Rabbit Renal Proximal Tubular Cells Research Articles

Formoterol PLGA-PEG Nanoparticles Induce Mitochondrial Biogenesis in Renal Proximal Tubules.

Formoterol is a long-acting β2 agonist (LABA). Agonism of the β2-adrenergic receptor by formoterol is known to stimulate mitochondrial biogenesis (MB) in renal proximal tubules and recover kidney function. However, formoterol has a number of cardiovascular side effects that limits its usage. The goal of this study was to design and develop an intravenous biodegradable and biocompatible polymeric nanoparticle delivery system that targets formoterol to the kidney. Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) nanoparticles containing encapsulated formoterol were synthesized by a modified single-emulsion solvent evaporation technique resulting in nanoparticles with a median hydrodynamic diameter of 442 + 17 nm. Using primary cell cultures of rabbit renal proximal tubular cells (RPTCs), free formoterol, encapsulated formoterol polymeric nanoparticles, and drug-free polymeric nanoparticles were biocompatible and not cytotoxic over a wide concentration range. In healthy male mice, polymeric nanoparticles were shown to localize in tubules of the renal cortex and improved the renal localization of encapsulated formoterol compared to the free formoterol. At a lower total formoterol dose, the nanoparticle localization resulted in increased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), the master regulator of MB, and increased electron transport chain proteins, markers of MB. This was confirmed by direct visual quantification of mitochondria and occurred with both free formoterol and the encapsulated formoterol polymeric nanoparticles. At the same time, localization of nanoparticles to the kidneys resulted in reduced induction of MB markers in the heart. These new nanoparticles effectively target formoterol to the kidney and successfully produce MB in the kidney.

Calpain 10 is required for cell viability and is decreased in the aging kidney

Aging is associated with abnormalities in kidney function, but the exact mechanisms are unknown. We examined calpains 1, 2, and 10 protein levels in kidneys from rats, mice, and humans of various ages and determined whether calpain 10 is required for cell viability. Calpain 10 protein expression decreased in the kidney, but not in the liver, of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. There was no change in calpains 1 or 2 levels in the kidney or liver in control and caloric-restricted aging rats. Aging mice also exhibited decreased calpain 10 protein levels. Calpain 10 protein and mRNA levels decreased linearly in human kidney samples with age in the absence of changes in calpains 1 or 2. Our laboratory previously found calpain 10 to be expressed in both the cytosol and mitochondria of rabbit renal proximal tubular cells (RPTC). Adenoviral-delivered shRNA to rabbit RPTC decreased mitochondrial calpain 10 expression below detectable levels by 3 days while cytosolic calpain 10 levels remained unchanged at 3 days and decreased to approximately 20% of control by 5 days. Knockdown of mitochondrial calpain 10 resulted in nuclear condensation and cleaved procaspase 3, markers of apoptosis. In summary, mitochondrial calpain 10 is required for cell viability and calpain 10 levels specifically decrease in aging rat, mice, and human kidney tissues when renal function decreases, suggesting that calpain 10 is required for renal function and is a biomarker of the aging kidney.

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na(+)-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na(+)-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na(+)-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na(+) pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na(+)-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A(2) (cPLA(2)). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA(2) specific inhibitor AACOCF(3). ATP depletion-induced alterations in Na(+)-dependent phosphate uptake and cell viability were prevented by AACOCF(3). Inhibition of Na(+)-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA(2) activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

Regulation of Dedifferentiation and Redifferentiation in Renal Proximal Tubular Cells by the Epidermal Growth Factor Receptor

