Endothelial cells regulate proximal tubule epithelial cell sodium transport

BACKGROUND: Proximal tubule epithelial cells are in close contact with the renal microvasculature, but the effect of endothelial cells (ECs) on proximal tubule epithelial cell (PTEC) function is not known. METHODS: To determine if ECs regulate PTECs, we coincubated ECs with PTECs in a system that permitted cross-talk between the two cell types and the vectorial transport of sodium. RESULTS: In the presence (but not absence) of ECs, adding bradykinin or acetylcholine increased cGMP and decreased sodium transport, as well as Na,K-ATPase in PTECs. Interleukin (IL)1B preconditioning of ECs also increased cGMP and decreased sodium transport and Na,K-ATPase in PTECs. Bradykinin, acetylcholine, and IL1B EC-dependent effects were reversed with the nitric oxide (NO) synthase inhibitor L-NNA. In the absence of ECs, the addition of NO donors to PTECs increased cGMP and decreased sodium transport and Na,K-ATPase. 8Br-cGMP also decreased PTEC sodium transport and Na,K-ATPase. CONCLUSION: Endothelial cells regulate PTEC function. This effect is mediated by NO synthase-dependent up-regulation of cGMP in PTECs.     

The paracellular permeability of opossum kidney cells, a proximal tubule cell line

The paracellular permeability of opossum kidney cells, a proximal tubule cell line. BACKGROUND: The regulation of the unusually leaky paracellular pathway of the proximal tubule is poorly understood partially because of the lack of an appropriate in vitro cell model. In this study, we determined whether the paracellular permeability of opossum kidney (OK) cells would resemble that of the in vivo proximal tubule epithelium. METHODS: The parental and subclonal OK cells and, for comparison, LLC-PK1 cells were cultured on permeable Transwell supports. The apparent paracellular permeability coefficient (Papp) for the extracellular marker 3H-mannitol was determined. RESULTS: The Papp of OK cell sheets (12.17 x10-6 cm/sec) was remarkably close to the previously reported Papp of rat proximal tubules. The Papp of LLC-PK1 cells, another proximal tubule cell line, however, was approximately 20-fold lower than that of both OK cells and the in vivo proximal tubule. Phorbol 12-myristate 13-acetate, a protein kinase C activator, enhanced the Papp of OK cell sheets. The characteristic response of paracellular permeability to Ca2+ switch was demonstrated in OK cell sheets. Slight variations of Papp among several OK subclones were observed. Basal to apical Papp was uniformly higher than apical to basal Papp, independent of cell subtype. This rectification was attenuated by inhibition of active transport. CONCLUSIONS: OK cell sheets cultured on Transwell supports possess a leaky paracellular pathway resembling that of the proximal tubule epithelium in vivo.     

Delayed apoptosis post-cadmium injury in renal proximal tubule epithelial cells

BACKGROUND: Accumulation of the widespread environmental toxin cadmium (Cd) in the kidney results initially in proximal tubule dysfunction. Exposure to Cd has been previously shown to induce apoptosis in LLC-PK (Lily Laboratory Culture, Porcine Kidney) cells, which are a model of proximal tubule epithelium. HYPOTHESIS: We postulated that modulation of the components of the apoptotic pathway triggered by Cd is amenable to therapeutic intervention. METHODS: We subjected confluent LLC-PK cells grown on two-compartment filters and on plastic to Cd (1-50 microM). Apoptosis and changes in components of the apoptotic pathway were measured by immunocytochemical and immunoblot analysis during the period of exposure and following Cd withdrawal. RESULTS: Insignificant apoptosis was seen during exposure to Cd and immediately after removal of this metal. Two waves of apoptosis were noted 6 and 48 h after the Cd was removed from the apical compartment. The apoptosis 48 h post-Cd exposure was accompanied by a decrease in cellular ATP levels and transepithelial resistance and preceded by an increase in p38 phosphorylation. Inhibition of p38 mitogen-activated protein kinase activity decreased the delayed apoptotic peak, without affecting the rate of recovery of the integrity of the renal epithelium. IGF-1 neither altered the delayed apoptosis nor facilitated the rate of recovery of the integrity of the renal epithelium. CONCLUSION: We demonstrate that following exposure to Cd, renal epithelial cells undergo significant apoptosis, which appears to involve p38 and is not amenable to IGF therapy.     

