Rat Proximal Tubule Cells Research Articles

Classical and channel-like urate transporters in rabbit renal brush border membranes

The precise mechanism by which urate is transported across rabbit renal proximal tubule luminal membranes has not been defined. To determine whether urate flux across this membrane represents simple diffusion or transport on specific carriers, urate uptake was examined in brush border membrane vesicles that were prepared by a Mg+(+)-aggregation technique and then exposed to CuCl2. Na(+)-independent, voltage sensitive urate transport was demonstrated in these Cu+(+)-exposed vesicles. Transport was trans-stimulated by urate and cis inhibited by pyrazinoic acid and oxonate. A small fraction of transported urate and urate in the extravesicular fluid was oxidized to allantoin. Kinetic analysis revealed the presence of two kinetically distinct transporters; a channel-like carrier that was inhibited by pyrazinoic acid and oxonate, and a high-affinity, classical, saturable carrier that was inhibited by higher concentrations of oxonate. These studies provide the first direct evidence for carrier-mediated urate transport in rabbit renal brush-border membranes and demonstrate that the rabbit transporter(s) share a number of properties with the urate uniporter in rat proximal tubule cell membranes.

Electron Energy-Loss Spectroscopical and Image Analysis of Experimentally Induced Rat Microliths II

Following a microlith-inducing diet of ethylene glycol plus ammonium chloride, intraluminal and intracellular crystals are observed in aldehyde-fixed rat proximal and distal tubule cells by light and electron microscopy. Qualitative, in-situ analysis with electron-probe X-ray microanalysis (EPMA) of these intraluminal and intracellular crystals shows the presence of calcium, a trace of magnesium, some chlorine and the virtual absence of phosphorus and sulphur. Electron energy-loss spectroscopical element (EELS) analysis and electron-spectroscopic imaging (ESI) confirm, at both sites, the presence of calcium. Selected area electron diffraction (a) confirmed the crystallinity of both the intracellular and intraluminal crystals; (b) produced identical diffractográms from intracellular crystals in proximal tubule cells and deliberately internalized exogenous COM-crystals in cultured LLC-PK1 cells and (c) produced mean dhkl-values, identical to the dhkl-values from calcium oxalate monohydrate (14-771) in the ASTM index, from 4 different intracellular crystals in proximal-tubule cells.

Uric acid transport in rat renal basolateral membrane vesicles

Pathways for urate transport across the basolateral membrane of rat proximal tubule cells were investigated using membrane vesicles isolated from rat renal cortex. The presence of an anion exchange mechanism(s) operative in the mode of α-ketoglutarate/urate; Cl −/urate and OH −(HCO 3 −)/urate as well as a mediated conductive mechanism was assessed from tracer flux measurements. In the presence of an inwardly directed Na + gradient an α-ketoglutarate dependent concentrative accumulation of PAH but not urate was observed suggesting an absence of the mediated exchange of α-ketoglutarate for urate. The imposition of an outwardly directed Cl − gradient stimulated urate uptake in the absence but not the presence of conditions designed to minimize membrane potential development suggesting an indirect electrostatic coupling of urate uptake to a Cl − gradient-induced diffusion potential. Conditions favoring the development of an inside-positive K + diffusion potential was observed to induce an inhibitor-sensitive, concentrative accumulation of urate in the absence of Cl −. The stimulation of urate uptake measured in the presence of an inside-alkaline pH gradient was not sufficient magnitude to suggest the apparent conductive urate uptake was secondary to a membrane voltage induced, inside alkaline pH gradient and the operation of an OH −(HCO 3 −)/urate exchanger. The evidence obtained from the present investigation suggests rat basolateral membrane urate transport occurs by a mediated, conductive mechanism and is not coupled to Cl −, α-ketoglutarate or HCO 3 −. This finding, when considered together with existing evidence supporting the presence of transport pathways mediating the active uptake of urate across the brush-border membrane, suggests the transtubular reabsorptive flux of urate in rat proximal tubules occurs by active uptake at the apical membrane and passive efflux at the basolateral membrane.

Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells.

