Electron microscopical observations on the brush border of proximal tubule cells of mammalian kidney

1972 ◽  
Vol 132 (4) ◽  
pp. 473-496 ◽  
Author(s):  
J. Rostgaard ◽  
L. Thuneberg
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Proximal Tubule Cells ◽  
Mammalian Kidney ◽  
Tubule Cells ◽  
Electron Microscopical

Immunolocalization of NHE8 in rat kidney

2005 ◽  
Vol 288 (3) ◽  
pp. F530-F538 ◽  
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.

scholarly journals Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

2010 ◽  
Vol 299 (6) ◽  
pp. F1496-F1506 ◽  
Author(s):  
Alan C. Pao ◽  
Aditi Bhargava ◽  
Francesca Di Sole ◽  
Raymond Quigley ◽  
Xinli Shao ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Proximal Tubule ◽  
Cell Surface Expression ◽  
Thick Ascending Limb ◽  
Proximal Tubule Cells ◽  
Mammalian Kidney ◽  
Tubule Cells ◽  
Exchange Activity

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na+) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na+/H+ exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na+/H+ exchange activity by Na+-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na+/H+ exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+ exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na+ transport through NHE3 in the proximal tubule.

scholarly journals Study of the mechanism by which the Na+-Pi co-transporter of mouse kidney proximal-tubule cells adjusts to phosphate depletion

1988 ◽  
Vol 252 (1) ◽  
pp. 105-109 ◽  
Author(s):  
M Jahan ◽  
P J Butterworth
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Mouse Kidney ◽  
Transport Activity ◽  
Normal Medium ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Kinetic Investigations ◽  
Stimulation Of

1. Proximal-tubule cells isolated from mouse kidney after digestion with collagenase take up Pi by an Na+-dependent and saturable process mediated by the Na+-Pi co-transporter of the brush-border membrane. 2. Pi depletion of the cells is accompanied by a stimulation of Pi-transport activity. Kinetic investigations reveal that Vmax. is increased by 90% and Km decreased by 50% after Pi depletion. Transport activity returns to normal values after incubation for 30 min at 37 degrees C of Pi-depleted cells in normal medium containing 1 mM-Pi, but the fall in transport activity under these conditions is inhibited by colchicine. 3. The energy of activation of Na+-Pi co-transport activity of depleted cells differs greatly from that found for normal replete cells. 4. The results provide evidence that stimulation of transport by Pi depletion arises from an increase in the number of carrier sites in the brush-border membrane. Additionally, changes in the properties of the transporter occur which may reflect altered phospholipid-carrier-protein interaction in the Pi-depleted condition.

scholarly journals Comparative study of ezrin phosphorylation among different tissues: more is good; too much is bad

2008 ◽  
Vol 295 (1) ◽  
pp. C192-C202 ◽  
Author(s):  
Lixin Zhu ◽  
Jason Hatakeyama ◽  
Cheng Chen ◽  
Aditi Shastri ◽  
Kevin Poon ◽  
...  
Keyword(s):  
Comparative Study ◽  
Proximal Tubule ◽  
Brush Border ◽  
Parietal Cells ◽  
Proximal Tubule Cells ◽  
Surface Plasticity ◽  
Phosphorylation Level ◽  
Tubule Cells ◽  
Gastric Parietal Cells ◽  
Different Tissues

