• The Korean Journal of Physiology
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• 제29권1호
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• pp.69-80
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• 1995

Renal proximal tubular hypertrophy and hyperfunction are known to be early manifestations of experimental and human diabetes. As the hypertrophy and hyperfunction have been suggested to be central components in the progression to renal failure, an understanding of their underlying causes is potentially important for the development of therapy. A primary rabbit kidney proximal tubule cell culture system was utilized to evaluate the possibility that the renal proximal tubular hypertrophy and hyperfunction observed in vivo in diabetes mellitus, can be attributed to effects of elevated glucose levels on membrane transport systems. Primary cultures of rabbit proximal tubules, which achieved confluence at 10 days, exhibited brush-border characteristics typical of proximal tubular cells. Northern analysis indicated $2.2{\sim}2.3$ and 2.0 kb Na/glucose cotransporter RNA species appeared in fresh and cultured proximal tubule cells after confluence, repectively. The cultured cells showed reduced Na/glucose cotransporter activity compared to fresh proximal tubules. Primary cultured proximal tubule cells incubated in medium containing 20 mM glucose have reduced ${\alpha}-MG$ transport compared to cells grown in 5 mM glucose. In the proximal tubule cultures incubated in medium containing 5 mM or 20 mM glucose, phlorizin at 0.5 mM inhibited 0.5 mM ${\alpha}-MG$ uptake by 84.35% or 91.85%, respectively. The uptake of 0.5 mM ${\alpha}-MG$ was similarly inhibited by 0.1 mM ouabain (41.97% or 48.03% inhibition was observed, respectively). In addition, ${\alpha}-MG$ uptake was inhibited to a greater extent when $Na^{+}$ was omitted from the uptake buffer (81.86% or 86.73% inhibition was observed, respectively). In cell homogenates derived from the primary cells grown in 5 mM glucose medium, the specific activity of the Na/K-ATPase $(6.17{\pm}1.27\;{\mu}mole\;Pi/mg\;protein/hr)$ was 1.56 fold lower than the values in cell homogenates treated with 360 mg/dl D-glucose, 20 mM $(9.67{\pm}1.22\;{\mu}mole\;Pi/mg\;protein/hr)$. Total $Rb^{+}$ uptake occurred at a significantly higher rate (1.60 fold increase) in primary cultured rabbit kidney proximal tubule cell monolayers incubated in 20 mM glucose medium $(10.48{\pm}2.45\;nM/mg\;protein/min)$ as compared with parallel cultures in 5 mM glucose medium. $Rb^{+}$ uptake rate in 5 mM glucose medium was reduced by 28% when the cultures were incubated with 1 mM ouabain. The increase of the $Rb^{+}$ uptake by rabbit kidney proximal tubule cells in 20 mM glucose could be attributed primarily to an increase in the rate of ouabain-sensitive $Rb^{+}$ uptake $(5\;mM\;to\;20\;mM;\;4.68{\pm}0.85\;to\;8.38{\pm}1.37\;nM/mg\;protein/min)$. In conclusion, the activity of the renal proximal tubular Na,K-ATPase is elevated in high glucose concentration. In contrast, the activity of the Nafglucose cotransport system is inhibited.

• BMB Reports
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• 제29권1호
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• pp.11-16
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• 1996

The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the $Ca^{2+}$ ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation. $GTP{\gamma}S$ also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.

• 대한약리학회지
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• 제31권1호
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• pp.85-93
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• 1995

Testosterone이 serum-free medium에서 배양한 토끼의 신장 근위세뇨관 상피세포의 세포성장과 기능에 미치는 영향을 관찰한 바 다음과 같은 결과를 얻었다. 1. 토끼의 신장 근위세뇨관 상피세포는 testosterone 1 nM의 농도에서 유의한 세포 성장 촉진 효과를 나타내었고, testosterone 10 nM이상의 농도에서는 세포성장이 억제되었다. 2. Testosterone은 serum-free medium에서 성장촉진인자의 하나인 hydrocortisone을 growth supplement로 넣어준 serum-free medium에서 토끼 신장의 근위세뇨관 상피세포의 성장을 촉진시키었다. 3. Testosterone은 hydrocortisone을 growth supplement로 넣어준 serum-free medium에서 토끼 신장의 근위세뇨관 상피세포의 성장을 촉진시키었다. 4. Testosterone은 Northern blot analysis에 의하여 확인한 토끼 신장의 근위 세뇨관 상피세포의 ${\beta}-actin$ mRNA level은 증가되었다. 이상의 결과로 미루어 보아, serum-free 그리고 hormonally defined media에서 testosterone이 토끼의 신장 근위세뇨관 상피세포의 성장 및 기능에 대하여 촉진적으로 작용하는 것은 cellular mecrofilament의 중요한 구성단백의 하나로 밝혀진 ${\beta}-actin$의 합성 증가에 기인하는 것으로 생각된다.

