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

Nowak, Graz ̇yna; Schnellmann, Rick G.

doi:10.1152/ajpcell.1996.271.6.c2072

Cited by 88 publications

(108 citation statements)

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“…RPTCs were isolated using the iron oxide perfusion method and grown in 35-mm tissue culture dishes under improved conditions as previously described (35). The culture medium was a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 medium (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 M pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate.…”

Section: Methodsmentioning

confidence: 99%

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Signaling of Mitochondrial Biogenesis following Oxidant Injury

Rasbach¹,

Schnellmann

2007

Journal of Biological Chemistry

142

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Mitochondrial dysfunction is a common consequence of ischemia-reperfusion and drug injuries. For example, sublethal injury of renal proximal tubular cells (RPTCs) with the model oxidant tert-butylhydroperoxide (TBHP) causes mitochondrial injury that recovers over the course of six days. Although regeneration of mitochondrial function is integral to cell repair and function, the signaling pathway of mitochondrial biogenesis following oxidant injury has not been examined. A 10-fold overexpression of the mitochondrial biogenesis regulator PPAR-␥ cofactor-1␣ (PGC-1␣) in control RPTCs resulted in a 52% increase in mitochondrial number, a 27% increase in respiratory capacity, and a 30% increase in mitochondrial protein markers, demonstrating that PGC-1␣ mediates mitochondrial biogenesis in RPTCs. RPTCs sublethally injured with TBHP exhibited a 50% decrease in mitochondrial function and increased mitochondrial autophagy. Compared with the controls, PGC-1␣ levels increased 12-fold on days 1, 2, and 3 post-injury and returned to base line on day 4 as mitochondrial function returned. Inhibition p38 MAPK blocked the up-regulation of PGC-1␣ following oxidant injury, whereas inhibition of calcium-calmodulindependent protein kinase, calcineurin A, nitric-oxide synthase, and phosphoinositol 3-kinase had no effect. The epidermal growth factor receptor (EGFR) was activated following TBHP exposure, and the EGFR inhibitor AG1478 blocked the up-regulation of PGC-1␣. Additional inhibitor studies revealed that the sequential activation of Src, p38 MAPK, EGFR, and p38 MAPK regulate the expression of PGC-1␣ following oxidant injury. In contrast, although Akt was activated following oxidant injury, it did not play a role in PGC-1␣ expression. We suggest that mitochondrial biogenesis following oxidant injury is mediated by p38 and EGFR activation of PGC-1␣.3 is a 92-kDa protein that was first identified as a regulator of adaptive thermogenesis in brown adipose tissue (1). PGC-1␣ is highly expressed in tissues with high metabolic demands, such as heart, skeletal muscle, and kidney (1). Ectopic expression of PGC-1␣ was later shown to stimulate the biogenesis of mitochondria by increasing the expression of nuclear respiratory factors 1 and 2 and enhancing the transcriptional activity of nuclear respiratory factor 1 on the promoter for mitochondrial transcription factor A (1). In addition to cold exposure, expression of PGC-1␣ is responsive to various physiological stimuli, such as exercise, caloric restriction, and exposure to lipopolysaccharide (1-4), demonstrating the ability of PGC-1␣ to alter the metabolic state of the cell in response to changes in the cellular or extracellular environment. What has not been examined is the signaling pathway responsible for mitochondrial biogenesis following cellular and mitochondrial injury, particularly oxidative stress.A variety of signaling mechanisms have been proposed to regulate the expression of PGC-1␣ and mitochondrial biogenesis including nitric oxide-soluble guanylate cyclase (5-12), ␤-adren...

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Section: Methodsmentioning

confidence: 99%

“…Basal and uncoupled (FCCP) RPTC QO 2 was measured polarographically by using a Clark-type electrode as described previously (35).…”

Section: Methodsmentioning

confidence: 99%

Signaling of Mitochondrial Biogenesis following Oxidant Injury

Rasbach¹,

Schnellmann

2007

Journal of Biological Chemistry

142

View full text Add to dashboard Cite

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“…RPTCs were isolated using the iron oxide perfusion method and grown in 6-well or 35-mm tissue culture dishes under improved conditions as previously described (37). The culture medium was a 1:1 mixture of Dulbecco's modified Eagle's medium/ Ham's F-12 (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 M pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate.…”

Section: Methodsmentioning

confidence: 99%

p38 Kinase-mediated Transactivation of the Epidermal Growth Factor Receptor Is Required for Dedifferentiation of Renal Epithelial Cells after Oxidant Injury

Zhuang¹,

Yan

Han

et al. 2005

Journal of Biological Chemistry

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Renal proximal tubular cell (RPTC) dedifferentiation is thought to be a prerequisite for regenerative proliferation and migration after renal injury. However, the specific mediators and the mechanisms that regulate RPTC dedifferentiation have not been elucidated. Because epidermal growth factor (EGF) receptor activity is required for recovery from acute renal failure, we examined the role of the EGF receptor in dedifferentiation and the mechanisms of EGF receptor transactivation in primary cultures of RPTCs after oxidant injury. Exposure of confluent RPTCs to H 2 O 2 resulted in 40% cell death, and surviving RPTCs acquired a dedifferentiated phenotype (e.g. elongated morphology and vimetin expression). The EGF receptor, p38, Src, and MKK3 were activated after oxidant injury and inhibition of the EGF receptor or p38 with specific inhibitors (AG1478 and SB203580, respectively) blocked RPTC dedifferentiation. Treatment with SB203580 or adenoviral overexpression of dominant negative p38␣ or its upstream activator, MKK3, inhibited EGF receptor phosphorylation induced by oxidant injury, whereas AG1478 had no effect on p38 phosphorylation. Inhibition of Src with 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) blocked MKK3 and p38 activation, and inhibition of MKK3 blocked p38 activation. In addition, inactivation of Src, MKK3, p38, or the EGF receptor blocked tyrosine phosphorylation of ␤-catenin, a key signaling intermediate that is involved in the epithelialmesenchymal transition and vimentin expression. These results reveal that p38 mediates EGF receptor activation after oxidant injury; that Src activates MMK3, which, in turn, activates p38; and that the EGF receptor signaling pathway plays a critical role in RPTC dedifferentiation.In many cases, kidneys subjected to an ischemic or toxic insult can completely recover. For example, surviving RPTCs 1 are thought to be crucial for the restoration of renal function after acute renal failure. During the recovery process, surviving RPTCs convert to a dedifferentiated phenotype and then proliferate and migrate to replace lost cells (1, 2). Finally, regenerating RPTCs differentiate and resume normal structure and functions. Because dedifferentiation of RPTCs is a prerequisite for migration and proliferation, it is important to understand the molecular mechanisms of RPTC dedifferentiation. In turn, this information may contribute to the development of novel treatments that stimulate RPTC regeneration and decrease recovery time of acute renal failure.

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“…Anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase were supplied by Kirkegaard & Perry Laboratories (Gaithersburg, MD) and Supersignal Chemiluminescent Substrate by Pierce. The sources of the other reagents and cell culture hormones have been described previously (34).…”

Section: Methodsmentioning

confidence: 99%