Renal proximal tubule sodium reabsorption is enhanced by apical or basolateral angiotensin II (AII). Although AII activates phospholipase C (PLC) in other tissues, AII coupling to PLC on either apical or basolateral surfaces of proximal tubule cells is unclear. To determine if AII causes PLC activation, and the differences between apical and basolateral AII receptor function, receptors were unilaterally activated in rat proximal tubule cells cultured on permeable, collagen-coated supports. Apical AII incubation resulted in concentration- and time-dependent inositol trisphosphate (IP3) formation. Basolateral AII caused greater IP3 responses. Apical AII-induced IP3 generation was inhibited by DuP 753, suggesting that the type 1 AII receptor subtype mediated proximal tubule PLC activation. Apical AII signaling did not result from paracellular ligand leak to basolateral receptors since AII-induced PLC activation occurred when basolateral AII receptors were occupied by Sar-Leu AII or DuP 753. Inhibition of endocytosis with phenylarsine oxide prevented apical (but not basolateral) AII-induced IP3 formation. Cytoskeletal disruption with colchicine or cytochalasin D also prevented apical AII-induced IP3 generation. These results demonstrate that in cultured rat proximal tubule cells, AII is coupled to PLC via type 1 AII receptors and cytoskeleton-dependent endocytosis is required for apical (but not basolateral) AII receptor-mediated PLC activation.

A POSTNATAL INCREASE IN CONTACT INHIBITION REDUCES HYPERPLASTIC GROWTH IN RENAL EPITHELIAL CELLS

During postnatal maturation, the proximal tubule undergoes rapid growth, initially mainly hyperplastic, later hypertrophic. The mechanism behind the reduction of hyperplastic growth is unknown. Proliferation was studied in proximal tubule cells (PTC) from infant (I) and adolescent (A) rats after 2 days in primary culture and analyzed by 3H-thymidine autoradiography. The rate of proliferation was determined as the percentage labelled nuclei (LI).The cells grew in colonies with contact inhibited central cells and rapidly growing peripheral cells. The growth rate was determined in each layer of cells from periphery (layer 1) to center (layers 5-6). In layer 1 I and A cells proliferate at the same rate (44 ± 4% vs 41 ± 4% NS). Moving into the center proliferation in A cells fell faster than in I cells. Compared to layer 1, proliferation in layers 5-6 was inhibited by 87 ± 4% in A cells whereas in I cells by 59±5% (p<0.05).To investigate possible mechanisms explaining the low degree of contact inhibition in I cells, we tested their sensitivity to growth factors. Removing serum from the culture medium lead to a more marked inhibition of I than A central cells (84±5% vs 54 ± 5% p<0.05). TGFβ1 (0.1 pM), a well characterized inhibitor of epithelial growth, inhibited I central cells by 66 ±7% whereas no significant effect was seen in A cells.Conclusion: An increasing contact inhibition in rat PTC may explain the postnatal reduction in hyperplastic growth. A reduced sensitivity to growth stimulatory factors rather than an increased sensitivity to growth inhibitors is suggested.

Effects of angiotensin II and nonpeptide receptor antagonists on transduction pathways in rat proximal tubule.

Because the presence of the angiotensin II (ANG II)-dependent phosphoinositide hydrolysis has been questioned from studies in proximal cells in culture, we looked for this transduction pathway in suspension of freshly isolated rat proximal tubule fragments. ANG II-receptor activation induced a prompt (within 15 s) and sustained increase in [3H]inositol phosphates (IPs; inositol trisphosphate, inositol bisphosphate, and inositol monophosphate). In fura-2-loaded tubules, it elicited a rapid and biphasic rise in cytosolic free calcium ([Ca2+]i) with an early peak (within 15 s) followed by a plateau. The peak was maintained in the absence of extracellular calcium. ANG II-induced inositol trisphosphate and [Ca2+]i rises showed a similar dose dependency, with a 50% effective concentration (EC50) of 2.9 and 5.5 nM, respectively. We checked that ANG II inhibited basal (EC50 4.4 nM) and parathyroid hormone- and forskolin-stimulated cAMP production, the latter effect being inhibited by pertussis toxin pretreatment. The effects of ANG II on IPs and [Ca2+]i were inhibited by the ANG II receptor subtype 1 (AT1) antagonist losartan and not by the ANG II receptor subtype 2 (AT2) antagonists PD 123177 and PD 123319. The effect of ANG II on forskolin-stimulated cAMP was inhibited by losartan and not by PD 123319. In agreement with these results, specific binding of 125I-[Sar1,Ile8]ANG II was markedly inhibited by losartan, whereas PD 123319 had no effect. These results demonstrate that AT1 receptor subtypes are present in intact rat proximal tubule cells and are coupled to both IPs-Ca2+ and cAMP signaling pathways. No evidence for AT2 receptor subtype is found.

