The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings.

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.


INTRODUCTION
The cells of duct systems which perform a transepithelial transport of solutes are characterized by a morphological polarity of the membranes surrounding the lumen of the duct on one side, and by a contact with the interstidum on the other side. Thus, in the proximaI tubuIe of kidney, the luminal surface of the cell is coated with microviUi (the so-called brush border), whereas, on the contraluminal side, plasma membranes form the basal infoldings of the cell.
In order to clearly determine the molecular basis for transepithelial transport, it is essential to isolate and characterize both membranes in pure homogeneous form. The fact that there are differences in the cell envelope due to the polarity of the ceil has been overlooked, particularIy for the kidney, and plasma membrane fractions have been isolated which did not allow a localization of bio-chemical processes in one special region of the cell membrane. Thuneberg and Rostgaard (1) and Kinne and Kinne-Saffran (2) tried to characterize the luminal membrane by isolating membrane fractions which are enriched in brush-border fragments. This approach was also folIowed by Berger and Sacktor (3), Wilfong and Neville (4), and Binkley et al. (5). In all of these membrane fractions, the alkaline phosphatase, a marker enzyme for microvilli, is enriched. There is also an enrichment of Na-K-ATPase activity in these preparations as compared with the starting material (6). Since this enzyme is present in the basal part of the cell, the activity of Na-K-ATPase in these preparations could be expiained as very likely being due to the presence of plasma membranes from the basal infoldings In accordance with this assumption, electron micrographs of those mere-

232
T~E JoIm~TAL OF CELL BIOLOGY • VOLUME 54, 1972 • pages ~32--~5 brane fractions show that, in addition to brushborder fragments, vesicular structures are present which are similar in morphology to classical plasma membranes (7) In this paper, the isolation and separation of microvilli (from the apical part) and of plasma membranes (from the basal part) from the proximal tubule cell of rat kldney cortex is described. After several centrifugation steps, a fraction rich in alkaline phosphatase and Na-K-ATPase (crude brush-border fraction) is obtained from the homogenate which is electrophorized in the preparative free-flow electrophoresis apparatus developed by Hannig (8). On the basis of their different electrical surface charges, and the comparison with the activitms in the crude brush-border fraction, the following is obtained. In the fraction closest to the anode, the Na-K-ATPase activity is enriched by a factor of 16 whereas the alkaline phosphatase is markedly reduced. In the membrane fraction closest to the cathode, the alkaline phosphatase activity is enriched by a factor of 14, whereas the Na-K-ATPase is reduced. Electron microscopy shows that the fraction rich in alkahne phosphatase contains nearly pure microvilli In the fraction with higher Na-K-ATPase activity, tvpicaI plasma membranes with characteristic junctional complexes predominate. These results support the utility of alkaline phosphatase as a marker enzyme for microvilli, and of Na-K-ATPase for membranes derived from the basal infoldings of the proximal tubule cell from rat kidney.

Preparation of Biological Material
All the experiments were carried out between 0 ° and 4°C. Male Wlstar rats, 130 g in weight, were sacrificed by a blow on the neck and the kidneys were removed immediately The renal cortex (4-5 g from 10 rats) was removed with a razor blade, Niembranes were then prepared in a refrigerated Sorvall RC9-B centrifuge according to the isolation diagram shown in Fig 1 The fractmn thus obtained, called crude brush-bordcI fraction, was used for the separation in the FF4. A buffer which contained 1 X 10 -2 ~,t triethanolamine-HC1 and 2.5 X 10 -1 M sucrose, pit 7.6 (1 N NaOH) was used as an isolation medium.

