Proximal Tubule-like Cells Research Articles

CAMP activation of phosphoenolpyruvate carboxykinase transcription in renal LLC-PK1-F+ cells.

Phosphoenolpyruvate carboxykinase (PCK) is a key regulatory enzyme in renal ammoniagenesis and gluconeogenesis. LLC-PK1-F+ cells are porcine renal proximal tubule-like cells that express significant levels of the cytosolic PCK. Treatment of subconfluent LLC-PK1-F+ cells with 0.1 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) for 8 h causes a 21-fold increase in PCK mRNA. This response is very rapid and is not inhibited by 0.5 mM cycloheximide, indicating that ongoing protein synthesis is not required. Similarly, cells transfected with PCK(-490)CAT exhibit an 8- to 10-fold increase in chloramphenicol acetyltransferase (CAT) activity when treated with cAMP for 24 h. The addition of okadaic acid, a protein phosphatase inhibitor, both stimulated the CAT activity and potentiated the cAMP effect by twofold, suggesting that phosphorylation may contribute to the transcriptional activation. Assays using a series of PCK-CAT constructs containing specific deletions or block mutations established that the CRE-1 the P3(II) elements are required for the cAMP response. Cotransfection experiments using dominant negative expression vectors indicated that a CCAAT enhancer binding protein (C/EBP) transcription factor, and not CREB, mediates cAMP activation of transcription in LLC-PK1-F+ cells.

Differential expression of multiple glutaminase mRNAs in LLC-PK1-F+ cells.

LLC-PK1-F+ cells are porcine proximal tubule-like cells that have been used to model the renal ammoniagenic response to metabolic acidosis. A 3.2-kb porcine glutaminase (GA) cDNA (pGA201) containing 528 bp of coding sequence and 2.7 kb of 3'-untranslated region was cloned and sequenced. Probes derived from both porcine and rat GA cDNAs were used to characterize the expression of putative GA mRNAs in LLC-PK1-F+ cells. Two larger putative GA mRNAs (approximately 5.0 and 4.5 kb in length) were resolved and a smaller 2.5-kb species was also observed. The level of the 5.0-kb mRNA is detectable in freshly split LLC-PK1-F+ cells and increases as the cells reach confluence. In contrast, the amount of the 4.5-kb GA mRNA is greatest in freshly split cells and decreases gradually as the cells approach confluence. The levels of the 5.0- and 2.5-kb mRNAs are also affected by refeeding the cells, and the 2.5-kb mRNA accumulates to high levels if cells are retained in the same media for 4 days. Exposure to acidic media had little or no effect on the levels of GA mRNAs expressed in confluent or postconfluent cells, whereas, in growing and undifferentiated cells, this treatment did affect the level of the 4.5-kb mRNA. Thus the putative GA mRNA species are differentially expressed. Given this complexity, a careful assessment of GA mRNA species, of basal expression, and of growth conditions are essential for a meaningful analysis of GA mRNA levels in cultured cells.

Signaling and growth responses of LLC-PK1/Cl4 cells transfected with the rabbit AT1 ANG II receptor.

Angiotensin II (ANG II) receptors of the AT1 subtype are present on the apical and basolateral membranes of renal proximal tubule cells. Cells of the proximal tubulelike cell line, LLC-PK1/Cl4, were transfected with an expression plasmid containing cDNA encoding the rabbit AT1 ANG II receptor. In transfected cells, specific binding of 125I-ANG II was detected on both apical and basolateral membranes; wild-type LLC-PK1/Cl4 cells did not express ANG II receptors. In transfected cells, apical or basolateral ANG II increased both S6 kinase activity and incorporation of [3H]leucine. In cells pretreated with pertussis toxin, the stimulatory effect of apical or basolateral ANG II on [3H]leucine incorporation was abolished. In contrast, ANG II did not affect mitogenesis, determined by [3H]thymidine incorporation. Apical or basolateral ANG II (10(-6) M) stimulated phosphoinositide turnover by 13.4 +/- 4.4% (n = 8) and 16.3 +/- 4.2% (n = 9), respectively. The activity of protein kinase C, determined by phosphorylation of a specific protein kinase C peptide substrate, was also stimulated by ANG II in transfected cells. Apical or basolateral ANG II had no significant effect on cellular adenosine 3',5'-cyclic monophosphate levels. In permeabilized transfected cells, apical ANG II (10(-6) M) inhibited the phosphorylation of a specific peptide substrate of protein kinase A; lower apical concentrations or basolateral ANG II were without significant effect. These results indicate that AT1 ANG II receptors sort to both apical and basolateral membranes in renal epithelial cells and are coupled to activation of phospholipase C. ANG II stimulates protein synthesis by binding to either apical or basolateral receptors; this effect requires coupling to G proteins and may be mediated by activation of S6 kinase. Because high concentrations of ANG II exist in proximal tubule, binding to apical and basolateral receptors may regulate proximal tubule cell growth under physiological conditions.