Evidence that activation of protein kinase-C can stimulate 1,25-dihydroxyvitamin D3 secretion by rat proximal tubules.

PTH stimulates mammalian renal proximal tubule cell synthesis and secretion of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a Ca-dependent process. In the present study regulation of 1,25-(OH)2D3 secretion by PTH, phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the Ca ionophore A23187, and calcitonin was evaluated in perifused rat proximal tubule cells isolated by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low Ca diet secreted 1,25-(OH)2D3 at a rate 2.5 times that of tubule cells from rats fed a normal Ca diet. Perifusion of tubules with human PTH-(1-34) (10(-7) M) induced an immediate and sustained increase in 1,25-(OH)2D3 secretion. Perifusion with either A23187 or 12-O-tetradecanoylphorbol 13-acetate caused transient increases in hormone secretion, while both agents perifused simultaneously resulted in a sustained increase in 1,25-(OH)2D3 secretion. Perifusion of tubule cells with the protein kinase-C (PKC) inhibitor staurosporine blocked the PTH-induced increase in 1,25-(OH)2D3 secretion. Calcitonin had no effect on 1,25-(OH)2D3 secretion rates. The results of the present studies show that an activator of PKC increases 1,25-(OH)2D3 secretion by mammalian proximal tubule cells and suggest that the phospholipase-C/PKC signalling system may mediate PTH stimulation of 1,25-(OH)2D3 secretion.

Low K+ increases Na(+)-K(+)-ATPase alpha- and beta-subunit mRNA and protein abundance in cultured renal proximal tubule cells

Studies from this laboratory demonstrate that LLC-PK1/Cl4 cells, a cultured renal cell line, respond to incubation in low-K+ medium by coordinately increasing abundance of both alpha- and beta-subunits of Na(+)-K(+)-ATPase but increase only beta- and not alpha-mRNA levels (Lescale-Matys et al. J. Biol. Chem. 265: 17935-17940, 1990) and that alpha-abundance is likely increased as a result of increased efficiency of alpha-mRNA translation (L. Lescale-Matys and A. A. McDonough. J. Cell Biol. 111: 311A, 1990). The aim of this report was to determine if nontransformed kidney cells would respond to low K+ in a similar manner. We incubated primary cultures of rat proximal tubule cells in low K+ (0.25 mM) for up to 24 h to address this aim. Na(+)-K(+)-ATPase activity, measured enzymatically, and abundance of alpha- and beta-subunits, measured by immunoblot, were increased significantly and coordinately by 8 h of low K+, and, by 24 h of low K+, these parameters were increased to 2.17 +/- 0.34 (activity), 2.03 +/- 0.21 (alpha), and 2.39 +/- 0.48 (beta)-fold over control. Pretranslationally, beta-mRNA, measured by Northern blot analysis, increased to 1.76 +/- 0.35 after 3 h of low K+ and to 3.4 +/- 0.75-fold over control after 24 h of low K+. The increase in alpha-mRNA was smaller and delayed compared with the beta-mRNA response, but it was sufficient to account for the observed increase in alpha-protein and Na(+)-K(+)-ATPase activity at steady state: alpha-mRNA increased to 1.27 +/- 0.09 after 6 h and to 1.91 +/- 0.41-fold over control after 24 h in low K+. We conclude that the accumulation of sodium pumps in cultured renal proximal tubule cells, unlike LLC-PK1 cells, can be accounted for by increases in both alpha- and beta-subunit mRNA levels.