Preparative Free-Flow Eteetrophoresis
Preparative free-flow electrophoresls was carried out in the FF4 Electrophoresis Apparatus developed by Hanuig (8) (Desaga, Heidelberg, Germany, and Brinkmann Instruments Inc, tYestbury, N. Y.) The electrophoresis buffer (buffer in the separation chamber) was prepared from 1 X 10 -2 g triethanolamine, 1 X 10 -2 ~a acetic acid, and 3 3 X 10 -1 sucrose, pH 7.4 (2 N NaOH) The conductivity was 5 0-5.2 ><, 10 -2/zmhos. The buffer for the electrode vessels consisted of 1 X 10 -I }x triethanolamine and 1 X t0 -1 acetic acid, pH 7 4 (2 N NaOH). The condmons of the runs were as follows 90 =t= 10% v/cm, 110 ma, temperature 5°C, electrophoresis buffer flow 2 mt per hr and fracnon. All the samples were spun down for 10 rain at 500 g before the separation in order to remove aggregates The protein content of the super-Renal cortex of 10 rats (4-5 g) Homogenize in 10 vol of isolation medium with loose fitting glass homogenizer, 10 strokes by hand and three strokes at 800 rpm, centrifuge homogenate for 10 mm at 700 g (P1, S1). Discard P1 Recentnfuge S1 for 10 min at 700 g (P~, S£ Resuspend white fluffy top layer in 10 ml of isolation medium, homogemze gently with three strokes, add 10 ml of isolation medium Centrifuge for e0 rain at 16,000 g (PS, $5). Discald $5.
Resuspend white fluffy top layer in the kind and amount of buffer reqmred for further experiments. Crude brush-border fraction FmURE 1 Isolation scheme for the preparation o1 a crude brush-border fraction of rat kidney cortex.
H~rDam~t ~T ~L. The Polarity of the Proximal Tubule Cell zn Rat K~dney ha|ant thus obtained was brought to about 10 mg per ml with electrophoresis buffer, and this sample was then injected into the FF4 apparatus above fraction 70. Sample injection was 2 ml per hr. 92 fractions were collected at 0°C.

Electron Microscope
For preparing the thin sections for the microscope, FF4 fractions containing the membranes to be characterized were spun at 37,000 g for 10 rain at 4°C. The pellets were fixed with glutaraldehyde and OsO4, dehydrated, and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate. For negative staining, samples from the FF4 fractions were directly stained at 0°C either with 2% phosphotungstate, pH 7.2, or with 2 5% ammonium molybdate, pH 7.4 (droplet method). A JEM T7 electron microscope was used for viewing the specimens (60 kv).

STARTING
MATERIAL FOR THE FF4 EX-PERIMENTS: In order to preserve the structural and morphological integrity of the membranes~ an isotonic isolation and separation medium was used. Particularly, ethylenediaminetetraacetate (EDTA) was omitted from the buffer, since it was found in pilot experiments that EDTA-treated membranes have a much lower electrophoretic mobility than untreated ones. This agrees with similar findings on isolated mitochondrial membranes 1. In the crude brush-border fraction prepared with this buffer and according to the isolation scheme (Fig 1) Na-K-ATPase is already enriched by a factor of 5, and alkaline phosphatase by a factor of 8, when compared with the starting cortex homogenate (12). This indicates that in this fraction, brush borders as well as plasma membranes must be enriched. Almost all of the brush-border fragments described earlier (1), (3), (4), (13), (14), (15), (16), and almost all of the mitochondria were excluded by the centrifugation procedure. A very important condition for a reliable separation in the FF4 is the removal of adhesive, masking proteins from the material to be separated This was achieved by several washings during the preparation Finally, before electrophoresis, the crude brush-border suspension was centrifuged at low speed in order to remove aggregates formed during the isolation of the membrane fraction. The preparation thus obtained was suitable for an electrophoretic separation. ENZYMATIC DATA AFTER ELEGTROPI-IOREsis: After electrophoresis of the crude brushborder fraction, a protein distribution as is illustrated in Fig 2 a was obtained. A maximum can be seen in fraction 32 with a shoulder towards the anode. Both marker enzymes, Na-K-ATPase and alkaline phosphatase, show a completely different distribution in the fractions collected ( Fig.  2 b) The maximum for the Na-K-ATPase is in fraction 24, and for the alkaline phosphatase in fraction 32 The maxima are clearly separated from each other. The lack of a protein maximum for the Na-K-ATPase-carrying membranes in fr~cdon 24 is due to the relatively low amount of this type of membranous material in comparison to the membranes in fraction 32. In Fig. 2 c the distribution of Mg-ATPase is illustrated. This enzyme is present in both types of membranes Fractions 20-28 and 31-38 were pooled separately and reelectrophorized. Fig. 3 shows the protein and enzyme distribution after the run It can clearly be seen that this step leads to a further purification of both types of membranes, as is indicated by the broad protein distribution in comparison with the sharp peaks of the enzymes The slight shift of both enzyme maxima is due to the fact that for the reelectrophoresis run purer fractions were injected into the apparatus As a rule, this regularly influences the electropb_oretic mobility. After this second run the left membrane fraction was obtained with a Na-K-ATPase activity enriched by a factor of 16, whereas the specific activity of the alkaline phosphatase is only two-thirds of that in the starting suspension On the other hand, the alkaline phosphatase m fraction 33 is 15 times that in the starting material, and the Na-K-ATPase is reduced (Fig 4).
Contamination by other cellular components was generally very low, even in the crude brushborder fraction itself before electrophoresis (12). Table I shows that after this purification step, the amount ofmitochondria and endoplasmatic reticu-lum is reduced even more The acid phosphatase, however, was not significantly changed by electrophoresis, but this might be due to the reasons discussed below.   Crude brush-0 0032 0 08 0 199 border fraction 23 (from Fig 3) 0 00025 0 049 0.034 35 (from Fig 3) 0 00016 0.065 0 027

