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The development over the past 20 years of a variety of cultured renal tubule cell lines derived from different parts of the renal tubule has provided invaluable powerful cell systems for in vitro analyses of the various tubule segment-specific biochemical functions and ion transport processes. Immortalized cell lines have been established using different hybrid gene constructs, most of them carrying the immortalizing simian virus 40 large T antigen (Tag) gene. The development of transgenic mice carrying unregulated Tag, and of others in which the expression of Tag remains controlled, has made it possible to establish permanent cell lines derived from microdissected or immunoselected renal proximal, distal, and collecting duct tubules. This review summarizes the different strategies of cellular immortalization used and the most frequently used human, rabbit, rat, and mouse tubule cell lines. This review provides an overview of the use of immortalized mouse tubule cell lines for in vitro analyses of various tubule cell-specific functions and the regulation of ion transporters and membranous channels. The advantages of using primary cultures of isolated tubules dissected from physiopathological models of transgenic mice are also discussed.

Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in the genus Staphylococcus with the recognition sequence CCNGG.

This study describes the functional characterization of two cell lines derived from the proximal convoluted (PKSV-PCT cells) and proximal straight (PKSV-PR) tubules microdissected out from kidneys of transgenic mice harboring the simian virus 40 (SV40) large T and small t antigens placed under the control of the rat L-type pyruvate kinase (L-PK) 5′ regulatory sequence. Both cell lines exhibited cellular cyclic AMP stimulated by parathormone (PTH) and calcitonin (CT) and a sodium-dependent glucose transporter. Uptake of the fluid-phase marker [3H]inulin showed that both cell lines grown on filters exhibited biphasic apical and basolateral endocytic rates. Results from Northern blot analysis indicate that the expression of the T antigen gene (Tag) is dependent on the concentration of D-glucose in the medium and show that the L-PK construct has maintained its capacity for up- or down-regulation by carbohydrates. Replacement of D-glucose by neoglucogenic substrates (lactate, oxaloacetate) blunted the expression of Tag transcripts and induced arrest of cell growth. Compared to cell grown in D-glucose-enriched medium, the hormonal sensitivities to PTH and CT and the sodium-dependent glucose uptake were unchanged whereas quiescent cells exhibited increased hydrolase content. Thus the proximal function has been preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice. As the expression of Tag transcripts is controlled by D-glucose, the structural and physiological characteristics of these cell lines can be studied in either quiescent or active growth conditions.

In glomeruli isolated from adult rats, arachidonic acid (C20:4) is metabolized through at least two different pathways: the lipoxygenase and the cyclooxygenase pathway, resulting in the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) and four prostaglandins (PG) respectively. Because renal blood flow (RBF) and glomerular filtration rate (GFR) increase during development, and because C20:4 metabolites are implicated in their local regulation, the conversion of 3H-C20:4 was studied in 3 groups of rats; group A: 4 days old, 10 g; group B: 10 days old, 25 g; group C: 60 days old, 200 g. Glomeruli mechanically isolated from blanched kidneys were incubated with 5.4 × 10––8M3H-C20:4. Lipoxygenase and cyclooxygenase products were extracted and resolved by high-performance liquid chromatography (HPLC); quantitative determination of PGs was performed by radioimmunoassay (RIA). The results are: (1) conversion of C20:4 to lipoxygenase product is predominant in comparison to cyclooxygenase products; (2) conversion of labeled C20:4 into 12-HETE is constant with age; (3) identified cyclooxygenase products, PGE2, and particularly PGF2α are maximum in group B; (4) the variation of C20:4 metabolism during development suggest that these products may be involved in the maturation and the regulation of glomerular functions.

Because the oxygenated metabolites of arachidonate synthesized by macrophages, particularly prostaglandins (PG)I2 and E2, thromboxane (TX)A2 and 12-hydroxyeicosatetraenoic acid (12-HETE) have been shown to modulate the immune response of T-cells, we tested the effect of cyclosporine A (CsA), a potent immunosuppressor agent, on arachidonate (AA) metabolism in cultured peritoneal rat macrophages. Endogenous AA release and 12-HETE synthesis were estimated by radiometric HPLC after prelabelling of macrophages with [3H]AA whereas PG were determined either by radiometric HPLC or by direct radioimmunoassay in the culture mediums. Exposure of prelabelled cells for 16 hr to CsA led to a large increase in the release of AA itself and of its oxygenated metabolites, PG and 12-HETE, indicating stimulation of phospholipase activity. This effect was time- and dose-dependent at concentrations of CsA between 2 and 50 microM. There was also a marked increase in the ratio PGI2/TX, suggesting, in addition to activation of phospholipase, a partial blockade of TX synthase. When macrophages were triggered by A 23187 calcium ionophore (2 microM) or opsonized zymosan (1 mg/ml), the only detectable effect of CsA was a strong and specific inhibition (50%) of TX synthesis. Addition of an excess of exogenous AA (5 micrograms/ml) to cells treated by CsA confirmed the fact that CsA acted by specifically blocking the transformation of AA into TX without affecting PGI2 or 12-HETE synthesis. These results demonstrate that CsA acts at two different levels: it promotes phospholipase activation on resting cells but simultaneously induces a partial blockade of TX-synthase. This latter effect predominates when cells are stimulated. The resulting change in the ratio PGI2/TX promotes immunosuppression to the expense of immunostimulation. This may represent one of the factors underlying the potent immunosuppressive role of CsA.