• The Korean Journal of Physiology
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• 제30권2호
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• pp.249-256
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• 1996

The effect of probenecid on the transport of tetraethylammonium (TEA) was studied in renal cortical slices and isolated membrane vesicles to investigate the interaction of organic anion with the organic cation transport system in proximal tubule. Probenecid reversibly inhibited TEA uptake by renal cortical slices in a dose-dependent manner over the concentration range of 1 and 5 mM. The efflux of TEA was not affected by the presence of 3 mM probenecid. Kinetic analysis indicated that probenecid decreased Vmax without significant change in Km. Probenecid inhibited significantly tissue oxygen consumption at concentrations of 3 and 5 mM. However, probenecid did not significantly reduce TEA uptake in brush border and basolateral membrane vesicles prepared from renal cortex even at a concentration as high as 10 mM. These results indicate that probenecid reduces TEA uptake in cortical slices by inhibiting tissue metabolism rather than by an interaction with the organic ration transporter.

• 대한약리학회지
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• 제29권1호
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• pp.73-83
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• 1993

Steroid hormone의 하나인 ${\beta}-estradiol$이 serum-free medium에서 배양한 토끼의 신장 근위세뇨관 상피세포의 세포성장과 기능에 미치는 영향을 관찰한 바 다음과 같은 결과를 얻었다 1. Serum-free medium에서 토끼의 신장 근위세뇨관 상피세포는 ${\beta}-estradiol$ 1 nM의 농도에서 유의한 세포 성장 촉진 효과를 나타내었고, ${\beta}-estradiol$ 10 nM이상의 농도에서는 세포성장이 억제되었다. 2. ${\beta}-Estradiol$은 serum-free medium에서 성장촉진인자의 하나인 hydrocortisone을 뺀 조건하에서 세포 성장을 증가시키었다. 3. ${\beta}-Estradiol$은 hydrocortisone을 growth supplement로 넣어준 serum-free medium에서 토끼 신장의 근위세뇨관 상피세포의 성장을 촉진시키었다. 4. ${\beta}-Estradiol$은 Northern blot analysis에 의하여 확인한 alpha I (IV) collagen mRNA level에는 별다른 변화를 보이지 않으나, ${\beta}-actin$mRNA level은 증가되었다. 이상의 결과로 미루어 보아, serum-free 그리고 hormonally defined media에서 ${\beta}-estradiol이$ 토끼의 신장 근위세뇨관 상피세포의 성장 및 기능에 대하여 촉진적으로 작용하는 것은 cellular microfilament의 중요한 구성단백의 하나로 밝혀진 ${\beta}-actin$의 합성 증가에 기인하는 것으로 생각된다.

• The Korean Journal of Physiology
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• 제29권2호
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• pp.191-202
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• 1995

The purpose of this study was to compare effects of insulin and IGFs on growth, apical membrane enzyme activities and membrane transport systems of primary cultured rabbit kidney proximal tubule cells. Results were as follows: 1. Insulin and IGF-I produced significant growth stimulatory effects at $5{\times}10^{-10}M.\;IGF-II(5×10^{-10}\;M)$ did not stimulate significant cell growth. 2. Insulin stimulated the phosphorylation of a 97 KD protein. It was difficult to determine whether this band represents insulin and/or the IGF-I receptor. 3. The activities of apical membrane enzymes (alkaline phosphatase, leucine aminopeptidase, and ${\gamma}-glutamyl \;transpeptidase)$ were observed to be diminished after the cells were placed in the culture environment. 4. The uptake of ${\alpha}-MG,$ Pi and Na was significantly increased in cells incubated with insulin or IGF-I, IGF-II had no effect on the uptake of these substrates. 5. Na-pump activity, as assayed by Rb uptake, was significantly increased in cells treated with insulin or IGFs. In conclusion, insulin and IGF-I exert stimulatory effects on growth and membrane transporter(glucose, Na, Pi, and Na-pump) activities in primary cultured rabbit kidney proximal tubule cells. IGF-II had no effect on cell growth and membrane transporter(glucose, Na and Pi) activities.

