JOURNAL OF CELLULAR PHYSIOLOGY 150:243-250 (1992)

Growth and Function of Primary Rabbit Kidney Proximal Tubule Cells in Glucose-Free Serum-Free Medium JEE C H A N G JUNG, SANG-MOG LEE, N I N A KADAKIA, AND MARY TAUB* Biochemistry Department, State University of N e w York at Buffalo, Buffalo, N e w York 142 74 The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1 .O doublingsiday, both in glucosefree and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK i s a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucoseimg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium

Glucose has been observed to have dramatic effects on the functional properties of epithelial cells (Brownlee and Cerami, 1981; Lee, 1987). Under hyperglycemic conditions, epithelial basement membranes are altered, which, a s a consequence, affects cell function (Brownlee and Cerami, 1981; Martin and Timpl, 1987). Intracellular sorbitol levels are elevated (Brownlee and Cerami, 1981; Bylander and Sens, 1990), protein kinase C is activated (Lee et al., 19891, and Na+/K+ ATPase activity is altered (Lee et al., 1989; Ku et al., 1986). Hypertrophy and hyperplasia may occur (Brownlee and Cerami, 1981; Ku et al., 1986). Under conditions of glucose deprivation, cultured epithelial cells have been found to exhibit other types of structural and functional changes, including the development of a n elaborated brush border (Huet e t al., 1987) and the formation of multicellular domes (Taub et al., 1989). Dome formation is a n indication of transepithelial solute transport across epithelial monolayers in culture (Leighton et al., 1969; Lever, 1979). Investigations concerning the underlying mechanisms of these glucose effects have been limited to a large extent by the in vitro cell culture conditions. Tissue culture medium generally contains glucose, because most animal 0 1992 WILEY-LISS, INC

cells stringently require a n exogenous source of sugar for normal growth and function (Bettger and Ham, 1982). Animal cells in several tissues, including the liver and the kidney, do, however, possess the capacity for gluconeogenesis (Krebs et al., 19631, and thus should be capable of growing in vitro in culture medium lacking sugars. Indeed, primary rat hepatocytes synthesize DNA and retain functional capacity in glucose deficient medium (Leffert and Paul, 1972). However, gluconeogenic capacity has not been clearly demonstrated in normal kidney cells in culture. The kidney cells which possess this capacity are localized in the renal proximal tubule (Guder and Ross, 1984). The established porcine kidney epithelial cell line LLC-PK, retains a number of properties distinctive of the renal proximal tubule (Gstraunthaler, 1988),but does not survive in glucose-

Received April 22,1991; accepted August 28,1991.

“To whom reprint requestsicorrespondence should be addressed a t Biochemistry Department, 140 Farber Hall, State University of New York a t Buffalo, Buffalo, NY 14214.

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free medium (Gstraunthaler and Handler, 1987). The LLC-PK, cell line lacks a n intact gluconeogenic pathway (Gstraunthaler and Handler, 1987). LLC-PK, cells may conceivably have lost this capacity, as the consequence of genetic alterations which occurred during their establishment as a continuous cell line. Loss of gluconeogenesis also may possibly be a n immediate consequence of putting renal proximal tubule cells into the culture situation. In order to examine this latter possibility, we have employed a primary rabbit kidney proximal tubule cell culture system. Primary rabbit kidney cells grow without fibroblast overgrowth in serum-free medium supplemented with insulin, transferrin, and hydrocortisone (Chung et al., 1982). Confluent monolayers retain such proximal tubule transport systems as a sodium/ glucose cotransport system (Chung et al., 1982; Sakhrani et al., 1984), a sodium dependent phosphate transport system (Waqar et al., 1988), and a p-aminohippurate transport system (Yang et al., 1988). Unlike LLC-PK, cells, the primary rabbit kidney cells exhibit parathyroid hormone sensitive cyclic AMP production typical of the renal proximal tubule (Chung et al., 1982). A key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), is present in the proximal tubule cell cultures (Wang and Taub, 1991). Physiologic levels of insulin regulate the PEPCK activity in the primary cultures, suggestive of the presence of insulin receptors (Wang and Taub, 1991). In this report primary rabbit kidney proximal tubule cells were observed to grow in glucose-free medium supplemented with pyruvate and glutamine. Although no significant glucose could be detected in the culture medium, low levels of glucose were found intracellularly. The intracellular glucose may possibly originate from preexisting glycogen stores, rather than from gluconeogenesis. However, the glycogen content of the cells was observed to increase while being cultured in glucosefree medium. These results suggest that the intracellular glucose in the primary cells does indeed originate from gluconeogenesis and that the primary cells retain the capacity for gluconeogenesis in vitro.