Mechanism of proximal NaCl reabsorption in the proximal tubule of the mammalian kidney

In the mammalian proximal tubule NaCl reabsorption occurs by both passive and active transport processes. Passive NaCl reabsorption occurs in the presence of a high luminal chloride and a low luminal bicarbonate concentration. These anion gradients provide the driving forces for diffusive Na and Cl movement. Na is driven by the lumen positive PD effected by the greater permeability of the tubular wall to Cl than to HCO3. Cl is driven by its high tubular concentration. Passive NaCl reabsorption accounts for only about 10% to 15% of total proximal NaCl transport. The remaining proximal NaCl is reabsorbed by active transport processes and occurs both in the presence or absence of anion gradients reabsorption. Two mechanisms of active NaCl reabsorption participate in active NaCl reabsorption along the proximal tubule. Firstly, active NaCl reabsorption is electrogenic. In the early proximal tubule Na enters to cell coupled to organic solute transport. This Na reabsorption generates a lumen negative PD and effects "coupled" electrogenic NaCl reabsorption. This mechanism is limited by the supply of organic solutes and is blunted by the greater Na than Cl permeability in the proximal tubule; it probably can account for no more than 10% of proximal NaCl reabsorption. In the terminal proximal tubule, the proximal straight tubule, the apical membrane appears to possess a channel for Na entry. This Na reabsorption also generates a lumen negative PD and effects "simple" electrogenic NaCl reabsorption. This mechanism is limited by the low transport capacity of this segment and probably accounts for no more than 5% to 10% of total proximal NaCl reabsorption. The great bulk of proximal NaCl reabsorption occurs along the entire proximal tubule by active, transcellular electroneutral NaCl reabsorption. The precise cellular transport mechanisms responsible for this process are only recently being defined. At the apical membrane parallel ion exchangers are responsible for NaCl entry into the cell. Na enters via the apical membrane Na-H antiporter. Cl most likely crosses the apical membrane by some combination of Cl-OH and Cl-HCO2 exchangers but not via a Cl-HCO3 exchanger. The relative contributions of Cl-OH and Cl-HCO2 exchange have not been defined. There are two important considerations in this question. First is the availbility of OH versus HCO2. Although there is an infinite supply of OH and a small equilibrium supply of HCO2, it is possible that the luminal concentration of HCO2 could be increased by an USL that raises the concentration of HCO2 to a degree sufficient to supply H2CO2 recycling for physiological transcellular Cl transport rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

P-glycoprotein in HK-2 proximal tubule cell line

P-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell line from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available. The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150 kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP.     