Redistribution of microvilli and membrane enzymes in isolated rat proximal tubule cells

Isolated cells from the proximal tubule have previously been used to study specific renal physiological processes. Here we analysed the structural changes induced by mechanical isolation on rat proximal tubule cells. As major modifications we detected: 1) a redistribution of brush border microvilli, which spread out over all the cell surface immediately after isolation; 2) a rapid redistribution of the apical membrane enzyme markers aminopeptidase N and isomaltase; and 3) a striking decrease of membrane surface amplification factor. The results show that cells isolated from the proximal tubule rapidly loose their characteristic polarized membrane structure. Loss of epithelial polarity implies loss of several vectorial transports and should thus be cautiously taken into account when working with this model system.

Renal growth in infancy and childhood ?experimental studies of regulatory mechanisms

During the peri- and early postnatal period, nephrogenesis is completed and kidney growth is accomplished both by cellular proliferation and enlargement. The number of nephrons in a given species is predetermined, whereas cellular growth can be influenced by environmental factors in an age-dependent manner. Unilateral nephrectomy or a high-protein diet stimulates renal growth more in the young than in the adult. Conversely, pyelonephritis inhibits renal growth in infancy but not in adulthood. The relative importance of hyperplasia and hypertrophy for renal growth also changes with renal maturation. The mechanisms behind these developmental changes in regulation of renal growth are largely unknown, but age-dependent changes in the expression of several proto-oncogene products have been demonstrated. These include growth factor receptors as well as components of the intracellular system that transfers the signal from an activated growth factor receptor to the cell nucleus. Studies on rat proximal tubule cells in primary culture might be of great value in expanding our knowledge of growth regulation in the developing kidney. Such studies have already shown that under identical environmental conditions the basal proliferative rate is age dependent, that the proliferative response to growth stimulation changes postnatally, and that this is associated with changes of both the response of the Na+/H(+)-exchanger and the expression of the c-fos proto-oncogene.

Short-Term Primary Cultures in Studies of Growth Regulation in Rat Proximal Tubule Cells

Recent methodological developments have made primary cultures of renal cells a promising model for studies of renal growth regulation. By minimal culture time and single-cell techniques, homogenous populations of highly differentiated cells can be studied. This report reviews the model and recent studies on growth regulation in rat proximal tubule cells (PTC). It is demonstrated that no correlation exists between proliferative rate and intracellular pH (pHi) in PTC, a topic of previous debate. The rate of proliferation in cultured PTC is similar to in vivo and dependent on the postnatal age of the animal. Recent evidence demonstrates that the model is an important tool for studies of postnatal changes in renal growth regulation.

Immunolocalization of 15-kDa membrane proteins in the kidneys of normal and acidotic rats

Proteins with apparent molecular masses between 15 kDa and 17 kDa were enriched from rat renal brush-border membranes by preparative gel electrophoresis and used for immunization of rabbits. The serum of one of the rabbits reacted in Western blots of separated renal brush-border proteins with a single 15-kDa band. A comparably strong reaction is seen with a 15-kDa band of renal endosomal proteins. Basolateral membranes show a much weaker reaction. In light- and electron-microscopic studies the serum stains brush-border membranes and endosomes in rat proximal tubule cells, but not mitochondria and basolateral membranes. In cortical collecting ducts, principal cells are not stained with the antiserum. alpha-type (H(+)-secreting) intercalated cells bind the antibodies at apical tubulovesicles. The luminal membrane is scarcely labelled. Conversely, beta-type (HCO3(-)-secreting) intercalated cells exhibit antibody binding to their basolateral membrane. Thus, the antiserum detects 15-kDa proteins differently sorted in alpha- and beta-intercalated cells. After induction of an acute (6 h) metabolic acidosis, the antibody-binding pattern changes only in intercalated cells, type alpha, and occurs at the markedly enlarged luminal plasma membrane. The amount of alpha-type intercalated cells with enlarged luminal membrane ("secreting cell") increases at the expense of alpha cells with apical tubulovesicles ("resting cell"). Taken together, the antiserum detects 15-kDa proteins, the localization and adaptive changes to metabolic acidosis of which are similar to H(+)-ATPases. The functional role of the 15-kDa proteins needs to be established in further studies.