• The Korean Journal of Physiology
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• 제25권2호
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• pp.171-177
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• 1991

This study was carried out to determine effect of acute renal ischemia on transport function of organic cation, tetraethylammonium (TEA), in rabbit kidney proximal tubule. Clamping of the renal artery for 30 and 60 min produced a polyuria which was accompanied by an increase in $Na^+$ excretion. The capacity of kidney cortical slices to accumulate TEA was increased after 30 and 60 min of ischemia. When blood flow was restored for 30 min after 30 and 60 min of ischemia, the augmented TEA uptake was recovered to the control values. Oxygen consumption of cortical slices was stimulated after 30 min of ischemia, whereas it was not altered by 60 min of ischemia. A 90-min ischemia produced a significant inhibition of TEA uptake and tissue oxygen consumption. These results suggest that the basolateral transport system for organic cation persists after ischemic periods of 60 min despite evidence that tubular reabsorptive mechanism of $Na^+$ and water is markedly impaired. This may indicate that the active secretory systems of proximal tubule are more resistant to ischemic injury than the reabsorptive systems.

• Biomolecules & Therapeutics
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• 제2권1호
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• pp.39-46
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• 1994

The present study was undertaken to demonstrate whether or not bradykinin activates a phospholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [$^3$H]phosphatidic acid and [$^3$H]phosphatidylethanol we could elucidate the direct stimulation of phospholipase D by bradykinin. Bradykinin leads to a rapid increase in [$^3$H]phosphatidic acid and [$^3$H]diacylglycerol, and [$^3$H]phosphatidic acid formation preceded the formation of [$^3$H]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine by the action of phospholipase D, not from diacylglycerol by the action of diacylglycerol kinase. In addition, the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activated and regulated by extracellular calcium ion and pertussis toxin-insensitive G protein, respectively. It has also been shown that bradykinin may activate phospholipase D through protein kinase C-dependent pathway. In conclusion, we are now, for the first time, strongly suggesting that bradykinin-induced activation of phospholipase D in the rabbit kidney proximal tubule cells is mediated by a pertussis toxin-insensitive G protein and is dependent of protein kinase C.

• The Korean Journal of Physiology
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• 제28권2호
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• pp.233-240
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• 1994

In this study, it was investigated whether immortalized proximal tubule cells transformed with pRSVT could survive through the numerous passages. Results were as follows: 1. The cells transfected with pRSVT formed rapidly growing, multilayered colonies within 2 weeks in a hormone defined medium. Domes were also observed in some of the cultures. 2. r-glutamyl transpeptidase activity was equivalent to that observed in primary renal proximal tubule cell cultures. 3. Transformed cells with pRSVT form tubules in matrigel following 20 passages. 4. Genomic DNA of transformants was digested with either the restriction enzyme Xba or BamH1. A band of approximately 7.5kb was detected with Xba. Three BamH1 bands were detected at approximately 15 kb, 6.5 kb, and 3 kb.

• Archives of Pharmacal Research
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• 제22권4호
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• pp.367-371
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• 1999

Effects of several durgs on rabbit renal proximal tubules were examined for the applicability of renal dipeptidase (RDPase, EC 3. 4. 13. 11) release as a model system to study nephrotoxicity. The proximal tubule prepared by the method of Taub (1990) released RDPase spontaneously in the control experiment which was confirmed by Western blotting. RDPase was also released form cisplatin, lipopolysaccardie (LPS), and indomethacin-treated tubules. Gentamicin inhibited RDPase release in a concentration-dependent manner. This RDPase release system may not be a general model to screen nephrotoxicity but could be a useful source of RDPase purification in a simple and inexpensive way.