Repair of injured renal epithelium is thought to be mediated by surviving renal proximal tubular cells (RPTC) that must dedifferentiate to allow the proliferation and migration necessary for epithelial regeneration. RPTC then redifferentiate to restore tubular structure and function. Current models suggest that epidermal growth factor receptor (EGFR) activation is required for dedifferentiation characterized by enhanced vimentin expression, decreased N-cadherin expression, spindle morphology, and loss of apical-basal polarity after injury. Because an in vitro model of RPTC redifferentiation has not been reported, and the mechanism(s) of redifferentiation has not been determined, we used rabbit RPTC in primary cultures to address these issues. H2O2 induced the dedifferentiated phenotype that persisted >48 h; redifferentiation occurred spontaneously in the absence of exogenous growth factors after 72 to 120 h. Phosphorylation of two tyrosine residues of EGFR increased 12 to 24 h, peaked at 24 h, and declined to basal levels by 48 h after injury. EGFR inhibition during dedifferentiation restored epithelial morphology and apical-basal polarity, and it decreased vimentin expression to control levels 24 h later. In contrast, exogenous epidermal growth factor addition increased vimentin expression and potentiated spindle morphology. p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta receptor inhibitors did not affect redifferentiation after H2O2 injury. Similar results were observed in a mechanical injury model. These experiments represent a new model for the investigation of RPTC redifferentiation after acute injury and identify a key regulator of redifferentiation: EGFR, independent of p38 MAPK and the TGF-beta receptor.

Identification of calcium-independent phospholipase A2γ in mitochondria and its role in mitochondrial oxidative stress

Oxidant-induced lipid peroxidation and cell death mediate pathologies associated with ischemia-reperfusion and inflammation. Our previous work in rabbit renal proximal tubular cells (RPTC) demonstrated that inhibition of Ca(2+)-independent phospholipase A(2) (iPLA(2)) potentiates oxidant-induced lipid peroxidation and necrosis, implicating iPLA(2) in phospholipid repair. This study was conducted to identify a RPTC mitochondrial PLA(2) and determine the role of PLA(2) in oxidant-induced mitochondrial dysfunction. iPLA(2) activity was detected in Percoll-purified rabbit renal cortex mitochondria (RCM) and in isolated mitochondrial inner membrane fractions from rabbit and human RCM. Immunoblot analysis and inhibitor sensitivity profiles revealed that iPLA(2)gamma is the RCM iPLA(2) activity. RCM iPLA(2) activity was enhanced in the presence of ATP and was blocked by the PKCepsilon V1-2 inhibitor. Oxidant-induced mitochondrial lipid peroxidation and swelling were accelerated by pretreatment with R-BEL, but not S-BEL. Furthermore, oxidant treatment of isolated RCM resulted in decreased iPLA(2)gamma activity. These results reveal that RCM iPLA(2) is iPLA(2)gamma, RCM iPLA(2)gamma is regulated by phosphorylation by PKCepsilon, iPLA(2)gamma protects RCM from oxidant-induced lipid peroxidation and dysfunction, and that a strategy to preserve or enhance iPLA(2)gamma activity may be of therapeutic benefit.

H2O2-induced transactivation of EGF receptor requires Src and mediates ERK1/2, but not Akt, activation in renal cells.

Although oxidative stress activates epidermal growth factor receptor (EGFR), ERK1/2, and Akt in a number of cell types, the mechanisms by which oxidative stress activates these kinases are not well defined in renal epithelial cells. Exposure of primary cultures of rabbit renal proximal tubular cells to hydrogen peroxide (H(2)O(2)) stimulated Src, EGFR, ERK1/2, and Akt activation in a time-dependent manner as determined by the phosphorylation of each protein. The Src inhibitor PP1 completely blocked EGFR, ERK1/2, and Akt phosphorylation following H(2)O(2) exposure. In contrast, blockade of the EGFR by AG1478 inhibited phosphorylation of ERK1/2 but not Src or Akt phosphorylation following H(2)O(2) exposure. Exogenous EGF stimulated EGFR, ERK1/2, and Akt activation and the EGFR inhibitor blocked phosphorylation of ERK1/2 and Akt. The presence of PP1, but not AG1478, significantly accelerated H(2)O(2)-induced cell death. These results suggest that Src mediates H(2)O(2)-induced EGFR transactivation. H(2)O(2)- and EGF-induced ERK1/2 activation is mediated by EGFR, whereas Akt is activated by Src independent of EGFR following H(2)O(2) exposure. Src-mediated EGFR transactivation contributes to a survival response following oxidative injury.