In a comparison of three different tissues, the membrane cytoskeleton linker protein ezrin was found to assume high levels of phosphorylation on threonine-567 (T567) in the brush border membranes of renal proximal tubule cells and small intestine enterocytes, in contrast to the apical canalicular membrane of gastric parietal cells. Together with an earlier observation that increased T567 phosphorylation is associated with more elaborate microvilli in parietal cells, this comparative study suggested a higher phosphorylation level requirement for the denser and more uniform distribution of microvilli at brush border surfaces. Using a kinase inhibitor, staurosporin, and metabolic inhibitor, sodium azide, relatively high turnover of ezrin T567 phosphorylation was observed in all three epithelia. Aiming to understand the role of phosphorylation turnover in these tissues, detergent extraction analysis of gastric glands and proximal tubules revealed that an increased phosphorylation on ezrin T567 greatly enhanced its association with F-actin, while ezrin-membrane interaction persisted regardless of the changes of phosphorylation level on ezrin T567. Finally, expression of Thr567Asp mutant ezrin, which mimics the phospho-ezrin state but does not allow turnover, caused aberrant growth of membrane projections in cultured proximal tubule cells, consistent with what had previously been observed in several cell lines and gastric parietal cells. These results fit into a model of surface plasticity, which posits that the turnover of phosphorylation on T567 empowers ezrin to relax and reposition membrane to the underlying cytoskeleton under varying conditions of filament growth or rapid membrane expansion (or depletion).

Role of dipeptidyl peptidase IV in regulating activity of Na+/H+exchanger isoform NHE3 in proximal tubule cells

2004 ◽  
Vol 287 (5) ◽  
pp. C1238-C1245 ◽  
Author(s):  
Adriana C. C. Girardi ◽  
Felix Knauf ◽  
Hans-Ulrich Demuth ◽  
Peter S. Aronson
Keyword(s):  
Tyrosine Kinase ◽  
Proximal Tubule ◽  
Brush Border ◽  
Kinase Inhibitor ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Tubule Cells

We recently reported that NHE3 exists in multimeric complexes with dipeptidyl peptidase IV (DPPIV) in renal brush-border membranes. To examine the possible role of DPPIV in modulating NHE3 activity, we evaluated whether specific competitive inhibitors that bind to the active site of DPPIV affect NHE3 activity in the OKP line of opossum kidney proximal tubule cells. The DPPIV inhibitors diprotin A and P32/98 significantly reduced NHE3 activity, whereas the inactive isomer P34/98 had no effect. DPPIV inhibitors did not reduce the activity of another brush-border transport process, Na-phosphate cotransport. Effects of DPPIV inhibitors on NHE3 activity were not associated with detectable changes in amount or apparent molecular weight of NHE3 or in NHE3 surface expression. To investigate the signaling mechanisms involved in modulation of NHE3 activity by DPPIV, we used inhibitors of protein kinase pathways known to regulate NHE3. Whereas the PKA inhibitor H-89 failed to block the effect of DPPIV inhibitors, the tyrosine kinase inhibitor genistein alone caused a decrement in NHE3 activity very similar in magnitude to that caused by P32/98. We also found that the effects of genistein and P32/98 on NHE3 activity were not additive. In contrast, forskolin/IBMX and P32/98 had additive inhibitory effects on NHE3 activity. These findings suggested that the effect of DPPIV inhibitors to reduce NHE3 activity results from inhibition of a tyrosine kinase signaling pathway rather than by activation of PKA. We conclude that DPPIV plays an unexpected role in modulating Na+/H+exchange mediated by NHE3 in proximal tubule cells.

scholarly journals Redistribution of villin to proximal tubule basolateral membranes after ischemia and reperfusion

1997 ◽  
Vol 273 (6) ◽  
pp. F1003-F1012 ◽  
Author(s):  
Dennis Brown ◽  
Richard Lee ◽  
Joseph V. Bonventre
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Actin Binding ◽  
Ischemia And Reperfusion ◽  
Proximal Tubule Cells ◽  
Basolateral Plasma Membrane ◽  
Tubule Cells ◽  
Renal Tubular