Cell culture environment Primary rabbit kidney proximal tubule cells were maintained in a 37"C, 5% CO, humidified environment in a serum-free basal medium supplemented with 3 growth supplements, 5 pgiml insulin, 5 pgiml transferrin and 5 x lops M hydrocortisone (Chung et al., 1982). The basal medium, a glucose-free modified DMEiF12 medium, pH 7.4, was a 50:50 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 lacking glucose, hypoxanthine, thymidine, pyruvate, L-glutamine, L-glutamic acid, L-serine, and linoleic acid. The basal medium was further supplemented with 15 mM HEPES buffer (pH 7.4) and 20 mM sodium bicarbonate. Immediately prior to the use of the medium, the 3 growth supplements were added. Water utilized in medium preparation was purified by means of a Millique deionization system.

MATERIALS AND METHODS Materials Hormones, transferrin, and other chemicals were purchased from Sigma Chemical Corp. (St. Louis, MO). Powdered medium and soybean trypsin inhibitor were from Life Technologies (Grand Island, NY). Class IV collagenase was from Worthington (Freehold, NJ). Amyloglucosidase and glucokinase (Bacillus stearothermophilus) were from Sigma. Gamma 32P-ATP, alpha 32P dCTP (3,000 Ciimmol), and random priming Iabelling kits were purchased from DupontiNEN. Restriction endonucleases were obtained from Promega, 1 kb DNA ladder from Life Technologies, Inc., and Zeta Probe Blotting Membrane from Biorad. Liquiscint was obtained from National Diagnostics (Parsippany, NY). Iron oxide was prepared by the method of Cook and Pickering (1958). Stock solutions of iron oxide in 0.9% NaCl were sterilized using a n autoclave and diluted with PBS prior to use.

Cell growth studies Primary proximal tubule cell cultures were initiated in 35 mm dishes for cell growth studies. Generally, tubules were inoculated a t one-fourth the normal inoculum (the normal inoculum being 0.5 mg proteinidish measured by the method of Bradford, 1976; Chung et al., 1982). Periodically, cells were removed from the dishes using phosphate buffered saline (PBS) containing 0.05% trypsin and 0.5 mM EDTA. The cells were counted utilizing a Coulter Model ZF particle counter. Values are the average of triplicate determinations.

Primary rabbit kidney proximal tubule cell culture Primary rabbit kidney proximal tubule cell cultures were prepared by a modification of the method of Chung et al. (1982). To summarize, the kidneys of a male New Zealand white rabbit (2 to 2.5 kg) were perfused via the renal artery, first with phosphate buffered saline (PBS), and subsequently with DMEiFl2 containing 0.5% iron oxide (wtivol), such that the kidney turned grey-black in color. Renal cortical slices were homogenized with 4 strokes of a sterile Dounce homogenizer (type A pestle), and the homogenate was poured first through a 253p and then a 83p mesh filter. Tubules and gomeruli on top of the 83p filter were transferred into sterile glucose-free modified DMEiF12 medium containing a magnetic stir bar. Glomeruli (containing iron oxide) were removed with the stir bar. The remaining purified proximal tubules were briefly incubated in glucose-free modified DMEiFl2 containing 0.125 mgiml collagenase (class IV) and 0.025% soybean trypsin inhibitor. The tubules were then washed by centrifugation, resuspended in glucose-free modified DMEiFl2 containing the 3 supplements, and transferred into tissue culture dishes. Medium was changed one day after plating and every two days thereafter.

Glucose assay Primary proximal tubule cells were grown to confluence in 100 mm dishes containing either glucose-free or glucose-containing modified DMEiFl2 supplemented with insulin, transferrin, hydrocortisone, pyruvate, and glutamine. The medium was changed in confluent monolayers. After 24 hours the medium was removed

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and replaced with PBS. The cells were removed from each of three dishes using a rubber policeman, transferred into a n Eppendorf tube, and centrifuged for 5 minutes a t 500 rpm. The cell pellets were quickly frozen on dry ice, and stored a t -70°C prior to use for glucose assays, Cellular glucose levels were determined by the method of Bradley and Kaslow (1989). To summarize: Cellular samples were incubated at 37°C for 30 minutes in a reaction mixture containing 50 mM triethanolamine hydrochloride, pH 9, 1 Uiml glucokinase, 2 mM MgCl,, and 40 pM ly-32P]-ATP (30,000 cpmitube). To terminate the glucokinase reaction, excess 1N HC104 containing 0.1 mM H3P0, was added. The reaction mixtures were incubated at 95°C for 40 minutes. Unincorporated 32Piwas then precipitated using 13 mM ammonium molybdate and 16.7 mM triethanolamine. The precipitate was removed by centrifugation a t 3,OOOg for 20 minutes. The supernatant containing 32P-glucose-6phosphate was assayed in a Packard scintillation counter. The quantity of glucose in unknowns was determined using a glucose standard curve. Data were the mean of triplicate determinations t the standard deviation.