Linoleic acid epoxide promotes the maintenance of mitochondrial function and active Na + transport following hypoxia

Low concentrations of arachidonic acid monoepoxides protect against ischemia/reperfusion injury. This study examined whether low concentrations of the linoleic acid monoepoxide, cis-12,13-epoxy-9-octadecenoic acid (12,13-EOA), protect renal cells against decreases in mitochondrial and transport functions induced by hypoxia/reoxygenation. Primary cultures of rabbit renal proximal tubular cells (RPTC) were pretreated with diluent or 1, 5, or 10 μM 12,13-EOA for 1 h and exposed to 2 h hypoxia/0.5 h reoxygenation in the absence of 12,13-EOA. Basal respiration, oligomycin-sensitive oxygen consumption (QO 2), and ATP content decreased 31, 35 and 65%, respectively, following hypoxia/reoxygenation. Hypoxia/reoxygenation also increased mitochondrial membrane potential (Δ Ψ m). Pretreatment with 12,13-EOA prevented decreases in basal and oligomycin-sensitive QO 2s and increases in Δ Ψ m. Despite the protection against decreases in mitochondrial function, 12,13-EOA pretreatment did not prevent the initial decrease in intracellular ATP content following hypoxia. However, pretreatment did accelerate the recovery of intracellular ATP levels during reoxygenation. Pretreatment with 12,13-EOA also prevented hypoxia-induced decreases in active Na + transport. Ouabain-sensitive QO 2 (a marker of active Na + transport) decreased 38% following hypoxia/reoxygenation but was maintained in RPTC pretreated with 1, 5 or 10 μM 12,13-EOA prior to hypoxia. Pretreatment of RPTC with the hydrolyzed product of 12,13-EOA, 12,13-dihydroxyoctadecenoic acid, did not have any protective effects against mitochondrial dysfunction and decreases in active Na + transport. Thus, this is the first report demonstrating that preconditioning of RPTC with low concentrations of 12,13-EOA, but not its hydrolyzed product, maintains mitochondrial respiration, accelerates restoration of ATP levels, and prevents decreases in active Na + transport following hypoxia/reoxygenation.

Ginsenosides inhibit EGF-induced proliferation of renal proximal tubule cells via decrease of c-fos and c-jun gene expression in vitro.

Recent epidemiological studies have demonstrated that ginseng intake is associated with a reduced risk for environmentally related cancers. However, the effects of ginsenosides on the proliferation of renal proximal tubule cells have not yet elucidated. This study investigated the effect of total ginsenosides, protopanaxatriol (PT) saponin, and protopanaxadiol (PD) saponin fraction on epidermal growth factor (EGF)-induced renal cell proliferation and, furthermore, c-fos and c-jun gene expression. In the present study, total ginsenosides (10 -6 g/ml) completely blocked EGF-induced DNA synthesis and cell growth. In contrast, the PT and PD fractions partially blocked it. In addition, the EGF-induced increase of c-fos and c-jun gene expression was completely blocked by total ginsenosides and partially by PT and PD saponins. In conclusion, ginsenosides, in part, inhibit EGF-induced cell proliferation via decrease of c-fos and c-jun gene expression in primary cultured rabbit renal proximal tubular cells (PTCs). Abbreviations. EGF:epidermal growth factor PD:protopanaxadiol PT:protopanaxatriol PTCs:primary cultured renal proximal tubule cells

Leukotriene D4 Inhibits Na+ Uptake through cAMP and PLC Pathways in Primary Cultured Renal Proximal Tubular Cells