After ischemia and reperfusion, severe alterations in the cytoskeletal organization of renal tubular epithelial cells have been reported. These effects, accompanied by a modification in the polarized distribution of some membrane transport proteins, are especially evident in the proximal tubule. In normal proximal tubule cells, actin is concentrated in apical brush border microvilli, along with the actin-binding protein villin. Because villin plays an important role in actin bundling and in microvillar assembly but can also act as an actin-fragmenting protein at higher calcium concentrations, we examined the effects of ischemic injury and reperfusion on the distribution of villin and actin in proximal tubule cells of rat kidney. Using specific antibodies against villin and actin, we show that these proteins redistribute in parallel from the apical to the basolateral plasma membrane within 1 h of reperfusion after ischemia. Ischemia alone had no effect on the staining pattern. Repolarization of villin to the apical membrane begins within hours after reperfusion with enhanced apical localization over time during the period of regeneration. This apical repolarization of villin is accompanied by the migration of actin back to the apical membrane. These results show not only that villin may be involved in the initial disruption of the actin cytoskeleton during reperfusion injury but also that its migration back to the apical domain of these cells accompanies the reestablishment of a normal actin distribution in the brush border.

Increased functional differentiation of rabbit proximal tubule cells cultured in glucose-free media

1992 ◽  
Vol 263 (1) ◽  
pp. F152-F162
Author(s):  
A. Blais ◽  
F. Jalal ◽  
P. Crine ◽  
J. Paiement ◽  
A. Berteloot
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Primary Cultures ◽  
Morphological Differentiation ◽  
Functional Expression ◽  
Functional Differentiation ◽  
Rabbit Kidney ◽  
Proximal Tubule Cells ◽  
Tubule Cells

We have determined the influence of glucose (Glc)-free medium on the growth and differentiation of rabbit kidney proximal tubule cells (PTC) in primary cultures. The specific growth rates and the protein-to-volume ratios are shown to be independent of the culture conditions. In contrast, the functional expression of four brush-border membrane enzyme markers was found to decline steadily and in the same way from day 3 in culture up to late confluence in Glc-containing medium, and different evolution patterns and high expression levels were observed up to confluence in a Glc-free glutamine (Gln)-supplemented medium. Electron microscopy clearly showed, however, that the functional and morphological differentiation of the brush-border membrane is not correlated. Finally, by use of an indirect immunofluorescent technique in combination with flow cytometry, it is demonstrated that confluent cells grown in Glc and Gln media form homogeneous cell populations of PTC. It is thus concluded that the functional differentiation of rabbit kidney PTC in primary cultures is strongly dependent upon the energy source present in the culture medium.

The vitamin D receptor in the proximal renal tubule is a key regulator of serum 1α,25-dihydroxyvitamin D3

2015 ◽  
Vol 308 (3) ◽  
pp. E201-E205 ◽  
Author(s):  
Yongji Wang ◽  
Jinge Zhu ◽  
Hector F. DeLuca
Keyword(s):  
Vitamin D ◽  
Proximal Tubule ◽  
Vitamin D Receptor ◽  
Brush Border ◽  
Distal Tubule ◽  
Proximal Convoluted Tubule ◽  
Proximal Tubule Cells ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Tubule Cells

It is well established that the mitochondria of proximal convoluted tubule cells of the kidney are the site of production of circulating 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The production of 1,25(OH)2D3 at this site is tightly regulated. Parathyroid hormone markedly stimulates 1,25(OH)2D3 production, whereas 1,25(OH)2D3 itself suppresses production. The mechanism of suppression by 1,25(OH)2D3 has not yet been elucidated. We have now found that in the absence of vitamin D (vitamin D deficiency), the vitamin D receptor (VDR) is found in the interior of the apical brush border of the proximal tubule cells. This is unique for the proximal tubule cells, since this has not been observed in the distal tubule cells or in other epithelial cells, such as intestinal mucosa. Administration of 1,25(OH)2D3 to vitamin D-deficient rats results in the movement of VDR from the brush border to the cytoplasm and nucleus presumably bound to reabsorbed 1,25(OH)2D3. The VDR bound to 1,25(OH)2D3 suppresses expression of 25-hydroxyvitamin D3 1α-hydroxylase and stimulates the 25-hydroxyvitamin D3 24-hydroxylase. Thus, VDR in the apical brush border of the proximal convoluted tubule cells serves to “sense” the level of circulating 1,25(OH)2D3 and modulates the activity of the 1α-hydroxylase and the 24-hydroxylase accordingly.

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