Glycogen assay Primary proximal tubule cells were grown to confluence either in glucose-free modified DME/Fl2 medium supplemented with pyruvate, glutamine, and three growth supplements (insulin, transferrin, and hydrocortisone), or in glucose containing modified DMEIF12 medium with the same three growth supplements. Glycogen was assayed by the method of Bradley and Kaslow (1989). The culture medium was removed by aspiration, and primary cells were washed twice with ice cold PBS. The cells were removed from the dish into PBS, using a rubber policeman, and centrifuged at 500g. The cell pellet was digested by a 20-minute incubation a t 100°C in 30% KOH. Glycogen was precipitated by the addition of ethanol and LiBr to final concentrations of 60% and 0.3%, respectively, and the glycogen precipitate was centrifuged at 14,000 rpm for 10 minutes. The pellet was washed twice by resuspension in 60% ethanol/0.3% LiBr and centrifugation. The pellet was finally resuspended either in 200 pl of 23 mM sodium acetate, pH 5, containing 0.7 mgiml amyloglucosidase, or 23 mM sodium acetate, pH 5 alone (no enzyme control). Glycogen was digested for one hour at 55"C, and then assayed for glucose, using the radiometric glucose assay, Protein was determined by the Bradford method. Northern analysis of cellular RNA Total RNA was isolated by the guanidinium isothiocyanate/cesium chloride method (Chirgwin et al., 1979). RNA was isolated from primary rabbit kidney proximal tubule cells grown to confluence either in glucose-free modified DMEiF12 + insulin, transferrin, and hydrocortisone, or in the same modified DMEiFl2 supplemented with glucose, in addition t o the other 3 growth supplements. RNA (10 pgisample) was fractionated by electrophoresis in formaldehyde gels containing 0.8% agarose and was transferred to Zeta Probe Blotting Membranes. Duplicate RNA samples on the

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Fig. 1. Growth in glucose-free medium. Primary proximal tubule cells were cultured either in glucose-free modified DMEIFlZ supplemented with 5 factors (insulin, transferrin, hydrocortisone, pyruvate, and glutamine) (a), or in the modified DME/F12 further supplemented with 6.67 mM glucose, as well as the 5 factors ( 0 ) . Starting on day 3, cells were routinely counted every other day over a 14-day culture period. Values are averages ( 5 standard deviation) of triplicate determinations.

gel were stained with ethidium bromide to verify the quality and the quantity of the RNA. A restriction fragment containing mouse alpha I(1V) collagen cDNA obtained from plasmid pE123 (Boot-Handford et al., 1987) and a restriction fragment containing mouse laminin B1 cDNA obtained from plasmid pE386 (Boot-Handford et al., 1987) were utilized for making labelled probes. Plasmids pE123 and pE386 were kindly provided by Dr. Marceau Kirkinin. The restriction fragments were radiolabelled with alpha 32PdCTP by the random primer method and were utilized for hybridization following the method of Church and Gilbert (1984). Standard stringent hybridization conditions were utilized.

RESULTS The possibility was examined that primary rabbit kidney proximal tubule cells could grow in glucose-free modified DMEiFlZ medium supplemented with insulin, transferrin, and hydrocortisone. Figure 1 shows that during the initial 7 days in culture, the proximal tubule cells grew a t 1.0 doublingsiday both in glucosefree medium and in glucose-containing modified DMEi F12 medium supplemented with insulin, transferrin, and hydrocortisone. Subsequently, the cultures in glucose-containing medium achieved a saturation density which was substantially higher than the density achieved by cultures in glucose-free medium. The glucose concentration used in this study was 6.67 mM, which is within the concentration range present in blood under normal physiologic conditions. In the study described above, the glucose-free modified DME/F12 medium was supplemented with 2.5 mM

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Culture Time (Days) Fig. 2. Effect of nutrients on growth in glucose-free medium. Primary proximal tubule cells were grown in glucose-free modified DMEI F12 medium supplemented with insulin, transferrin, hydrocortisone, and a) palmitate (n), or b) acetate ( A ) , or c) glutamine (*), or d) pyruvate (A), or e) no further supplements (control (0)). Cells were routinely counted over a 13-day time interval. Values are averages of triplicate determinations.

Fig. 3. Effect of insulin on growth. Primary proximal tubule cells were grown in 35 mm dishes containing either glucose-free DMEiFl2 or DMEIF12 supplemented with 6.67 mM D-glucose. Both the glucosefree and glucose-containing culture medium was further supplemented either with 2 factors, transferrin and hydrocortisone (no glucose + 2F, and glucose + 2F, respectively), or with 3 factors, insulin, transferrin and hydrocortisone (no glucose + 3F, and glucose + 3F, respectively). The culture medium in all cases was also supplemented with glutamine and pyruvate. Cells were counted periodically over a 13-day time interval. Values are averages of triplicate determinations.