Leukotriene D₄ (LTD₄) is one of the slow–reacting substances of anaphylaxis and is reported to have a diverse response including the mediation of glomerular nephritis. However, little is known about the functions of LTD₄ and its mechanisms of action in primary cultured rabbit renal proximal tubular cells (PTCs). The purpose of this study is to investigate the effect of LTD₄ on Na⁺ uptake and its related signal transduction pathways in PTCs. LTD₄ (>10^–9 M) significantly inhibited the Na⁺ uptake after 15 min (in nmol/mg protein: controls 431.7±11.4 vs. LTD₄ (10^–9 M) 355.0±23.6; p<0.05); and its effect was blocked by MK–571 (10^–6 M), a leukotriene receptor antagonist, in PTCs. Preincubation with cilastatin, a renal dipeptidase inhibitor, and polyclonal antibody against renal dipeptidase potentiated the inhibitory effect of LTD₄ on Na⁺ uptake. SQ 22536 (10^–6 M), an adenylate cyclase inhibitor, and the myristoylated protein kinase A inhibitor amide 14–22 (PKI; 10^–5 M) blocked the effect of LTD₄ on Na⁺ uptake (in nmol/mg protein: LTD₄ 349.9±18.5 vs. SQ 22536+LTD₄ 476.5±22.0 and PKI+LTD₄ 440.3±19.3; p<0.05), and LTD₄ induced an increase in cyclic adenosine monophosphate (cAMP), suggesting the involvement of cAMP in the inhibition of Na⁺ uptake. In addition, U 73122 (10^–6 M) and neomycin (10^–4 M), phospholipase C (PLC) inhibitors, W–7 (10^–4 M), a calmodulin antagonist, and bisindolylmaleimide I, a protein kinase C (PKC) inhibitor, blocked the LTD₄–induced inhibition of Na⁺ uptake, strongly suggesting involvement of the PLC–PKC signal pathways in the effect of LTD₄. LTD₄ significantly increased [Ca²]_i by 49±7% as compared with baseline. TMB–8 (10^–5 M) and BAPTA/AM (10^–5 M), intracellular calcium mobilization blockers, completely blocked the LTD₄–induced inhibition of Na⁺ uptake (in nmol/mg protein: LTD₄ 347.6±19.0 vs. TMB–8+LTD₄ 436.4±22.3 and BAPTA/AM+LTD₄ 419.9±14.3; p<0.05); however, EGTA (1 mM), a calcium chelator, partially blocked the LTD₄–induced inhibition of Na⁺ uptake. In conclusion, LTD₄–induced inhibition of Na⁺ uptake may be involved in both cAMP and PLC–PKC signal pathways in PTCs. In addition, Ca²⁺, which comes from the intracellular Ca²⁺ mobilization, is primarily responsible for the LTD₄–induced inhibition of Na⁺ uptake.

Role of phospholipase A2 isozymes in agonist-mediated signaling in proximal tubular epithelium.

Angiotensin II in proximal tubule epithelium is known to stimulate the release of arachidonic acid after stimulation of phospholipase A2 (PLA2) independent of phospholipase C-mediated signaling. Furthermore, an angiotensin II type 2 receptor subtype has been linked to this signaling cascade. We investigated the regulation and differential stimulation of PLA2s by comparing the PLA2 activities associated with the membranes and cytosol of rabbit renal proximal tubular epithelial cells after stimulation with angiotensin II, epidermal growth factor, and bradykinin. Both fractions demonstrated PLA2 activity that was dithiothreitol insensitive, required micromolar concentrations of Ca2+ for optimal activity, and was inhibited in a dose-dependent manner by an antiserum to a cytosolic PLA2 with a molecular mass of 85 kD. However, membrane-associated PLA2 did not demonstrate significant substrate specificity, whereas 1-steroyl-2-[14C]arachidonylphosphatidyl choline was the preferred substrate for cPLA2. An antiserum generated against mastoparan, a known PLA2 activator, inhibited membrane- but not cytosol-associated PLA2 activity. Membrane fractions showed a broad pH range (7.5 to 8.5) for optimal PLA2 activity, whereas cytosol was maximum at pH 9.5. Angiotensin II stimulated membrane-associated PLA2 activity by 88%, whereas bradykinin and epidermal growth factor inhibited activity by 54% and 41%, respectively. The three agonists stimulated cPLA2. Moreover, angiotensin II-induced activation of membrane-associated PLA2 preceded the activation of cPLA2. These results demonstrate differential localization and regulation of proximal tubular epithelial PLA2 isozymes, which may determine the pattern of subsequent arachidonic acid metabolism by the cytochrome P450 system.

Advantages of a two-chamber culture system to test drug nephrotoxicity: the example of cephaloridine.

