Role of microfilaments in maintenance of proximal tubule structural and functional integrity

1990 ◽  
Vol 259 (2) ◽  
pp. F279-F285 ◽  
Author(s):  
P. S. Kellerman ◽  
R. A. Clark ◽  
C. A. Hoilien ◽  
S. L. Linas ◽  
B. A. Molitoris
Keyword(s):  
Proximal Tubule ◽  
Tubular Reabsorption ◽  
Cytochalasin D ◽  
Apical Surface ◽  
Proximal Tubule Cells ◽  
Terminal Web ◽  
Initial Decrease ◽  
And Function ◽  
Subsequent Stabilization

To determine the selective effect of microfilament disruption on both cellular structure and function, microfilament-specific doses of cytochalasin D (10 microM) were used in an isolated perfused kidney system. Structurally, cytochalasin D resulted in extensive disruption of the apical surface with blebbing, vacuolization, and patchy loss and fusion of microvilli. Functionally, cytochalasin D resulted in an initial decrease in glomerular filtration rate (300.8 +/- 29.9 vs. 541.6 +/- 51 microliters.min-1.g-1, P less than 0.05) with subsequent stabilization throughout the duration of the perfusion. In contrast, the tubular reabsorption of sodium decreased significantly in a linear fashion from 97.1 +/- 0.7 to 64.3 +/- 7.0% over the duration of the perfusion. Similarly, the tubular reabsorption of lithium decreased linearly from 74.8 +/- 2.6%, before the addition of cytochalasin, to 33.6 +/- 6.8% by the end of the perfusion. Correlation of the decrements in percent tubular reabsorption of sodium and lithium for individual kidneys was 0.87 (P less than 0.01), suggesting the effect of microfilament disruption on tubular reabsorption of sodium was localized primarily to the proximal tubule. Because ischemic injury is characterized by time-dependent structural alterations in the apical membrane of proximal tubule cells, we set out to determine whether microfilament disruption occurs during ischemic acute renal failure. Utilizing indirect immunofluorescence with an anti-actin antibody, control kidneys demonstrated intact circumferential apical immunofluorescence representing brush-border and terminal web actin staining. Fifteen minutes of ischemia resulted in multiple large gaps in the terminal web, and 50 min of ischemia caused diffuse redistribution of actin immunofluorescence throughout the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Abstract P244: Role of the Na-H Exchanger Regulatory Factor 1 (NHERF1) in Hypertension of Aging Animals

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Sathnur Pushpakumar ◽  
Corey J Ketchem ◽  
Michelle T Barati ◽  
Utpal Sen ◽  
Pedro J Jose ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Sodium Intake ◽  
Dietary Sodium ◽  
Brown Norway ◽  
Proximal Tubule Cells ◽  
Left Femoral Artery ◽  
Old Rats ◽  
Dopamine Signaling ◽  
Old Mice

Aging animals develop hypertension when challenged with high salt diet due, in part, to desensitization of dopamine receptors (DR) in renal proximal tubules (RPT). We have demonstrated that NHERF1 associates with DR1 and Na-K ATPase (NKA) and is important for regulation of NKA in RPT. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 results in increased blood pressure (BP) and lack of natriuretic response to dopamine (DA) in aging animals. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed diet containing 1% or 8% NaCl for one week and, BP was measured in anesthetized animals using an indwelling left femoral artery catheter. 8% NaCl did not increase BP in 1m or 4 month old rats. By contrast, 8% NaCl diet increased BP in 12m (84.3±3.5 vs 90.8±2.36) and 24m (73.5±7.58 vs 104±1.6) old animals. To determine if lack of NHERF1 is responsible for the increase in BP, we measured BP in 12 m old WT and NHERF1 KO mice. By contrast to WT mice, 8% NaCl diet did not increase BP in NHERF1 KO mice (84±4.9 vs 96.5±3.56 (WT) and 78.2±3.89 vs 81.8±9.2 (NHERF1 KO mice)). To confirm that NHERF1 is required for DA-mediated inhibition of NKA, NKA activity in primary proximal tubule cells (PTC) from young and old mice in culture was measured in the presence or absence of DA. DA decreased NKA activity in PTC from young animals (67.2±3.8 vs 32.7±5.3) but not in PTC from old animals. Transfection of NHERF1 restored NKA regulation by DA in PTC from old rats (58.4±4.2 vs 64.4±4.3 (in untransfected cells) 54.2±3.8 vs 31.1±3.4 (in NHERF1 transfected cells)). We conclude that NHERF1 regulates DA-mediated proximal tubule sodium handling; however, other factors modulate BP response to dietary sodium intake in young and old animals. The contribution of NHERF1 and dopamine signaling to sodium homeostasis requires further study.

Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells

1998 ◽  
Vol 274 (5) ◽  
pp. F897-F905 ◽  
Author(s):  
Thomas J. Thekkumkara ◽  
Rochelle Cookson ◽  
Stuart L. Linas
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Epithelial Tissue ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Binding Studies ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
And Function ◽  
In Cells

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1A are present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood, because specific binding of ANG II to intact PTEC has not been found and because a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells, which stably express AT1A( K d = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of nontransfected OK cells revealed no evidence of AT1A mRNA (reverse transcriptase-polymerase chain reaction analysis) or protein (125I-labeled ANG II binding studies) expression. In cells stably expressing AT1A, ANG II binding was saturable, reversible, and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions that allowed selective access to AP or BL surfaces. AT1A were expressed on both AP ( K d = 8.7 nM, Bmax = 3.33 pmol/mg protein) and BL ( K d = 10.1 nM, Bmax = 5.50 pmol/mg protein) surfaces. Both AP and BL AT1Areceptors underwent agonist-dependent endocytosis (AP receptor: t 1/2 = 7.9 min, Ymax = 78.5%; BL receptor: t 1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, ANG II caused time- and concentration-dependent increases in transepithelial22Na transport (2-fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line that expresses AT1A on both AP and BL surfaces, undergoes agonist-dependent receptor endocytosis, and is functional, as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC.

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

2004 ◽  
Vol 287 (2) ◽  
pp. C517-C526 ◽  
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.

Role of MAPK pathways in light chain-induced cytokine production in human proximal tubule cells

2003 ◽  
Vol 284 (6) ◽  
pp. F1245-F1254 ◽  
Author(s):  
Sule Sengul ◽  
Craig Zwizinski ◽  
Vecihi Batuman
Keyword(s):  
Proximal Tubule ◽  
Light Chain ◽  
Cytokine Production ◽  
Tubulointerstitial Injury ◽  
Proximal Tubule Cells ◽  
Mapk Pathways ◽  
Cytokine Responses ◽  
Tubule Cells ◽  
Human Proximal Tubule

We previously demonstrated that light chain (LC) endocytosis by human proximal tubule cells (PTCs) leads to production of cytokines through activation of NF-κB. Here, we examined the role of MAPK pathways in these responses using four species of myeloma LCs (κ1, κ2, κ3, and λ1) previously shown to induce cytokine production by PTCs. Among these, κ1-LC, which yielded the strongest cytokine responses, was selected for detailed studies. Activation of MAPKs was probed by Western blot analysis for the active kinases, ERK 1/2, JNK 1/2, and p38 in κ1-LC-exposed human PTCs. To evaluate the functional role of MAPKs in LC-induced cytokine responses, we tested the effects of U-0126, an ERK inhibitor; SP-600125, an inhibitor of JNK; SB-203580, a p38 inhibitor; and curcumin, a JNK-AP-1 inhibitor, all added to media before 4-h exposure to 1.5 mg/ml κ1-LC. IL-6 and monocyte chemotactic protein-1 (MCP-1) were determined by ELISA. Both LC and human serum albumin (HSA) activated ERK, although the HSA effect was weaker. κ1-LC stimulated all three MAPKs, although phosphorylation of ERK was more pronounced and sustained than others. Inhibitors of ERK, JNK, and p38 reduced LC-induced IL-6 and MCP-1 production. These findings suggest that activation of MAPKs plays a role in LC-induced cytokine responses in PTCs. Activation of MAPKs may be involved in cytokine responses induced by other proteins as well as LCs and may be pivotal in the pathophysiology of tubulointerstitial injury in proteinuric diseases.

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