Serum Factors Induce C-fos Expression and Rapid Cell Proliferation in Adolescent But Not in Infant Rat Proximal Tubule Cells

Kidney epithelial cells in short-term primary culture have been studied with regard to proliferative rate and expression on the c-fos protooncogene. The experiments were performed on subconfluent renal proximal tubule cells isolated from infant and adolescent rats. Proliferation was determined by 3H-thymidine autoradiography and nuclear content of c-fos protein by semiquantitative immunofluorescence. The basal proliferative rates in infant and adolescent renal proximal tubule cells were the same after 48 h of primary culture in Dulbecco's modified Eagle's medium with 10% FCS. Serum deprivation for 24 h caused a significant growth inhibition in both infant and adolescent cells. C-fos was expressed to the same extent in infant and adolescent serum-deprived cells. The rapid response to the addition of serum was markedly different in infant and adolescent cells. In adolescent cells, addition of serum led to a transient significant increase in the nuclear expression of c-fos protein, reaching a peak at 60 min. No increase in c-fos was seen in infant cells. In adolescent cells, the rate of proliferation increased 11-fold and 3H-thymidine labeling index reached 26.7 +/- 4.3%. In infant cells, the proliferative response to serum addition was significantly lower; the labeling index reached only 4.2 +/- 1.2%. It could be excluded that the attenuated response in infant cells was due to cell death or impaired metabolic function. The results imply that the principles of growth regulation change postnatally.

Post‐natal changes in growth of rat proximal tubule cells: a study of cells in short primary culture

Etude visant a examiner les changements post-nataux de croissance de cellules tubulaires proximales renales de rats âges de 10 a 80 jours. Utilisation de methode autoradiographique

Influence of Na+ intake on dopamine-induced inhibition of renal cortical Na(+)-K(+)-ATPase

The enzyme L-amino acid decarboxylase (L-AADC), found in abundance in rat proximal tubule cell cytosol, converts L-dopa to dopamine. Dopamine, in turn, suppresses proximal tubule sodium transport by inhibiting Na(+)-K(+)-ATPase activity. We sought to determine whether changes in dietary sodium intake in rats lead to adaptation of dopamine formation and dopamine-induced Na(+)-K(+)-ATPase inhibition. In rats on a high-salt (HS) diet, the maximal velocity (Vmax) of renal cortical L-AADC was 78 +/- 19% higher than that in rats on a low-salt (LS) diet. The Michaelis constant (Km) of the enzyme remained unchanged. In renal cortical tubule cell suspensions the L-dopa-induced inhibition of ouabain-sensitive oxygen consumption (QO2) was significantly greater in rats on HS diet than in rats on LS diet. Furthermore, L-dopa completely inhibited the nystatin-induced rise in QO2 in the HS but not in the LS group. Carbidopa, an inhibitor of L-AADC, abolished the L-dopa-induced inhibition of nystatin-stimulated QO2 in cells from HS rats and was without significant effect in cells from LS rats. L-Dopa-stimulated K+ efflux was greater in cells from HS rats at 28 +/- 1 nmol.min-1.mg protein-1, compared with 7 +/- 6 nmol.min-1.ng protein-1 in cells from LS rats. By contrast, ouabain-stimulated K+ efflux did not differ between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Dog and rat kidney proximal tubule cells in culture: Responses to parathyroid hormone

We have described and compared culture systems for proximal tubule cell (PTC) preparations from dog and rat kidney. Cells were prepared from kidney cortex by enzyme digestion and purified on Percoll density gradient. The dog PTC and rat PTC differed in their growth characteristics in culture. Although the dog PTC tended to overgrow the contaminating fibroblastic cells, the rat PTC tended to be overgrown by the latter cells when cultured in medium containing 15% fetal calf serum (FCS). Cultures of rat PTC in serum-free medium or medium containing only 2% FCS yielded only epithelium-like cells exhibiting characteristics of cells that are proximal tubular in origin. These properties include protrusion of microvilli, high alkaline phosphatase activity, ability to transport sugar, and responsiveness to parathyroid hormone (PTH) in terms of cAMP production. The time course and dose-response curves of PTH-stimulated cAMP accumulation were studied in dog and rat PTC. The estimated half-maximal concentrations (Kact) for PTH in dog and rat PTC were 1.2 and 100 nM, respectively. Both values are within the range reported in the literature for the respective renal membrane preparations. In addition to our previously reported data on dog PTC, this study revealed the presence of PTH-inhibitable 25-hydroxyvitamin D3-24-hydroxylase in dog PTC. These two model cell culture systems should prove useful in studying PTH action in renal cells.