Rabbit renal proximal tubular cells, cultured to confluency on a permeable collagen film in a two-chamber system, were exposed for 72 h to various concentrations of the nephrotoxic antibiotic, cephaloridine (CLD). A decrease in cellular proteins, leakage of lactate dehydrogenase and morphological changes appeared at CLD concentrations of 0.1, 1.0, and 0.5 mg/ml, respectively. The permeability of the monolayer to Lucifer yellow (LY), a dye that does not cross cell membranes, was increased by 1 or 2 mg/ml but not by lower concentrations of CLD. The large basolateral/apical glucose concentration gradient established by the cells was decreased by CLD. However, the fact that, at the CLD concentration of 1 mg/ml, LY totally equilibrated by diffusion across the monolayer, whereas the injured monolayer was still able to maintain a detectable glucose gradient, shows that damage caused by CLD to the diffusion barrier prevails over that affecting glucose uptake. Consistent with the data in the literature concerning the mechanism of CLD accumulation in renal cells, our results show that CLD was more toxic when it was added at the basolateral than at the apical side of the cultured cells. These results illustrate the advantages of using a two-chamber system of cell culture in nephrotoxicity studies.

L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.

The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.

Hypoxia and proliferation are primarily responsible for induction of lactate dehydrogenase activity in cultured cells.

In vitro to in vivo extrapolations require cellular models that closely mimic cells in vivo. Typically, cultured cells revert to glycolysis, have increased lactate dehydrogenase (LDH) activity, have decreased oxidative metabolism, and dedifferentiate. This study examined the role of hypoxia and proliferation in the regulation of LDH activity, and the temporal relationships among induction of glycolysis, LDH activity, and proliferation in primary cultures of rabbit renal proximal tubular cells (RPTC). LDH activity in RPTC grown under standard conditions (STILL) did not increase during the first 3 d of culture, but increased 18-fold by d 7. LDH activity in RPTC grown under conditions of increased oxygen supply (SHAKE) did not increase during the first 3 d of culture, but increased 5.5-fold by d 7. Hypoxia and proliferation were responsible for 73% and 27%, respectively, of the increase in LDH activity in STILL RPTC. Hypoxia had no effect on RPTC proliferation. Neither medium glucose nor insulin concentrations had any effect on LDH activity in SHAKE RPTC. Supplementation of the culture medium with ribose 5-phosphate or ribose diminished the increase in LDH activity in SHAKE RPTC to 62% and 52% of controls but had no effect on monolayer DNA content. Two-day treatment of confluent SHAKE RPTC with epidermal growth factor (EGF) resulted in 1.6-, 1.4-, and 1.9-fold increases in LDH and G6PDH activities and monolayer DNA content, respectively. The stimulatory effect of EGF on LDH and G6PDH activities, but not monolayer DNA content, was abolished by ribose or ribose 5-phosphate. In contrast, transforming growth factor-beta 1 (TGF-beta 1) treatment stimulated lactate production but had no effect on LDH and G6PDH activities, or proliferation of SHAKE RPTC. These results show that (1) both hypoxia and proliferation are primarily responsible for the induction of LDH activity in cultured cells, (2) LDH activity is not an accurate indicator of glycolysis, (3) induction of LDH activity is secondary and due, in part, to the induction of the pentose phosphate pathway, (4) EGF stimulates glycolysis, LDH activity, and proliferation, and (5) TGF-beta 1 stimulates glycolysis but has no effect on LDH activity or proliferation.

Metalloprotease-mediated cleavage secretion of pulmonary ACE by vascular endothelial and kidney epithelial cells.

The pulmonary isozyme of angiotensin-converting enzyme (ACEP) is present in the body both as a cell-associated protein in endothelial, epithelial, and monocytic cells and as a soluble protein in various body fluids including serum. The mechanism by which soluble ACEP is produced in vivo is unknown. Using in vitro transfected cell culture systems, we previously demonstrated that the rabbit testicular isozyme of ACE (ACET), which shares extensive homology with ACEP, is first synthesized as a plasma membrane-anchored ectoprotein and then secreted to the culture medium by cleavage removal of its COOH-terminal membrane-anchored tail. Here, using in vitro cultures of arterial endothelial cells and acutely isolated renal epithelial cells, we demonstrate that ACEP is also cleavage secreted from their natural producer cells. Biochemical and immunological characterization of the in vitro secreted ACEP protein revealed that it is missing the COOH-terminal membrane-anchored region of the cell-associated ACEP. Similar analysis of ACEP proteins present in rabbit serum, lung, and kidney established that ACEP secretion in vivo is also caused by the cleavage removal of the COOH-terminal region of the cell-associated protein. To characterize the proteolytic enzyme responsible for ACEP secretion, we employed rabbit renal proximal tubular epithelial cells and demonstrated significant inhibition of secretion by compound 3, a hydroxamic acid-based inhibitor of specific metalloproteases. In contrast, the inhibitors of chymotrypsin, trypsin, serine, aspartate, and cysteine proteases were ineffective. These results indicate that soluble ACEP production by vascular endothelial and renal epithelial cells, both in vitro and in vivo, is achieved by cleavage removal of its membrane-anchoring COOH-terminal tail by a metalloprotease.