Hyperglycemia-induced changes in Na+/myo-inositol transport, Na(+)-K(+)-ATPase, and protein kinase C activity in proximal tubule cells.

In many tissues, hyperglycemia alters the activities of the Na(+)-dependent myo-inositol (Na/MI) transporter, Na(+)-K(+)-ATPase, and protein kinase C (PKC). However, little is known concerning adaptive changes in renal proximal tubular function after acute or chronic hyperglycemia. We examined hyperglycemia-induced changes in Na/MI transport, Na(+)-K(+)-ATPase activity, and PKC activity using three proximal tubule-like cell lines (JTC12, LLC-PK1, and OK/E cells) and primary cultures of human proximal tubular epithelium (HK cells) cultured for varying periods in low- or high-glucose media, myo-Inositol (MI) transport was mediated by a high-affinity (Km approximately 50 mumol/l) Na(+)-dependent saturable process in the four cell lines. Hyperglycemia produced a time-dependent and persistent increase in Na/MI transport in all cell lines. Chronic hyperglycemia increased the Km for MI transport in LLC-PK1 cells and increased the Vmax in both LLC-PK1 and JTC12 cells. Glucose competitively inhibited Na/MI transport in all low-glucose cells and in high-glucose HK, JTC12, and OK/E cells but had no effect on transport in high-glucose LLC-PK1 cells. Acute hyperglycemia also produced time-dependent increases in Na(+)-K(+)-ATPase activity in all cell lines, a change that persisted only in HK cells. A 24-h exposure to high glucose had no effect on PKC activity in any of the cell lines but increased Ca/phospholipid-dependent PKC activity in membrane fractions from chronically high-glucose LLC-PK1 and OK/E cells. These data suggest that hyperglycemia causes acute changes in proximal tubule function and long-lived adaptive responses in Na/MI transport and the PKC signaling pathway.

NMR-visible intracellular P(i) and phosphoesters during regulation of Na(+)-P(i) cotransport in opossum kidney cells.

There is an inverse relationship between intracellular concentration of P(i) ([P(i)]i) in the kidney and maximum velocity (Vmax) of Na(+)-P(i) cotransport in brush-border membrane vesicles both in P(i)-deprived and growing animals. However, at any given [P(i)]i, the Vmax is substantially higher in growing than in P(i)-deprived animals. This suggests that growth and P(i) depletion act on P(i) transport via different mechanisms. We tested this hypothesis by measuring the nuclear magnetic resonance-visible phosphate and the Vmax of Na(+)-P(i) cotransport in proximal tubule-like cells [opossum kidney (OK) cells] cultured in vitro. OK cells incubated in 1 mM extracellular P(i) had a [P(i)]i of 1.1 +/- 0.2 mM and a P(i) uptake of 1.47 +/- 0.06 nmol/mg in 5 min. Exposure of OK cells to P(i)-free medium decreased [P(i)]i by 80 +/- 7% (P < 0.01) and stimulated P(i) transport by 34 +/- 7% (P < 0.05). Exposure of OK cells to 10(-8) M insulin-like growth factor I (IGF-I) increased P(i) transport by 25 +/- 8% (P < 0.05) but did not affect [P(i)]i. The stimulation of Vmax produced by IGF-I was additive to that due to P(i) restriction. In addition, P(i) deprivation decreased the phosphomonoesters by 0.66 +/- 0.04-fold (P < 0.05) and increased the phosphodiesters by 2.5 +/- 0.5-fold (P < 0.01). Treatment with IGF-I increased both the phosphomonoesters (1.2 +/- 0.1-fold) and the phosphodiesters (4.1 +/- 0.6-fold). These results support the assumption that low P(i) supply and IGF-I stimulate Na(+)-P(i) cotransport by independent mechanisms.