Fibrate prevents cisplatin-induced proximal tubule cell death

BACKGROUND: In previous studies we have shown that cisplatin inhibits peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity and consequently fatty acid oxidation, and these events precede proximal tubule cell death. In addition the use of fibrate class of PPAR-alpha ligands ameliorate renal function by preventing both inhibition of fatty acid oxidation and proximal tubule cell death. METHODS: LLC-PK1 cells were treated with cisplatin and apoptosis was established by the presence of nuclear fragmentation and by cell cycle analysis. Proximal tubular cells treated with cisplatin and bezafibrate were subjected to sub cellular fractionation and the presence of Bax, Bcl-2, cytochrome c, and active caspase-3 in the cytosolic and mitochondrial membrane fractions was determined by Western blot analysis. PPAR-alpha activity was measured by determining luciferase activity after transfection of LLC-PK1 cells with TK-Luc 3x PPAR response elements (PPRE), and the accumulation of nonesterified free fatty acids was measured in lysates obtained from cells treated with cisplatin and bezafibrate. RESULTS: Incubation of LLC-PK1 cells with 25 micromol/L cisplatin for 18 hours induced 41.5% apoptosis measured by cell cycle analysis. Cisplatin-induced apoptosis was significantly suppressed by bezafibrate, a fibrate class of PPAR-alpha ligand. Bezafibrate treatment of LLC-PK1 cells prevented cisplatin-induced translocation of proapoptotic Bax from the cytosol to the mitochondrial fraction, and increased the expression of antiapoptotic molecule Bcl-2. Cisplatin-induced inhibition of PPAR-alpha activity was accompanied by increased accumulation of nonesterified free fatty acids. Pretreatment with bezafibrate prevented both the inhibition of PPAR-alpha activity and the accumulation of nonesterified free fatty acids induced by cisplatin. Finally, bezafibrate prevented cisplatin-induced release of cytochrome c from the mitochondria to the cytosol, and the cleavage of procaspase-3 to active caspase-3. CONCLUSION: Bezafibrate treatment inhibits cisplatin-mediated tubular injury by preventing the activation of various cellular mechanisms that lead to proximal tubule cell death. These findings support our previous observations where the use of fibrates represents a novel strategy to ameliorate proximal tubule cell death in cisplatin-induced acute renal failure.     

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

To determine the role of epidermal growth factor receptor (EGFR) activation in renal functional and structural recovery from acute kidney injury (AKI), we generated mice with a specific EGFR deletion in the renal proximal tubule (EGFR(ptKO)). Ischemia-reperfusion injury markedly activated EGFR in control littermate mice; however, this was inhibited in either the knockout or wild-type mice given erlotinib, a specific EGFR tyrosine kinase inhibitor. Blood urea nitrogen and serum creatinine increased to a comparable level in EGFR(ptKO) and control mice 24?h after reperfusion, but the subsequent rate of renal function recovery was markedly slowed in the knockout mice. Twenty-four hours after reperfusion, both the knockout and the inhibitor-treated mice had a similar degree of histologic renal injury as control mice, but at day 6 there was minimal evidence of injury in the control mice while both EGFR(ptKO) and erlotinib-treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. Additionally, renal cell proliferation was delayed due to decreased ERK and Akt signaling. Thus, our studies provide both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after acute kidney injury.     

Inhibition of Na,K-ATPase by dopamine in proximal tubule epithelial cells

In the current report we review the results that lay grounds for the model of intracellular sodium-mediated dopamine-induced endocytosis of Na,K-ATPase. Under conditions of a high salt diet, dopamine activates PKCzeta, which phosphorylates NKA alpha1 Ser-18. The phosphorylation produces a conformational change of alpha1 NH2-terminus, which through interaction with other domains of alpha1 exposes PI3K- and AP-2-binding domains. PI3K bound to the NKA alpha1 induces the recruitment and activation of other proteins involved in endocytosis, and PI3K-generated 3-phosphoinositides affect the local cytoskeleton and modify the biophysical conditions of the membrane for development of clathrin-coated pits. Plasma membrane phosphorylated NKA is internalized to specialized intracellular compartments where the NKA will be dephosphorylated. The NKA internalization results in a reduced Na+ transport by proximal tubule epithelial cells.     

Inhibition of brush border dipeptidase with cilastatin reduces toxic accumulation of cyclosporin A in kidney proximal tubule epithelial cells.