Coupling of Na-H exchange and Na-K pump activity in cultured rat proximal tubule cells

Na-H exchange was studied using electron probe analysis and microfluorescent pH analysis of individual cells, in 3-day primary cultures of rat proximal tubule cells (RPTC) obtained from 40- to 50-day-old Sprague-Dawley rats. After Na-K pump inhibition, the initial rate of net Na influx was inhibited 87% by 1 mM amiloride. K influx, an estimate of Na-K pump activity, was increased approximately three times in cells containing high Na (0.114 mM K X mM P-1 X min-1) compared with control cells containing low Na (0.038 mM K X mM P-1 X min-1). Single cell measurements of RPTC loaded with the cytoplasmic pH indicator 5- (and -6) carboxy-4',5'-dimethylfluorescein indicated that there was reversible intracellular acidification in the absence of external Na or in the presence of amiloride. When intracellular acidification was induced by the addition and subsequent removal of NH4Cl, recovery of intracellular pH was inhibited in the absence of external Na or in the presence of amiloride. Using a similar protocol, it was found that after intracellular acidification, the rate of Na influx increased at least 5.9 times, and intracellular Na content was increased 3.15 +/- 0.64 times at 60 s. There was an initial 50% inhibition of Na-K pump activity within the first 60 s compared with control (nonacidified) RPTC, secondarily followed by an increase in Na-K pump activity. Amiloride (0.5 mM) inhibited the acidification-induced increase in Na influx, and persistent acidification led to a persistent inhibition of Na-K pump activity compared with control. In summary, these results demonstrate that Na-H exchange mediates the majority of net Na influx into RPTC under our basal conditions and is necessary for maintenance of intracellular pH homeostasis. In RPTC, Na-H exchange is further activated by intracellular acidification, leading to a net increase in intracellular Na content, which secondarily stimulates Na-K pump activity. The initial inhibition of Na-K pump activity may be due to a direct effect of intracellular acidification.

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media.

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.

Use of monoclonal antibodies to culture rat proximal tubule cells.

Current renal cell culture techniques are limited by either a low yield of cells or by heterogeneity of cell types. We have used monoclonal antibodies to microvillus membrane proteins to isolate and culture a pure population of proximal tubule cells. The cells were characterized as proximal tubule cells by phase microscopy, enzyme histochemistry for alkaline phosphatase, butyrate esterase, and gamma-glutamyltransferase, electron microscopy, and specific reactivity with a variety of monoclonal antibodies for proximal tubule cells. Growth over 2-7 days yielded cell numbers up to 1,000-fold greater than obtained by single tubule microdissection. Dome formation was observed, suggesting intact fluid transport. In addition, Na+-H+ exchange and Na+-dependent D-hexose transport, known transport processes of the proximal tubule, were demonstrated by microfluorimetry of single cells and methyl-alpha-D-glucopyranoside uptake, respectively. Our results indicate that large numbers of homogeneous, cultured rat proximal tubule cells that maintain characteristics of in vivo proximal tubule cells can be obtained using a monoclonal antibody technique of isolation.

Simultaneous comparison of techniques for x-ray analysis of proximal tubule cells

Major differences in elemental and water content in cells of rat papillae in antidiuresis have been reported using x-ray microanalysis. The reason for these reported differences in unknown. In order to determine if the differing microprobe techniques used in these reports could account for the differences measured, we analyzed elemental concentrations in rat proximal tubule cells using both methods simultaneously on the same cells. Both methods provided comparable results. Furthermore, no differences in element or water content of proximal tubule cells cryosectioned with albumin at -53 degrees C and those cells cryosectioned at -80 degrees C were seen. Therefore, the differing values previously reported for elemental analysis of rat kidney cannot be ascribed to either cryosectioning at a warmer temperature or to the analytical algorithm used by either group.