TABLE 1. Effects of nutrients on the growth of proximal tubule cells' Added substrate

Concentration mM

Cell number

Percentage of control

fied DMEiFl2 medium, then insulin may possibly inhibit, rather than stimulate, proximal tubule cell None (Control) 32 f 6 100 20 growth in medium lacking glucose. However, Figure 3 Pyruvate 0.5 68 f 10 210 f 31 shows that primary proximal tubule cell growth was Lactate 0.3 85 f 12 262 f 38 stimulated by insulin, both in glucose-free and in gluHydroxybutyrate 0.033 71 f 8 220 f 26 Acetoacetate 0.009 51 f 3 157 f 10 cose-containing modified DME/F12 medium. Thus, inGlucose 6.7 137 z t 19 426 f 58 sulin very likely elicits its effects on growth in glucoseGlucose 20 146 f 31 453 f 95 free medium via a means other than gluconeogenesis. IPrimary rabbit kidney proximal tubule cells were initiated as described in In order to examine initially the possibility that conMaterials and Methods. The cells were cultured in glucose-free modified DME/FlZ fluent monolayers of primary proximal tubule cells supplemented either with 5 fig/ml insulin, 5 pg/ml transfemn, and 5 X M hydrocortisone alone, or with an additional nutritional substrate, as indicated have gluconeogenic capacity, the glucose content of above. The cell number present in each growth condition was determined after 7 confluent monolayers of primary rabbit kidney proxidays in culture using a Coulter counter. Values are the average (*std deviation) of triplicate determinations. mal tubule cells was determined 24 hours after a medium change (see Materials and Methods for details). The glucose level in the primary proximal tubule cells glutamine and 0.5 mM pyruvate, as well as the 3 was 5 2 1 pmoles D-glucose per mg protein. However, growth supplements-insulin, transferrin, and hydro- no significant glucose was detected in the culture mecortisone. The dependence of the growth of primary dium. The intracellular glucose in the primary cell cultures rabbit kidney proximal tubule cells upon a number of metabolic substrates other than glucose was examined. may possibly also originate from preexisting glycogen As illustrated in Figure 2, 0.4 mM pyruvate, 2.5 mM stores, rather than from gluconeogenesis. In this case, glutamine, and 0.01 mM palmitate were all individu- glycogen stores would gradually be depleted in the culally growth stimulatory in glucose-free modified DMEi tures over time. In order to evaluate this possibility, F12 supplemented with insulin, transferrin, and hydro- glycogen levels were determined both in the initial cortisone. Other growth stimulatory substrates for proximal tubule preparation and in the resulting priproximal tubule cells in the glucose-free medium were mary cell cultures. As illustrated in Figure 4, the glycolactate, beta hydroxybutyrate, and acetoacetate (Ta- gen levels increased significantly after the proximal ble 1).Of these substrates lactate, pyruvate and glu- tubule cells were cultured for 7 days in glucose-free tamine are utilized for renal gluconeogenesis (Krebs modified DMEiF12 medium, a s compared with the levet al., 1963; Mennes et al., 1978; Nagata and Rasmus- els detected in the original tissue. Even higher glycosen, 1970).Acetate, beta hydroxybutyrate, and acetoac- gen levels were present in cultures in glucose containing modified DMEiFlZ medium. The increase in etate are utilized for oxidative metabolism. Previously, we reported that primary renal proximal glycogen content in the primary rabbit kidney proxitubule cells possess a key regulatory enzyme in the mal tubule cells, while being cultured in glucose-free gluconeogenic pathway, phosphoenolpyruvate carbox- medium, can only be explained by the incorporation of ykinase (PEPCK) (Wang and Taub, 1991).We also ob- newly synthesized glucose into glycogen. Glucose has been reported to modulate the expresserved that the PEPCK activity of the primary cells was inhibited by insulin (Wang and Taub, 1991). If sion of the genes for basement membrane proteins (ZiyPEPCK activity (and the rate of gluconeogenesis) is adeh et al., 1990). The effect of exogenous glucose on rate limiting for cell growth in the glucose-free modi- the levels of alpha IUV) collagen and laminin B1 chain x 10-3

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Fig. 4. Glycogen content of proximal tubule cells. Purified rabbit kidney proximal tubules were either frozen or utilized to initiate primary proximal tubule cell cultures either in glucose-free DMEiFl2 supplemented with 5 supplements (insulin, transferrin, hydrocortisone, glutamine, and pyruvate) or in DMEiFl2 supplemented with 6.67 mM D-glucose, as well as these same 5 supplements. The glycogen content of the purified proximal tubules, as well as of confluent monolayers in either glucose-free or glucose-containing medium, was determined as described in Materials and Methods. Determinations were made with tubules and cultures derived from the same kidney preparation. Values are the average ( 2 std. deviation) of triplicate determinations.

mRNA's in primary rabbit kidney proximal tubule cells was examined by Northern analysis using mouse cDNA probes. Figure 5a shows that the level of alpha I(1V) collagen mRNA was significantly elevated in primary cultures in modified DME/F12 medium containing 6.67 mM glucose (GL), a s compared with glucosefree (GF) modified DME/F12 medium. Elevating the glucose concentration from 6.67 mM (GL) (normal blood levels) to 20 mM (GH) (blood levels in diabetes) had no significant effect on the alpha I(1V) collagen mRNA level. In contrast, laminin B1 chain mRNA levels did not differ significantly either in glucose-free or in glucose-containing modified DME/F12 medium (Fig. 5b).