BACKGROUND: Cilastatin reduces nephrotoxicity associated with cyclosporin A (CyA) in solid organ and bone marrow transplantation. This appears to be unrelated to changes in renal haemodynamics or CyA metabolism. How cilastatin induces this protection is unclear, but it could result from changes on accumulation of CyA proximal cells. METHODS: We investigated the effects of cilastatin on primary cultures of pig kidney proximal tubule epithelial cells (PTECs) treated with CyA and FK506. Cell membrane fluidity and membrane-bound cholesterol-rich raft (MBCR) distribution were evaluated by fluorescence microscopy, and CyA transport by radioimmunoassay. Changes in CyA- and FK506-induced apoptosis were also evaluated by electron and light microscopy, flow cytometry, and detection of cytoplasmic nucleosones by enzyme-linked immuosorbent assay. RESULTS: CyA caused a dose-dependent reduction of cell membrane fluidity, which was prevented by pre-treating PTECs with cilastatin. Cilastatin also inhibited CyA transport across membranes and reduced recovery of CyA in mitochondria and membrane-bound fractions from cilastatin-treated PTECs. This effect was not related to an altered distribution of MBCRs, which are essential for CyA transport. Cilastatin protected against CyA- and FK506-induced apoptosis. CONCLUSIONS: Prevention of CyA-induced reduction of cell membrane fluidity and inhibition of CyA transport are features of cilastatin's direct effects on PTECs. Unaltered distribution of MBCRs in the presence of cilastatin suggests that cilastatin binding to raft-bound dipeptidases, rather than MBCR modifications, causes interference with CyA transport. These results provide additional insight into the mechanisms and scope of cilastatin nephroprotection.     

Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation

Aristolochic acid contamination in herbal remedies leads to interstitial fibrosis, tubular atrophy, and renal failure in humans. To study the cellular mechanisms contributing to the pathophysiology of this renal disease, we studied Wistar rats treated with aristolochic acid and measured tubular and interstitial cell proliferation, epithelial/mesenchymal cell marker expression, tubular membrane integrity, myofibroblast accumulation, oxidative stress, mitochondrial damage, tubular apoptosis, and fibrosis. Oxidative stress, a loss of cadherin concomitant with vimentin expression, basement membrane denudation with active caspase-3 expression, and mitochondrial injury within tubular cells were evident within 5 days of administration of the toxin. During the chronic phase, interstitial mesenchymal cells accumulated in areas of collagen deposits. Impaired regeneration and apoptosis of proximal tubular cells resulted in tubule atrophy with a near absence of dedifferentiated cell transmembrane migration. We suggest that resident fibroblast activation plays a critical role in the process of renal fibrosis during aristolochic acid toxicity.     

Hypoxic injury in the proximal tubule of the isolated perfused rat kidney

The effect of hypoxia on the morphology of the proximal tubule was examined in isolated rat kidneys perfused with Krebs-albumin medium, gassed with 95% N2/5% CO2 for 45, 90 and 190 min. The major findings were heterogeneity of types of cell injury and definable topographical zones of protection from damage. The characteristic injury in S1 and S2 was that of mitochondrial swelling and brush border alterations that progressed to complete disorganization of this zone. S3 manifested two distinct lesions, one characterized by cell swelling and the other by tubular epithelial fragmentation. Protection from these injuries was seen in tubules located in periarterial zones, apparently due to gradients of oxygenation. The same types of injury but without zones of protection were seen after perfusion with cyanide (10 mM) in oxygenated medium. A "reflow" period of oxygenated perfusion did not alter the fundamental character of these lesions but affected tubules showed progression of damage while there was preservation of tubules in periarterial areas. Thus the responses of the proximal tubule to hypoxic injury depends both on segment type (S1, S2 versus S3) and on tubular location in relation to O2 availability. Additional factors appear to determine the type of response in S3 tubules.     

Activation of mitogenic pathways by albumin in kidney proximal tubule epithelial cells: implications for the pathophysiology of proteinuric states.