Electron Microscope
The contammatmn of the purified membrane fraction wath other cellular particles is extremely low, as is indicated by the specific activities.
tures can be seen at higher magnification Whether or not these structures resemble the filaments of the inner side of the microvilli described by Mukhel]ee and Staehelin for the rat intestine (17) has yet to be clarified. Freeze-etching experiments are presently under way in an effort to determine the character of these structures In thin sections, the timiting membrane can only occasionally be seen as a trilaminar membrane, but not very clearly nor conclusively. The external fuzzy coat is always preserved. Only very few vesicles from the endoplasmic reticulum are present as contamination.
In order to establish the optimal conditions for negative staining, experiments were conducted with different reagents Microwlli could be contrasted much better with 2 5 c) ammonium molybdate, pit 7 4, than with the normally used phosphotungstate. With this stain the subunits of the external coat of the microvilli could be recognized as knobs of about 60 A, whereas with phosphotungstate the coat was seen only as a cloudy layer (I 3). The electrophoretic mobility of the microvitli appears to be determined by a relation between the length of the microvillus and the amount of adhering membrane base The "cleaner" the microvilli, the less they move toward the anode The more dominant the base in relation to the length of the villus, the more ths particle moves toward the anode.  Fig. 2) This fraction was shown to contain a high specific activity of Na-K-ATPase, which is a typical plasma membrane marker The electron mmrographs of the negatively stained specimen showed, almost exclusively, large membranous sheets with bright borders In some specimens a regular pattern on the surface could vaguely be discerned, and on rare occasions, regularly arranged knobs on the limiting border In contrast to the microvilli, the membranes from this fraction could be stained much better with 2 % phosphotungstate, pH 7 2, than with ammonium molybdate. Staining experiments at different temperatures have not been conducted with this membrane (18). Micrographs of thin sections of this membrane also presented the typical morphology which is known for plasma membranes i e large open trilammar membranes with occasional granular content and with junctional complexes Relatively short tight junctions occurred quite often, but not desmosomes Again, no other cell particles could be found, ontv very few microvilli were  l~G v~ 7 Membranes of the basal infoldings of the proximal tubule cell purified by preparative freeflow electrophoresis (fraction ~0--~28 from ~'ig. 2). The insert in ~ig. 7 a shows a relatively short tight junction. Fig. 7 a, thin section. X 48,000; insert, X 70~000. ~ig. 7 b~ negative staining X 50~000. present, and only those with relatively large base m e m b r a n e pieces in comparison to their length

U N I D E N T I F I E D VESICLES:
In some experiments, large empty vesicles could be detected in FF4 fractions 35-38 (Fig 8). These vesicles were slmltar in size to lysosomes (19). No external surface layer could be detected, but at the inside ot the m e m b r a n e , a coat was present which varied in thickness In these "vesicle positive" experiments, acid phosphatase activity was posiuve not only in those fracuons in which lysosomes were present b u t also in fractions 35-38 (Fig. 8) However, histochemical experiments which could possibly demonstrate the acid phosphatase a c m u W within the vesicles have not yet been conducted T h e image of these vesicles resembled t h a t of outer mitochondrial membranes which could have been formed during the preparation of the crude brushborder fraction This possiblhty can be excluded since the ratio of the specific activities for succinate dehydrogenase/monoamine oxydase was 4 X 10 -4. Thls is the same ratio found in intact mitochondria, b u t the activities were, of course, extremely low. In addition, outer m i t o c h o n d n a t m e m b r a n e s never show any kind of coats, either on their outer or on their inner side.