DISCUSSION Glucose is not considered to be a major energy source for renal proximal tubule cells in vivo (Klein et al., 1981; Lee et al., 1981). However, when renal proximal tubule cells are grown in vitro in glucose containing culture medium, glucose is apparently utilized to a much greater extent (Sakhrani et al., 1984). The effects of glucose metabolism on proximal tubule function in culture have not been clearly defined. However, a n abnormally high usage of this substrate is very likely deleterious to normal physiologic function, as occurs in

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diabetes (Ku et al., 1986). Diabetes is associated with a number of changes (Ziyadeh and Goldfarb, 1991), including proteinuria and glucose secretion, as well a s a n increase in renal Na+ reabsorption and in renal blood flow. In the renal proximal tubule the activity of individual Na+/glucose cotransporters is reportedly reduced in diabetes. An increase in the activity of the Na+/H+ antiport system, and the Na+/K+ ATPase presumably results in increased Na+ reabsorption, as well as hypertrophy. Hyperglycemia has been proposed to interfere not only with tubular reabsorption (and normal cell volume control), but also to cause tubular basement membrane thickening. The changes in tubular basement membrane composition observed in diabetes may be responsible in part for altered proximal tubule function. In order to better define the effects of glucose on primary renal proximal tubule cells, glucose-free culture conditions were employed. The basal medium, DMEIF12, was modified such that glucose, hypoxanthine, thymidine, pyruvate, L-glutamine, L-glutamic acid, L-serine and lineolic acid were deleted. Primary rabbit renal proximal tubule cells grew a t equivalent rates in glucose-free medium, and in glucose-containing modified DME/F12 medium supplemented with insulin, transferrin, hydrocortisone, glutamine, and pyruvate. In the absence of a n external sugar source, growth can only occur if the primary cells themselves provide the required sugars. Although the primary cells did not secrete any detectable glucose in the culture medium, a small quantity of free glucose was detected intracellularly . The glucose may originate from gluconeogenesis or preexisting glycogen stores. The possibility that this intracellular glucose originates from preexisting glycogen stores seems unlikely, however, a s cellular glycogen levels increased, rather than decreased, after the proximal tubule cells were cultured in glucose-free medium. Many types of cultured animal cells, including Hela, mouse L-cells, normal human diploid fibroblasts, chick embryo fibroblasts, and rat hepatoma cells (Wice et al., 1981; Wice and Kennell, 1983), have also been observed to grow in glucose-free medium. The growth of such animal cell lines has been found to occur in a sugar-free medium, provided that a specific nucleoside, such as uridine, is present. Wice et al. (1981) presented evidence indicating that the uridine was being utilized not only for the biosynthesis of nucleotides, but also the biosynthesis of sugars via the pentose phosphate shunt. The essential function of sugars under these culture conditions was not to provide energy. Rather in these cases, the oxidation of glutamine by the citric acid cycle was sufficient to provide for the energy needs of the cells. The function of ribose-1-phosphate (derived from nucleoside phosphorylase activity) was to act as a substrate for the biosynthesis of nucleotides, as well a s for other molecules essential for cell survival. In vivo, the kidney has a very high rate of oxygen consumption (Al-Awqati et al., 1986). The energy produced by the aerobic oxidative metabolism in the kidney is utilized to support renal transport (Al-Awqati et al., 1986; Harris et al., 1981). In the renal proximal tubule, the reabsorption of a number of metabolic substrates, including glucose, occurs. These metabolic substrates are reabsorbed from the lumen of the nephron,

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Fig. 5. Northern blot analysis of alpha I(IV) collagen and laminin B1 mRNA from primary kidney cultures. RNA was isolated from primary cultures grown to confluence in DMEiFl2 with the 3 supplements, and glucose at either 0 mM (GF),or 6.67 mM (GL), or 20 mM (GH). Total RNA (10 Fgilane) was subjected to electrophoresis in 0.8% agarose gels, Duplicate sets of samples were run on each of two gels, gel I and gel 11. One set of samples both from gel I and from gel I1 was transferred to Zetabind blotting membrane. Blots were hybridized with

either an alpha UIV) collagen cDNA probe in the case of gel I or a laminin B1 chain cDNA probe in the case of gel 11. The blots were washed and exposed to X-ray film for 24 hours. The results with the alpha UIV) collagen probe (gel I) and the laminin B1 probe (gel 11) are illustrated in a and b, respectively. The other set of RNA samples both on gel I and gel I1 was stained with ethidiurn bromide, as illustrated in c and d, respectively.

in many cases by sodium-dependent transport systems, which are driven by a sodium electrochemical gradient across the plasma membrane (Ullrich, 1979). The sodium gradient is maintained by the activity of the Na+/K+ATPase. The activity of the Na+/K+ATPase is increased by the addition of metabolic substrates to previously substrate-depleted rabbit renal cortical tissue, which then apparently results in a n increase in the activity of Na+-cotransport systems (Harris et al., 1981). The metabolic substrates reabsorbed by renal

proximal tubules are either transported across the epithelium to the blood side, metabolized tc other substrates, or utilized a s energy sources (Harris et al., 1981). However, isolated rabbit renal proximal tubules have been observed to utilize particular substrates as energy sources (e.g., short chain fatty acids) more effectively than others (Balaban and Mandel, 1988). Glucose is a poorly metabolized substrate for isolated proximal tubules (Balaban and Mandel, 1988; Lee et al., 1971).