Glomeruli isolated from murine and human renal cortex metabolize arachidonic acid to prostaglandins via the cyclooxygenase pathway but whether such glomeruli can also metabolize arachidonic acid via the lipoxygenase pathway is controversial. [1-14C]Arachidonic acid was incubated with glomeruli or glomerular fractions isolated from rat and human renal cortex. The products were extracted, purified by high performance liquid chromatography, and identified by comparison of their retention times with those of authentic hydroxyeicosatetraenoic acid (HETE) standards and by gas chromatography-mass spectrometry. At low substrate concentrations, human glomeruli synthesized in equivalent amounts 12- and 15-HETE, whereas rat glomeruli synthesized only 12-HETE and in larger quantities than in man. At higher substrate concentrations, both species synthesized 12- and 15-HETE and the rate of synthesis for both products was higher in human glomeruli. No other HETE was detected in either species. The lipoxygenase products were stored within the glomeruli and recovered almost equally in the 10,000 x g pellet and in the 100,000 X g supernatant of the homogenized glomeruli. The properties of the lipoxygenase system were the following: the enzyme was distributed equally in the membranes and the cytosol; 12-HETE accumulation was linear with time over 15 min; and 12-HETE production correlated linearly with the amount of glomerular protein. 12-Lipoxygenase activity was maximum at pH 7.5 (rat) or 9.0 (human) and at 40-42 degrees C (both species). Km values calculated at low concentrations of substrate (10-200 microM) were for 15-HETE, 125 and 667 microM with murine and human glomeruli, respectively, and for 12-HETE, 44 microM with the glomeruli of both species. This study demonstrates lipoxygenase activity in murine and, for the first time, in human glomeruli. The products of such enzymatic activity, 12- and 15-HETE, may mediate the glomerular inflammatory response in various experimental or spontaneous glomerular diseases.

Abnormalities in glomerular function have been observed frequently in the early stages of both clinical and experimental diabetes mellitus. Because prostaglandins (PGs) are present in the glomerulus and have profound effects on glomerular hemodynamics, and because abnormalities of PG metabolism have been noted in other tissues from diabetics, we studied PG biosynthesis in glomeruli obtained from rats in the early stages of experimental diabetes mellitus. Streptozotocin, 60 mg/kg, was administered intravenously to male Sprague-Dawley rats. Control rats received an equal volume of the vehicle. Glomeruli were isolated 9-23 d later. Production of eicosanoids was determined by two methods: by direct radioimmunoassay after incubation of glomeruli under basal conditions and in the presence of arachidonic acid (C20:4), 30 microM, and by radiometric high-performance liquid chromatography (HPLC) after incubation of glomeruli with [14C]C20:4. When assessed by radioimmunoassay, mean basal production of both prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was twofold greater in the diabetic animals whereas production of thromboxane B2 (TXB2) was not significantly greater than control. In response to C20:4, both PGE2 and PGF2 alpha were also greater in the diabetic animals, but these differences were not statistically significant. The increased rate of basal PG production did not appear to be related directly to the severity of the diabetic state as reflected by the degree of hyperglycemia at the time of sacrifice. In fact, the rates of glomerular PG production in the individual diabetic animals correlated inversely with the plasma glucose concentration. The increased rate of PG synthesis did not appear to be due to a nonspecific effect of streptozotocin inasmuch as glomerular PG production was not increased significantly in streptozotocin-treated rats which were made euglycemic by insulin therapy. Furthermore, addition of streptozotocin, 1-10 mM, to the incubation media had no effect on PGE2 production by normal glomeruli. PGE2 production by normal glomeruli was also not influenced by varying the glucose concentration in the incubation media over a range of 1-40 mM. When metabolism of [14C]C20:4 was evaluated by high-performance liquid chromatography conversion to labeled PGE2, PGF2 alpha, TXB2, and hydroxyheptadecatrienoic acid by diabetic glomeruli was two- to threefold greater compared with that in control glomeruli, whereas no significant difference in conversion to 12- and 15-hydroxyeicosatetraenoic acid occurred. These findings indicate that glomerular cyclooxygenase but not lipoxygenase activity was increased in the diabetic animals. A concomitant increase in glomerular phospholipase activity may also have been present to account for the more pronounced differences in PG production noted in the absence of exogenous unlabeled C20:4. These abnormalities in PG biosynthesis by diabetic glomeruli may contribute to the altered glomerular hemodynamics in this pathophysiologic setting.

International audience; The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.

Cyclosporine (CsA) reduces nitric oxide (NO) production in medullary thick ascending limb (mTAL) cells. We postulated that CsA affected NO secretion in a vectorial manner in polarized renal epithelial cells. The experiments were performed in a model of mTAL subcultured cells. The expression of iNOS in mTAL cells was confirmed by RT-PCR. The cells were grown on a non-permeable filter. Nitrite was measured by the modified Griess method. Transepithelial resistance was measured to ensure the integrity of the tight junction. CsA (100 ng/ml) reduced NO production by 22% in mTAL cells. The inhibitory effect was limited to the basolateral side (control: 165 +/- 11; plus CsA: 93 +/- 17 nM/10(6) cells, P0.001) without affecting apical NO secretion. The transepithelial resistance through the epithelial monolayer remained unchanged in CsA-treated cells. CsA reduced basolateral NO secretion without affecting apical secretion. The results suggest that CsA might affect intrarenal hemodynamics at the peritubular level.