Flow Diagram
In addition to the investigations on the distribu-Uon of mlcrovilli a n d plasma m e m b r a n e s in the FF4, the elecu-ophoretic behavior of other cell organelles in kidney was studmd on the basis of their marker enzymes and morphology. It should be emphasized that all the mltochondria, lysosomes, a n d vesmles from the endoplasmic reticulum demonstrated the same electrophoredc mobflities as those found for the rat hver organelles (20). Therefore the results on rat kidney are presented only schematically (Fig. 9). Although a strong overlap of organelles and membranes can be recognized, it is apparent that a large part of the plasma membranes of the basal infoldings and a still larger part of the microvilli must be considered as being homogeneous. Also, the plasma membranes of rat liver have the same electrophorefic properties as the plasma membranes of the basal infoldings from the renal proximal tubule cell. 8

DISCUSSION
It has been shown in recent investigations (11), (20), (21), (22) 3 that the technique of preparative free-flow electrophoresis is capable of solving problems which cannot be treated by other methods One important condition for a successful application of this relatively new separation technique is a Heidrich, H -G. To be published. different electrical behavior of the materials to be purified. Therefore, in the separation of biological membranes, it is necessary that the membranes have different electrical surface charges. In most cases, this varying surface behavior appears to be connected with a functional difference. For theoretical reasons, differences in the functional behavior have been postulated for the brushborder membrane and the membrane of the basal infoldings (23). Corresponding structural differences have been found by microdissection (94) and by histochemical (25) and radioautographic (26) studies. However, satisfactory isolation and characterization of the functionally different areas of the cell membrane have not been achieved. Nearly all the preparations described previously and designated as brush-border fraction also contained membranes from the basal part of the cell.