GLUCOSE-FREE MEDIUM

Primary renal proximal tubule cells have a number of available substrates to use a s energy sources during their growth in glucose-free modified DME/Fl2 medium. Although the culture medium contains a number of amino acids which can be utilized as energy sources, significant growth of the primary proximal tubule cells did not occur in the glucose-free culture medium unless a n additional substrate, such as palmitate, lactate, or glutamine, was added. Palmitate, lactate, and glutamine are utilized a t a significant rate in the kidney and are present in normal blood at levels which are sufficient to make a substantial contribution to the energy requirements of the kidney (Balaban and Mandel, 1988). Glutamine is a major amino acid utilized by the kidney for ammoniogenesis, oxidative metabolism, and gluconeogenesis (Burch et al., 1978). Pyruvate and lactate are substrates of the gluconeogenic pathway, a s well as oxidative metabolism. Pyruvate, acetoacetate, and beta-hydroxybutyratewere also found to be growth stimulatory to primary rabbit kidney proximal tubule cells. However, these substrates are normally present in blood at concentrations which are too low to satisfy the energy needs of proximal tubule cells. The rate of glucose production by the primary rabbit kidney proximal tubule cells in culture is apparently much lower than isolated rabbit renal cortical tubules. Gullans et al. (1984) reported that rapidly shaking, oxygenated suspensions of rabbit renal cortical tubules secrete glucose at a rate of 70 nmoles/mg proteinihour. In contrast, our primary rabbit kidney proximal tubule cell cultures did not secrete any detectable glucose into the culture medium. Nevertheless the rate of gluconeogenesis is not clearly a limiting factor for the growth of primary rabbit kidney proximal tubule cells in glucose-free medium. The glucose-free medium contains three growth supplements-insulin, transferrin, and hydrocortisone. One of these growth supplements, insulin, was found to be growth stimulatory to the primary rabbit kidney proximal tubule cells in glucose-free medium, despite the inhibitory effect of this supplement on the cells’ phosphoenolpyruvate carboxykinase (PEPCK) activity (previously reported by Wang and Taub, 1991). PEPCK is a gluconeogenic enzyme, which is present in our primary renal proximal tubule cells (Taub et al., 1989). Thus, the effects of insulin on a set of a s yet unidentified processes are more critical to the control of proximal tubule cell growth in glucose-free medium than the effects of insulin on PEPCK activity (and gluconeogenesis) are. When present at physiological dosages, insulin acts on appropriate target cells via its interaction with specific insulin receptors. Previously, primary proximal tubule cells were shown to respond to physiologic levels of insulin (i.e., 5 ng/ml) by a decrease in their PEPCK activity (Wang and Taub, 1991). In this report the dosage of insulin utilized in the growth studies in glucosefree medium was 5 pg/ml, nearly a thousand-fold higher than the insulin levels found under physiologic conditions, Although we did not examine the dosage dependence of the effect of insulin on growth in glucosefree medium here, we have previously shown that a n insulin dosage of 50 ngiml was required to stimulate proximal tubule cell growth in glucose containing medium (Wang and Taub, 1991). Despite the elevated in-

249

sulin dosage required to elicit a growth response in primary proximal tubule cells in glucose-containing medium, this growth stimulatory effect of insulin may nevertheless be mediated by insulin receptors. A high rate of degradation of insulin by primary proximal tubule cells has been observed (Wang and Taub, 1991). Similarly, insulin has been found to be rapidly internalized and degraded by intact renal proximal tubules (Yagil et al., 1988). Insulin may nevertheless conceivably also stimulate proximal tubule cell growth via its interaction with either insulin-like growth factor I (IGF I) or IGF I1 receptors, explaining the higher dosage requirement. Indeed, receptors for IGF I, IGF 11, and insulin have all been detected in isolated canine proximal tubules (Hammerman and Rogers, 1987). Glucose-free medium has been utilized as a selective condition for isolating gluconeogenic animal cells in culture. One of the initial reports of this selective method utilized rat hepatoma x mouse lymphoblastoma somatic cell hybrids (Bertolotti, 1977). Liver specific functions are initially extinguished in such hybrid cells. However, segregated hybrid cells, which lose mouse chromosomes, may in some cases reexpress gluconeogenic enzymes and grow in glucose-free medium. Graunthaler and Handler (1987) have selected colonies from cultured LLC-PK, cells t h a t can grow in glucose-free medium supplemented with 10 mM pyruvate, unlike the original LLC-PK, cell population. The isolated colonies possess fructose bis-phosphatase activity, unlike the original parental population. Thus, these variant cells were designated LLC-PK,-FBPase+. As with primary rabbit kidney proximal tubule cells, LLK-PK,-FBPase+ cells did not secrete any detectable glucose into the culture medium. However, inhibition of their PEPCK activity with a specific inhibitor, 3-mercaptopicolinic acid, inhibited their glucose-free growth, suggesting a dependence upon gluconeogenesis. Unlike the parental LLC-PK, cell line, primary rabbit kidney proximal tubule cells grow without a lengthy lag period in glucose-free medium. As the rabbit kidney cells have just been removed from the animal, their initial phenotype in culture presumably still closely resembles that of proximal tubule cells in vivo. The results described in this report indicate that when primary rabbit kidney proximal tubule cells are grown in glucose-free medium, cells are obtained which possess nutritional requirements typical of the renal proximal tubule. Other types of renal cells, including fibroblasts, would not proliferate in the absence of a significant sugar source in the medium. Previously, we have shown t h a t the primary rabbit kidney proximal tubule cells retain hormone responses typical of the renal proximal tubule, including responsiveness to parathyroid hormone (Chung et al., 1982), insulin, IGF I, and IGF I1 (Wang and Taub, 1991). Thus, these primary cultures are appropriate for future investigations concerning the interplay among hormones and nutrients in modulating renal proximal tubule cell growth and function.