Transforming growth factor-beta 1 inhibits regeneration of renal proximal tubular cells after oxidant exposure.

Transforming growth factor-beta (TGF-beta), a regulatory cytokine expressed in the kidney, plays a role in nephrogenic repair. This study utilized a chemical model of renal proximal tubule cellular injury and regeneration to investigate the effects of TGF-beta 1 on regeneration. Confluent monolayers of rabbit renal proximal tubular cells (RPTC) in primary culture exposed to the oxidant t-butylhydroperoxide (800 microM TBHP) for 1.5 hours were 24% confluent after 24 hours. Confluency increased to 50% 4 days after TBHP exposure. Recovery of monolayer confluency was associated with increased monolayer protein but not with DNA content. Daily treatment of injured monolayers with TGF-beta 1 inhibited the recovery of monolayer confluency and inhibited recovery of protein content in a concentration-dependent manner (0.02-1 ng/mL). DNA content of injured monolayers was not altered by TGF-beta 1. A single treatment of injured monolayers with 0.2 ng/mL (8 pM) TGF-beta 1 inhibited recovery of monolayer confluency and protein content without altering monolayer DNA content. These data show that a single 24 hour exposure to a low concentration (8 pM) of TGF-beta 1 inhibits regeneration of renal proximal tubule cell monolayers following oxidative injury by inhibiting, in part, cellular migration/spreading.

Growth of cultured rabbit renal tubular cells does not require exogenous glutamine

Following the pioneering work of Eagle et a! [1], glutamine, which is the most abundant amino acid in plasma (0.5 to 0.6 mM) [2] and an important source of energy and nitrogen for cell metabolism and growth [3], is considered to be a mandatory component of cell culture media. In renal epithelial cell cultures, it is added often at a final concentration of 2 to 5 mrvi [4—10]. However, this is not necessarily useful in primary cultures of renal proximal tubular cells from the rabbit, the most widely used species for renal cell cultures. Indeed, glutamine synthetase is present in the renal cortex of this species; in addition, the activity of phosphate-activated glutaminase, the enzyme which initiates glutamine metabolism and yields glutamate and ammonium [3], is low in the rabbit kidney [2]. Moreover, freshly isolated rabbit proximal tubules do not or hardly remove glutamine in net amounts when they are incubated in the presence of glutamine concentrations varying from 1 to 5 mtvi [11]. In an attempt to clarify this point, we cultured rabbit proximal tubular cells on a permeable collagen membrane and investigated whether the addition of glutamine to the culture medium influences cellular growth and glycolysis. The results obtained show that omission of exogenous glutamine does not alter the growth of rabbit renal proximal tubular cells in primary culture, and that the provision of glutamine by the glutamine synthetase reaction is sufficient and mandatory for their growth and DNA synthesis. Addition of glutamine, which stimulated glycolysis in freshly isolated proximal tubules, failed to do so in cultured rabbit renal cells.

In Vitro Comparative Study on Nephrotoxicity of Cyclosporine A, its Metabolites M1, M17, M21, and its Analogues Cyclosporines C and D in Suspensions of Rabbit Renal Cortical Cells

The potential nephrotoxicity of cyclosporine A (CsA), its three main metabolites: M1, M17 and M21, and its two analogues: cyclosporines C and D (CsC, CsD) was evaluated in vitro in suspensions of freshly isolated rabbit renal proximal tubular cells. This assessment involved the measure of enzyme release in the incubation media and the determination of Na+/K(+)-ATPase activity and glutathione content directly in the tubular cells. In vitro nephrotoxicity results of the six compounds tested could be respectively schematized as: CsA > CsD > CsC > M21 > M17, M1 It would be interesting to promote the study of promising CsC because of its low nephrotoxicity and its high immunosuppressive potency as previously reported.