T~E JOVR~A~ OV C~LL BIOLOGY • VOLU~E 54, 1972
In these preparations, the enrichment of alkaline phosphatase and Na-K-ATPase occurs at almost the same rate. However, intimations of a possible separation of these two enzyme activities have recently been presented by Kinne et aI. (10), using density gradmnt techniques, and also by M¢ller and Rostgaard. 4 In both investigations, clear but unsatisfactory separations of the two marker enzymes present an the brush-border fractions have been shown. The density difference of the membranes carrying the two enzymes is perhaps too small to accomplish a complete separation via density gradient techniques. With these considerations in mind, the preparation method described above was devised which met all the requirements for electrophoretic separation The presence of larger particles, such as brush-border fragments, in the starting suspension was avoided as much as possible. Almost only single microvilli and free plasma membranes from the proximal tubule cell were used. Apparently, this kind of preparation is possible in rat kidney since a "lifting-off" from the apical and the basal part of the cell can occur quite easily in this organ (1) in comparison to the intestine The surface charges of these microvilh and membranes from the basal infoldings appear to be sufficiently different to permit a separation via electrophoresis. Somehow these differences in the membranes can be already recognized in the electron microscope by previous staining of the membranes with ammonium molybdate Microvilli are irregularly coated with particles. Similarly conspicious particles cannot be detected with ammonium molybdate in the basal plasma membranes of the proximal tubule cell.
In addition, the marker enzymes for the renal brush border have been more completely specified by-this work. Alkaline phosphatase and Na-K-ATPase are, in general, useful as markers for a brush-border fraction. The results presented here have clearly shown that two main groups of particulate material are obtained out of a brushborder fraction with electrophoresis. Since these two fractions correspond to two markedly different morphologies and to two different enzyme distributions, a specification of the enzyme markers can be made. Alkaline phosphatase and Na-K-ATPase have been established as markers for the different areas of the cell envelope of the renal proximal tubule cell. Alkaline phosphatase is re-4M¢ller, O. J., and J l~ostgaard. July 1971. Personal communication.
tained only in the microvilli, whereas Na-K-ATPase is a constituent of the basal infoldings of this cell The question whether it is useful to consider alkaline phosphatase as a marker for renal brush border in animals other than rat was not investigated. Berger and Sacktor (3), for example, have shown that, in rabbit kidney, trehalase is a better marker than alkaline phosphatase. This enzyme was not studied in the present paper.
The lack of alkaline phospharase or Na-K-ATPase in one of the two fractions discussed here cannot be due to isolation methods which resulted in extraction of the enzymes. The homogenization methods, and particularly the isotonic isolation and electrophoresis medium, are considered to be gentle tools for cell fractionation and have been proven, in several previous studies (11), (20), (22) "~ not to alter membranes. In the present work, this was verified by reelectrophoresis experiments, in wbich the resulting membranes were found to still have the same surface properties as in the first run. In addition, the morphological appearance, as seen in the electron microscope, was not altered by the separation methods. That is, the microvilli kept their coats, were closed at their upper ends, and were not broken or squeezed. Also, the membranes of the basal infoldings looked unaltered and represented the morphology which is described for the classical plasma membranes (7) The sharp triple-layered membrane of the large open membrane sheets, and especially the tight junction, are typical features of a plasma membrane well preserved during the purification procedure Again, desmosomes and mixed junctions were not very often present in the material investigated here.
An important question to be discussed is whether these plasma membranes were realIy the basel part of the proximal tubule cell or whether they might have been derived from other cells of the kidney. For the preparation of the membranes, only the renal cortex was used, and since this structure can easily be separated from the medullary parts of the organ, contamination with other parts of the kidney can be excluded. Consequently, m the cortex homogenate, only glomeruli and connective tissue with capillaries were present in addition to the tubular elements. Since the connective tissue and the glomeruli were not disintegrated by the homogenization methods used, all these structures are lost in the first centrifugation steps of the isolation procedure. The tubular elements generally consist of the proximal and the distal tubules, and the proximal tubule is four times more abundant than the distal tubule (27). A similar ratio can be calculated on the basis of morphometric data. There can be no doubt therefore, that a major portion of the material consisted of proximal tubule cell elements.
The nature and function of the vesicles described above should atso be considered. It is clear that these vesicles represent a type of membrane which is different from microvllli and membranes of the basal infoldings They are most hkely derived from the proximal tubule cells. Thoenes (28) has described particles of similar size and morphology in earlier studies and has designated them as prelysosomal vesicles, functioning in the pinocytotic processes. These particles are positive in acid phosphatase activity, as were those found in the work reported here. The reason that these elements appear in larger amounts in some experiments and not others is still an unanswered question. There might be a chance of obtaining higher amounts of these vesicles via FF4 after inducing the formation of these particles by an application of dextran to the animals (29). The other acid phosphatasecontaining particles (the lysosomes) function as storage organelles and can also be found m the kidney, but to a much lesser extent than in other organs. However, those which have been isolated during this study have the same morphologicai appearance and electrophoretic mobility as those derived from rat liver. This means that they are completely different from the vesicles of interest in this investigation.
The specific localization of two different marker enzymes onto different plasma membrane regions of the same cell (the proximal tubule cell) leads to the consideration of the specialization and differentiation of this celI envelope It has been described that tbe plasma membrane in mammalian ceUs can vary in thickness. 5 Still more strlking and of more importance for the function of the cell and its membrane must be the variation of the surface. Cell coats of a thickness between 100 and 5000 A, especially in microvilli, have been reported (30). In the present study, a clear indication for the specialization within the cell envelope has been obtained, but no information is available concern-5 For a diagrammatical represer~tation of several cell membrane structures see P. ing at what level of the cell development this occurs The whole enveloping membrane contains Mg-ATPase This may be considered as a hint for a common genotyplc precursor of this membrane This precursor must develop into a membrane of different phenotype, in which tile apical part (microvllli) possesses alkaline phosphatase and the above mentioned coat, whereas the basal part (membranes of the basal infoldings) contains the Na-K-ATPase, a key enzyme for active transport (31), (32). The latter enzyme is being considered as a general marker and constituent of a mammalian plasma membrane Almost nothing is known about the ontogenetic development of this enzyme in kidney. It was shown in a study by Beyth and Gutman (33) that during development of mice the renal cortex contains an almost constant level of Mg-ATPase ,activity, whereas the Na-K-ATPase is continuously increased and doubled at the end of the development. This is strong evidence for a specialization of the plasma membrane in this part of the organ. Whether Na-K-ATPase is synthesized in all the regions of the cell envelope, whether it has been possibly lost from the region of the microvilli during cell development, or whether it is still present in an inactive, repressed form should be clarified As to the coat on the surface of the microvilli, this has been found to be created during cell development by the cell itself via the Golgi apparatus (34). However, since alkaline phosphatase has been shown to be contained not only m the coat but also in the membrane of the microvilli (35) as a real constituent, investigation should be carried out to determine if this enzyme is prohferated only into the apical part of the cel] Such differentiation processes of the membrane should be triggered, either by a direct enzymatic induction (repression or derepression of active protein molecules) or by a very complex heterotypic cellular interaction (with alterations in morphogenesis, proliferation, and protein synthesis as consequences). Upon those two phenomena a study of membrane differentiation should be focused. The present blologmal system in combination with the techniques used might make it possible to obtain answers on the embryologic (ontogenefic) development of the renal proximal tubule cell envelope (23)