ACKNOWLEDGMENTS We thank Mr. William Pudlak and Mr. James U11rich for preparation of figures. This work was supported

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by National Institutes DK4028607 to M.T.

of Health

grant

9R01

LITERATURE CITED Al-Awqati, Q., Chase, H.S., and Kleyman, T.R. (1986) Cellular mechanisms of renal transport and metabolism. In: The Kidney, Vol. I. (B.M. Brenner, and F.C. Rector, eds.) W.B. Saunders, Philadelphia, Pa., pp. 61-92. Balaban, R.S., and Mandel, L.J. (1988)Metabolic substrate utilization by rabbit proximal tubule. An NADH fluorescence study. Am. J . Physiol., 254:rF407-F416. Bertolotti, R. (1977) Expression of differentiated functions in hepatoma cell hybrids: Selection in glucose-free media of segregated hybrid cells which reexpress gluconeogenic enzymes. Somat. Cell Genet., 3t579-602. Betteer. W.J.. and Ham. R.G. (1982) The nutritional reauirements of cukured mammalian 'cells. In: Advances in Nutritional Research. Vol. IV. H.H. Draper, ed. Plenum Publishers, New York, pp. 249286. Boot-Handford, R.P., Kurkinen, M., and Prockop, D.J. (1987) Steady state levels of mRNAs coding for the type IV collagen and laminin polypeptide chains of basement membranes exhibit marked tissuespecific stoichiometric variations in the rat. J. Biol. Chem., 262:12475-12478. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utillizing the principle of proteindye binding. Anal. Biochem., 72r248-254. Bradley, D.C., and Kaslow, H.R. (1989) Radiometric assays for glycerol, glucose, and glycogen. Anal. Biochem., 180:ll-16. Brownlee, M., and Cerami, A. (1981) The biochemistry of the complications of diabetes mellitus. Annu. Rev. Biochem. 50t385432. Burch, H.B., Narins, R.G., Chu, C., Fagioli, Choi, S., McCarthy, W. and Lowry, O.H. (1978) Distribution along the rat nephron of three enzymes of gluconeogenesis in acidosis and starvation. Am. J . Physiol. 235:F245-F253. Bylander, J.E., and Sens, D.A. (1990)Elicitation of sorbitol accumulation in cultured human proximal tubule cells by elevated glucose concentrations. Diabetes, 39t949-954. Chirgwin, J.J., Przbyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18t5294-5299. Chung, S.D., Alavi, N., Livingston, D., Hiller, S., andTaub, M. (1982) Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium. J. Cell Biol., 95:11%126. Church, G.M., and Gilbert, W. (1984)Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A.,81:1991-1995, Cook, W.F., and Pickering, G.W. (1958) A rapid method for separating glomeruli from rabbit kidney. Nature, 182:1103-1104. Gstraunthaler, G.J.A. (1988) Epithelial cells in tissue culture. Renal Physiol. Biochem., l l t 1 4 2 . Gstraunthaler, G., and Handler, J.S. (1987) Isolation, growth, and characterization of a gluconeogenic strain of renal cells. Am. J. Physiol., 252tC232-238. Guder, W.G., and Ross, B.D. (1984) Enzyme distribution along the nephron. Kidney Int., 26:lOl-111. Gullans, S.R., Brazy, P.C., Dennis, V.W., and Mandel, L.J. (1984) Interactions between gluconeogenesis and sodium transport in rabbit proximal tubule. Am. J. Physiol., 246:F859-F869. Hammerman, M.R., and Rogers, S. (1987) Distribution of IGF receptors in the plasma membrane of proximal tubular cells. Am. J. Physiol., 253:F841-F847. Harris, S.I., Balaban, R.S., Barrett, L., and Mandel, L.J. (1981) Mitochondrial respiratory capacity and Na ' - and K ' -dependent adenosine triphosphate-mediated ion transport in the intact renal cell. J . Biol. Chem., 256:10319-10328. Huet, C., Sahuquillo-Mering, C., Coudrier, E., and Louvard, D. (1987) Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29)provide new models for cell polarity and terminal differentiation. J. Cell Biol. 105:345-357. Klein, K.L., Wang, M.S., Torikai, S., Davidson, W.D., and Kurokawa, K. (1981) Substrate oxidations by isolated nephron segments of the rat. Kidney Int., 20:29-35. Krebs, H.A., Bennett, D.A.H., de Gasquet, P., Gascoyne, T., and Yoshida, T. (1963) Renal gluconeogenesis. The effect of diet on the

