽

Solid Organ ◽

Proximal Tubule Cell ◽

Tubule Cell ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Glutathione Redox

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

Download Full-text