Angiotensin II stimulation of Na-H antiporter activity is cAMP independent in OKP cells

Angiotensin II has been reported to stimulate the proximal tubule Na-H antiporter by inhibition of adenylyl cyclase, and possibly by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism. We examined the effect of angiotensin II on Na-H antiporter activity (JNa-H) in opossum kidney (OKP) cells, a proximal tubule-like cell line, whose Na-H antiporter resembles that of the proximal tubule apical membrane. We found that angiotensin II regulates JNa-H in a concentration-dependent manner similar to the proximal tubule, with angiotensin II concentrations < 10(-8) M stimulating and > 10(-8) M inhibiting JNa-H. The stimulatory effect of angiotensin II was blocked by 10(-8) M losartan and was pertussis toxin sensitive, suggesting mediation through an angiotensin II (AT1) receptor coupled to a pertussis toxin-sensitive G protein. Acute treatment with 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibited JNa-H by 30% and blocked angiotensin II-induced stimulation. However, angiotensin II (10(-12)-10(-6) M) did not inhibit basal, dopamine-stimulated, or forskolin-stimulated cAMP production measured in the presence of 3-isobutyl-1-methylxanthine (IBMX). In addition, angiotensin II had no effect on cAMP levels measured in the absence of IBMX. We conclude that angiotensin II at physiological concentrations stimulates JNa-H in OKP cells via a cAMP-independent mechanism mediated by an AT1 receptor and a pertussis toxin-sensitive G protein.

Citrate transport in proximal cell line.

Citrate uptake into kidney proximal tubules occurs via an apical dicarboxylate transporter and a poorly characterized process in the basolateral membrane. We used OK cells, a cell line derived from opossum kidney, to study citrate transport in proximal tubule-like cells. Citrate uptake into cell monolayers was studied using [14C]citrate with [3H]mannitol as a volume marker. Citrate uptake into these cells was sodium dependent and saturable with increasing concentrations of citrate. In contrast to previous models, citrate transport was altered minimally by changes in pH from 6.2 to 7.0 and increased at pH 7.4 to 7.8. A variety of di- and tricarboxylates were tested for interaction with citrate transport. The dicarboxylates succinate, malate, and oxaloacetate at 1 mM concentration inhibited citrate uptake minimally (uptake at least 80% of control); one dicarboxylate, alpha-ketoglutarate, did inhibit citrate uptake significantly. In contrast, the tricarboxylates isocitrate and tricarballylate inhibited citrate uptake significantly, indicating probable competitive inhibition with the transport process. These characteristics are distinctly different from those of the apical membrane dicarboxylate transporter. 1,2,3-Benzenetricarboxylic acid, an inhibitor of the mitochondrial tricarboxylate transporter, did not alter citrate uptake. In conclusion, the OK proximal cell line exhibits a novel citrate transport process compared with the apical transport of citrate described in most proximal systems. This transport process probably involves the trivalent species of citrate in contrast to the usual predominant transport of divalent citrate. This transport process may represent a process similar to that in the basolateral membrane of the proximal tubule.

Characterization and cellular distribution of acidic peptide and oligosaccharide metal-binding compounds from kidneys

Two low-molecular-mass Ni-binding fractions first isolated from human kidneys [Templeton & Sarkar (1985) Biochem. J. 230, 35-42] are further characterized. Both components are acidic and are readily separated from each other by gel chromatography on Bio-Gel P-2. After equilibration with 63Ni the largest complex constitutes about 30% of the radioactive 63Ni and is an approx. 3.5 kDa peptide and the smallest species comprise short oligosaccharides containing 70% of the radioactivity. Both of these components are found in human, bovine and porcine kidneys as well as in a porcine proximal tubule-like cell line LLC-PK1. There is a small variation in amino acid composition between species. The oligosaccharides are reducing sugars and contain sulphate, glucosamine, glucuronic acid and iduronic acid with two to four overall negative charges. The monosaccharide composition was determined by h.p.l.c. with pulsed amperometric detection of the acid hydrolysates and by gas chromatography. In the LLC-PK1 cell line the acidic peptide is both intracellular and extracellular, whereas the oligosaccharides are only intracellular. The concentration of extracellular peptide, as measured by 63Ni binding, is found to increase after exposure of the cells to low micromolar concentrations of Ni, whereas the oligosaccharide concentrations, also measured by 63Ni binding, remain constant. The oligosaccharide component is decreased by 40% in the presence of NH4Cl, suggesting that is derived from degradation of internalized heparan sulphate.

The presence of proximal tubulelike cells in the kidney parietal epithelium in response to unilateral nephrectomy.

Five months following unilateral nephrectomy, the parietal epithelia in the remaining kidneys of Sprague-Dawley rats were examined by light and electron microscopy. Compared with controls, the kidneys from uninephrectomized rats exhibited a dramatic increase in mass characteristic of compensatory hypertrophy. Approximately 20% of the renal corpuscles in the hypertrophied kidneys had parietal epithelia lined by tall cells which possessed a brush border and other morphological characteristics of proximal tubule cells. In some instances proximal tubulelike cells made up over half of the cells lining the parietal epithelium. The possible significance of this finding is discussed.