gluconeogenic capacity of rat-kidney-cortex slices. Biochem. J., 86:22-27. Ku, D.D., Sellers, B.M., and Meezan, E. (1986) Development of renal hypertrophy and increased renal Na,K-ATPase in streptozotocindiabetic rats. Endocrinology, 119:672-679. Lee, A.S. (1987) Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. Trends Biochem. Sci., 12t20-23. Lee, J.B., Vance, V.K., and Cahill, G.F. (1981) Metabolism of 14Clabelled substrates by rabbit kidney cortex and medulla. Am. J . Physiol., 203:27-36. Lee, T.S., MacGregor, L.C., Fluharty, S.J., and King, G.L. (1989) Differential regulation of protein kinase C and (Na,K)-adenosine triphosphate activities by elevated glucose levels in retinal capillary endothelial cells. J. Clin. Invest., 83:90-94. Leffert, H.L., and Paul, D. (1972)Studies on primary cultures ofdifferentiated fetal liver cells. J. Cell Biol., 52.559-568. Leighton, J., Brada, Z., Estes, L.W., and Justh, G. (1969) Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science, 163:472-473. Lever, J.E. (1979) Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK).Proc. Natl. Acad. Sci. U.S.A., 76:1323-1327. Martin, G.R., and Timpl, R. (1987)Laminin and other basement membrane components. Annu. Rev. Cell Biol., 3:57-86. Mennes, P.A., Yates, J., and Klahr, S. (1978) Effects of ionophore A23187 and external calcium concentrations on renal gluconeogenesis. Proc. SOC. Expt. Biol. Med., 157:168-174. Nagata, N., and Rasmussen, H. (1970)Renal gluconeogenesis: Effects of Ca2' and H' , Biochim. Biophys. Acta., 215:1-16. Sakhrani, L.M., Badie-Dezfooly, B., Trizna, W., Mikhail, N., Lowe, A.G., Taub, M., and Fine, L.G. (1984) Transport and metabolism of glucose by renal proximal tubular cells in primary culture. Am. J . Physiol., 246tF757-F764. Taub, M.L. (1991) Renal tubule cells (Growth of different subpopulations of renal cells in defined medium): In: Protocols in Cell and Tissue Culture. (J.B. Griffiths, A. Doyle, and D.G. Newell, eds.) John Wiley and Sons, West Sussex, England (in press). Taub, M.L., Yang, IS., and Wang, Y. (1989) Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions ~ ~ when cultured in a hormonally defined serum-free medium. In Vitro Cell. Dev. Biol. 25:770-775. Taub, M.L., Syracuse, J.A., Cai, J.W., Fiorella. P.. and Subieck. J.R. (1989) Glucose deprivation results in the induction of glucose-regulated proteins and domes in MDCK monolayers in hormonally defined serum-free medium. Expt. Cell Res., 182:105-113. Ullrich, K.J. (1979) Sugar, amino acid, and Na cotransport in proximal tubule. Annu. Rev. Physiol., 224:552-557. Wang, Y., and Taub, M. (1991) Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum-free medium. J. Cell. Physiol., 147r374382. Waqar, M.A., Seto, J., Chung, S.D., Hiller-Grohol, S., and Taub, M. (1985) Phosphate uptake by primary renal proximal tubule cell cultures grown in hormonally defined medium. J . Cell. Physiol., 124:411423. Wice, B.M., Reitzer, L.J., and Kennell, D. (1981) The continuous growth of vertebrate cells in the absence of sugar. J. Biol. Chem., 256:7812-7819. Wice, B.M., and Kennell, D.E. (1983)Sugar free growth ofmammalian cells on some ribonucleosides but not on others. J . Biol. Chem., 258t13134-13140. Yagil, C., Frank, B.H., and Rabkin, R. (1988) Internalization and catabolism of insulin by an established renal cell line. Am. J. Physiol., 254:C822-C828. Yang, I.S., Goldinger, J., Hong, S.K.,andTaub, M. (1988)Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells. J . Cell. Physiol., 135:481487. Ziyadeh, F.N., and Goldfarb, S. (1991)The renal tubulointerstitium in diabetes mellitus. Kidney Int., 39:464475. Ziyadeh, F.N., Snipes, E.R., Watanabe, M., Alvarez, R.J., Goldfarb, S., and Haverty, T.P. (1990) High glucose induces cell hypertrophy and stimulates collagen gene transcription in proximal tubule. Am. J. Physiol. 259:F70PF714.

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