Triiodothyronine enhances renal tubule cell replication by stimulating EGF receptor gene expression.

Thyroid hormone is known to accelerate renal function recovery following toxic acute renal failure. Because epidermal growth factor (EGF)-receptor activation is most likely critical in renal replicative repair, these studies were undertaken to assess whether triiodothyronine (T3), the most active form of thyroid hormone, may modulate EGF-induced renal proximal tubule cell proliferation by an effect on the EGF receptor. Rabbit renal proximal tubular cells were grown in primary culture and treated with or without T3 (0.1 or 1.0 nM) for 24 to 48 h. Compared with nontreated controls, T3 exposure led to significant increases in EGF-promoted DNA synthesis, as measured by [3H]thymidine incorporation, in renal tubule cells. Furthermore, T3 treatment resulted in increases in EGF receptor mRNA in proximal tubule cells compared with nontreated cells and was associated with elevated numbers of EGF receptors on the cell surface of proximal tubule cells. EGF binding studies demonstrated that T3 treatment had only modest effects on Kd values of both the high-affinity binding site (0.19 nM) and the low-affinity binding site (3.7 nM) but substantially increased the maximal number of high-affinity sites from 3.8 x 10(3) to 9.4 x 10(3) receptors/cell and the maximal number of low-affinity sites from 132 x 10(3) to 199 x 10(3) receptors/cell. These findings suggest that a T3 effect to increase EGF receptor gene expression with resulting increases in the number of cell surface EGF receptors on renal proximal tubule cells and a potentiated mitogenic response to EGF may be a mechanism for thyroid hormone to enhance renal function recovery following toxic acute renal failure.

Protein kinase C modulates cAMP content in proximal tubular cells: role of phosphodiesterase inhibition.

The present study was designed to evaluate whether protein kinase C (PKC) activation affects hormone-modulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rabbit renal proximal tubular cells in primary culture. When intracellular cAMP content was measured in the presence of Ro 20-1724, a selective inhibitor of type III phosphodiesterase (PDE), activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or by diacylglycerol kinase inhibitor R 59022 reinforced parathyroid hormone (PTH)- and forskolin-stimulated cAMP accumulation. During PKC activation, the inhibitory effect of norepinephrine on cAMP content persisted, whereas that of angiotensin II (ANG II) was blunted. In contrast, PKC activators had no effect on cAMP content during PDE blockade by the nonspecific inhibitor 3-isobutyl-1-methylxanthine (IBMX). These data suggested that PKC might affect cAMP degradation through inactivation of a Ro 20-1724-insensitive PDE. The possibility that the involved PDE was calcium sensitive was assessed; during PDE inhibition by Ro 20-1724, but not by IBMX, calcium ionophore A23187 inhibited PTH-stimulated cAMP accumulation and PMA abolished the effect of A23187. Finally, neither PKC inhibition by staurosporine nor its downregulation modified the magnitude of PTH-induced cAMP accumulation. In conclusion, 1) in proximal tubular cells PKC affects cAMP degradation rather than synthesis, possibly via inactivation of a calcium-sensitive PDE; 2) PKC modulates PTH-ANG II interaction; and 3) this pathway is likely to play a role in the fine tuning of the effect of PTH and ANG II in the proximal tubule.

A 13C NMR study on fluxes into the Krebs cycle of rabbit renal proximal tubular cells.

Suspensions of rabbit renal proximal tubular (PCT) cells were incubated with [2-13C] and [3-13C]pyruvate. The perchloric acid extracts of the cell pellets were examined by 13C NMR. All experiments showed that enriched lactate, alanine, glutamate, and glutamine were the main metabolic intermediates, and that enrichment to a minor extent was found in the glutamate residue of glutathione (GSH). From these experiments, it could be deduced that PCT cells show a highly glycolytic activity, whereas enrichment of glucose exhibits gluconeogenesis. The estimation by 13C NMR of the ratio of the flux into the Krebs cycle via pyruvate carboxylase to the flux via pyruvate dehydrogenase is discussed. From incubations with 10 mM 13C-labelled pyruvate, we calculated from the relative enrichments of the glutamate carbon atoms that the ratio of pyruvate carboxylase to pyruvate dehydrogenase is 1.44 +/- 0.04 in rabbit renal proximal tubules.