Control of phosphate transport in flounder renal proximal tubule primary cultures

Gupta, Arpita; Renfro, J. Larry

doi:10.1152/ajpregu.1989.256.4.r850

Cited by 21 publications

(16 citation statements)

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“…Flounder renal tubules were isolated and dispersed by cold trypsinization as previously described (12) and modified (13). Briefly, kidneys were perfused and teased apart in modified medium 199.…”

Section: Methodsmentioning

confidence: 99%

Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

Brown

Upender

Hightower

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Primary monolayer cultures of winter flounder renal proximal-tubule cells were used to determine whether transepithelial transport could be protected from the damaging effects of extreme temperature by previous mild heat shock. Renal tubule epithelial cells were enzymaticafly dispersed and reorganized as confluent monolayer sheets on native rat tail collagen. Transepithelial electrical properties (potential difference, resistance, short-circuit current, and Na+-dependent glucose current) and unidirectional [3SSsulfate fluxes were measured in Ussing chambers at 22°C. Examination of transepithelial electrical properties following acute 1-hr elevation of temperature over a range of 22-37C provided the basis for the "mild" versus "severe" thermal stress protocols. Severe elevation from 22°C to 32C for 1.5 hr followed by 1.5 hr at 22°C significantly decreased glucose current (7 ± 0.7 to 3 ± 0.8 MA/cm2) as well as net sulfate secretion [131 ± 11 to 33 ± 11 nmol/(cm2.hr)]. Mild heat shock of 27C for 6 hr prior to this severe heat shock completely protected both glucose transport (6 ± 0.7 MA/cm2) and sulfate flux (149 ± 13 nmol/(cm2 hr)].Scnning electron microscopy showed that the number of microvilli on the apical (l.nal) surface of the epithellum was decreased after a 32C heat shock. Monolayers exposed to 2rC for 6 hr prior to incubation at 32°C showed no loss ofmicrovilli. SDS/PAGE analysis of protein patterns from the cultures showed that three classes of heat shock proteins were maximally induced at 27C. Inhibition of protein synthesis by cycloheximide prevented the thermoprotective effect of mild heat shock. This suggests that certain renal transport functions can be protected from sublethal but debilitating thermal stress by prior mild heat shock and that heat shock proteins may play a role in this protection.Heat injury is observed in cells at temperatures only slightly higher than the normal range. In general, cells respond to sublethal thermal stress with alterations in cellular physiology that confer resistance to normally lethal temperatures. There appear to be two states of this acquired thermotolerance. One (a state) is unstable and independent of protein synthesis, whereas the other (,3 state) is longer lived and requires protein synthesis (1,2). Evidence that heat shock proteins (hsps) are involved in thermotolerance includes the expression of human 27-kDa hsp (hsp27) in rodent cells, which confers a permanent thermoresistant phenotype (3), and microinjection of rat cells with antibodies against hsp70, which blocks recovery from heat shock (4). Recent studies indicate that maintenance of protein homeostasis in the face of proteotoxicity is the primary activity of hsps (5).Most studies of acquired thermotolerance have measured cell survival by clonal assays. Some have identified specific processes that are enhanced or protected in thermotolerant cells, such as recovery of protein synthesis (6), repair of thermally damaged nucleoli (7,8), protection of photoreceptors in thermotolerant rat retina fr...

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Section: Methodsmentioning

confidence: 99%

Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

Brown

Upender

Hightower

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…During days 15-29, transepithelial electrical characteristics and P i transport were measured. The tissues were supported by 150-m mesh and mounted in Ussing chambers as previously described (13). The temperature was maintained at 39°C, and the saline bathing the luminal and basolateral sides of the tissue was continuously gassed (95% O 2-5% CO2) and stirred throughout the experiment.…”

Section: Methodsmentioning

confidence: 99%

Regulation of transepithelial phosphate transport by PTH in chicken proximal tubule epithelium

Dudas

Villalobos

Gocek-Sutterlin

et al. 2002

American Journal of Physiology-Regulatory, Integrative and Comp

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2001.-The effect of parathyroid hormone (PTH) and activation of protein kinase C (PKC) and protein kinase A (PKA) on transepithelial Pi transport was examined in monolayers of chick proximal tubule cells in primary culture (PTCs). Acute exposure of the PTCs to PTH (10 Ϫ9 M, basolateral side) significantly decreased the net reabsorption of Pi by ϳ66%. There was no effect after the addition of PTH to the luminal side. Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 0.1 M) dramatically decreased net P i reabsorption by ϳ60%. Bisindolylmaleimide I (BIM; 1 M), a highly selective PKC inhibitor, prevented PMA-induced inhibition. Activation of adenylate cyclase/PKA by forskolin (10 M) mimicked the effect of PTH by significantly reducing net P i reabsorption by one-half. Addition of H-89 (10 M), a potent inhibitor of PKA, abolished forskolin-induced inhibition. PTH inhibition was blocked by either BIM or H-89. Tissue electrophysiology remained stable after all treatments. There was a decreased immunoreactivity of the luminal Na ϩ -Pi cotransporter NaPi-IIa after PTH treatment. These data indicate that PTH inhibition of Pi reabsorption in this in vitro system is mediated by PKC and PKA. primary cultures; reabsorptive flux; secretory flux; luminal sodium-inorganic phosphate cotransporter NaPi-IIa; protein kinase C; protein kinase A THE STUDY OF RENAL FUNCTION in birds and other nonmammalian vertebrates has provided valuable insights into the regulation of renal phosphate (P i ) excretion. In mammals, filtration and reabsorption provide sufficient control of P i excretion; however, in birds, which generally have lower and more variable glomerular filtration rates (46), the capacity for net tubular P i secretion has been reported (3). P i can be excreted by the avian kidney in quantities severalfold higher than the filtered load (21). Because avian kidneys possess this additional level of control for renal P i handling, comparative studies of its regulation should further our understanding of P i homeostasis.In both the avian and mammalian renal proximal tubule, P i reabsorption occurs via secondary, Na ϩ -dependent electroneutral transport across the luminal membrane [brush-border membrane (BBM)] (15, 34) coupled to an undefined exit process in the basolateral membrane (BLM) (1). In both mammals and birds, parathyroid hormone (PTH) has been found to be the major regulator of this process (28,24). PTH decreases BBM Na ϩ -P i cotransport activity in both mammals and birds and, in the latter, additionally stimulates net P i secretion (1, 34), which likely occurs through a unique Na ϩ -independent, voltage-and K ϩ -dependent transporter in the BBM (1, 35).Studies with the opossum kidney cell line (OK cells) have shown that PTH exerts its effects through activation of both protein kinase A (PKA) and protein kinase C (PKC) (7, 32), resulting in inhibition of BBM Na ϩ -P i cotransport. Other studies indicated the phosphaturic effect of PTH involves endocytic retrieval followed by lysosomal degradation of the BBM Na ϩ -P...

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“…During days 15-29, transepithelial electrical characteristics and Si transport were measured. The tissues were supported by 150-m nylon mesh and mounted in Ussing chambers as previously described (19). The temperature was maintained at 39°C, and the saline bathing the luminal and basolateral sides of the tissue was continuously gassed (95% O 2-5% CO2) and stirred throughout the experiment.…”

Section: Methodsmentioning

confidence: 99%

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

Dudas

Renfro

2002

American Journal of Physiology-Regulatory, Integrative and Comp

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Control of phosphate transport in flounder renal proximal tubule primary cultures

Cited by 21 publications

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Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

Regulation of transepithelial phosphate transport by PTH in chicken proximal tubule epithelium

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

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