Albumin is filtered into the proximal tubule in large quantities in nephrotic states. It has been proposed that this protein may have a toxic effect on tubular epithelial cells and may be responsible for the initiation of interstitial inflammation and scarring. The mitogenic effect of recombinant human albumin in wild-type opossum kidney cells and in similar cells transfected with a dominant negative p85 subunit (deltap85) of phopshatidylinositide 3-kinase (PI 3-kinase) has been studied. This study demonstrates that recombinant human albumin stimulates proliferation of opossum kidney cells in culture. This effect is mediated via PI 3-kinase, and is inhibited by wortmannin and deltap85 expression. Albumin stimulates PI 3-kinase activity in opossum kidney cells as determined by three different experimental procedures. Recombinant albumin also stimulates pp70(s6) kinase activity in a kinase cascade downstream of PI 3-kinase. Activity of pp70(s6) kinase is essential for albumin-induced proliferation of opossum kidney cells. It is proposed that this mitogenic pathway may have a critical role in proximal tubular homeostasis and pathophysiology of proteinuric states.     

A cell cycle-dependent mechanism of renal tubule epithelial cell hypertrophy

The response of renal epithelial cells to injury can include hyperplasia (increase in cell number), apoptosis (cell death), antiproliferation (growth arrest), or hypertrophy (cells physically enlarge). Examining cell size and the protein:DNA ratio can differentiate between the growth response patterns, but it is proposed that the degree of activation of cyclin D kinase in the late G1 phase of the cell cycle differentiates between hyperplasia and hypertrophy.     

A high-affinity folate binding protein in proximal tubule cells of human kidney.

High-affinity 3H-folate binding in solubilized brush border membranes of human kidney cortex display characteristics such as apparent positive cooperativity typical of specific folate binding. The folate binding activity and the activity of the brush border membrane-marker enzyme, gamma-glutamyltransferase, were eightfold higher in brush border membranes compared to crude kidney homogenate. Ultrogel AcA 44 chromatography revealed a major (Mr approximately 100 kDa) and a minor (Mr approximately 25 kDa) folate binding protein in brush border membranes. The large molecular size form may represent a membrane-derived hydrophobic folate binding protein inserted in Triton X-100 micelles. This notion was supported by the identical molecular weights of the 100 kDa and 25 kDa folate binding peaks determined by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotting. The folate binding protein in renal brush border membranes cross reacted with rabbit antibodies against 25 kDa human milk folate binding protein, and showed immunoprecipitation in the presence of these antibodies. Paraffin embedded sections of kidney cortex showed immunostaining of cells in the convoluted proximal tubules after exposure to rabbit antiserum (1:8000 dilution). Glomeruli and distal tubules showed no immunostaining.     

Hyaluronan and proximal tubular cell migration

BACKGROUND: The ubiquitous polysaccharide hyaluronan has been associated with both acute renal injury and progressive renal disease. The aim of this study was to examine the effect of hyaluronan on proximal tubular cell migration. METHODS: The proximal tubular cell line, HK-2 cells, were grown in monolayer culture, and cell migration following addition of hyaluronan characterized in an in vitro model of injury that we have previously developed and characterized. RESULTS: Addition of well-defined preparations of exogenous hyaluronan increased cell migration; however, optimum enhancement of migration was seen with hyaluronan of high molecular weight. Activation of the mitogen-activated protein kinase (MAPK) signaling cascade, as assessed by increased expression of the dually phosphorylated active form of MAPK, could be demonstrated following addition of hyaluronan. This was blocked by the addition of a specific antibody to the hyaluronan receptor, CD44. Hyaluronan-dependent enhanced migration was also abrogated by addition the CD44 blocking antibody, and by inhibition of MAPK kinase (MEK) activity. Generation of a denuded area also led to increased synthesis of endogenous hyaluronan and activation of MAPK, and blockage of either CD44 or MAPK activation inhibited proximal tubule cell (PTC) migration and re-epithelialization under nonstimulated conditions. CONCLUSION: We have demonstrated that hyaluronan activation of the MAPK pathway through binding to its receptor CD44, enhances proximal tubule cell (PTC) migration. In addition, the results suggest that mechanical injury of PTC stimulated hyaluronan generation. These observations may have implications for both recovery from acute tubular injury and progressive renal fibrosis.