Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

Isolation of tubular proximal cells of the human kidney by affinity chromatography using Lotus tetragonolobus lectin

An affinity chromatography technique, using a lectin issued from Lotus tetragonolobus as ligand, and coupled to Sepharose 6MB support, has been chosen to enrich a cellular population in tubular proximal cells. Cell desorption was obtained with a competitive sugar, L Fucose. Intensive enzymatic brush border activity, shown in selected cells compared with initial cellular mixture, evidenced an enrichment in proximal tubular cells. These results prove that lectin use as ligand in affinity chromatography permits the separation of renal cell populations and presents the advantage that the bound cells can be recovered by a gentle competitive elution for further cultures.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.     

Ischemia in the isolated erythrocyte-perfused rat kidney. Protective effect of hypothermia

We have examined the effects of 25 min of ischemia in the isolated erythrocyte-perfused rat kidney (IEPK). We have previously shown that, in this model, perfusate flow rate is close to blood flow rates in vivo and morphology is normal. The functional and morphological consequences of both warm ischemia (at 37 degrees C) and ischemia induced during mild hypothermia (27 degrees C) were compared. (1) Warm ischemia resulted in a 51% increase in renal vascular resistance (RVR) during the reflow period, while glomerular filtration rate (GFR) was reduced to 24% of control levels. (2) Kidneys subjected to warm ischemia showed marked morphological damage localized to the proximal tubule. There was dilatation of the proximal segments and widespread loss of the proximal brush border due both to shedding into the lumen and interiorization into the cell. In contrast to the proximal tubular damage, the cells of the medullary thick ascending limb segments were intact. However, the lumena of many of these segments were filled with cytoplasmic blebs and necrotic cell debris. There was also pronounced vascular congestion of the capillary plexus in the inner stripe of the outer medulla. (3) Hypothermia to 27 degrees C resulted in almost complete protection against ischemic injury: RVR and GFR were not different from control values. Also, kidneys subjected to cold ischemia showed only isolated areas of mild brush border damage; no evidence of tubular obstruction or vascular congestion was present. (4) Thus, warm ischemia in the IEPK results in functional and morphological effects comparable to those found in vivo. Post-ischemic vasoconstriction as well as medullary congestion occur in the absence of systemic hormones and renal nerves. These consequences of ischemia are prevented by modest hypothermia.     

Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate

An iron chelate, ferric nitrilotriacetate (Fe3+-NTA), is nephrotoxic and also carcinogenic to the kidney in experimental animals. Iron-promoted lipid peroxidation in the proximal tubules is thought to be responsible for the pathologic process. In the present study, iron-promoted lipid peroxidation, with thiobarbituric acid (TBA) formation as an indication, in the tubular surface was simulated in vitro using rat kidney brush border membrane vesicles and the results were compared with those using linoleate micelles and rat liver microsomal lipid liposomes. Addition of ascorbate, cysteine, or dithiothreitol to the Fe3+-NTA solution resulted in consumption of dissolved oxygen and promoted the lipid peroxidation in the micelles and in the liposomes. In contrast, addition of glutathione to the Fe3+-NTA solution caused only sluggish oxygen consumption and far less peroxidation in these lipid systems. When the brush border membrane vesicles were used for the peroxidation substrate, Fe3+-NTA and glutathione could promote TBA formation at a rate comparable to that elicited by Fe3+-NTA with cysteine or dithiothreitol. Acivicin, a gamma-glutamyl transpeptidase inhibitor, suppressed the peroxidation of the brush border membrane vesicles promoted by Fe3+-NTA and glutathione. These results suggest the following mechanism of proximal tubular cell lipid peroxidation promoted by Fe-NTA: Fe3+-NTA filtered through glomeruli is rapidly reduced by cysteine and Fe2+-NTA starts lipid peroxidation at the site, leading to proximal tubular necrosis. Cysteine is amply supplied by the decomposition of glutathione within the lumen by the action of gamma-glutamyl transpeptidase and dipeptidase situated at the proximal tubular brush border membrane.     

Primary cultures of renal epithelial cells from X-linked hypophosphatemic (Hyp) mice express defects in phosphate transport and vitamin D metabolism.

Mutation in a gene (symbol Hyp) on the X chromosome causes hypophosphatemia in the mouse. The murine phenotype is a counterpart of X-linked hypophosphatemia in man. Both exhibit impaired renal reabsorption of phosphate in vivo. In vitro studies in the Hyp mouse have shown decreased Na+-dependent phosphate transport at the brush border membrane and abnormal mitochondrial vitamin D metabolism. To determine whether the mutant renal phenotype is intrinsic to the kidney or dependent upon putative extrinsic humoral factor(s) for its expression, we established primary cultures of renal epithelial cells from normal and Hyp male mouse kidneys. The cells are derived from proximal tubule. Initial uptake rates of phosphate and alpha-methyl-D-glucopyranoside (alpha-MG), a metabolically inert analogue of D-glucose, were measured simultaneously in confluent monolayers exhibiting epithelial polarity and tight junctions. The mean phosphate/alpha-MG uptake ratio in Hyp cultures was 82% of that in normal cells (P less than 0.01, n = 96). Moreover, the production of 24,25-dihydroxyvitamin D3 was significantly elevated in confluent cultures of Hyp cells relative to normal cells. These results imply that the Hyp gene is expressed in situ in renal epithelium and suggest that humoral factors are not necessary for the mutant renal phenotype in X-linked hypophosphatemia of mouse and man.     

Passive Heymann nephritis induced by rabbit antiserum to membrane antigens isolated from rat visceral yolk-sac microvilli

Passive Heymann nephritis (PHN) is an animal model of immune-complex-induced renal disease resembling human membranous glomerulonephritis. It was induced in rats by injecting rabbit antiserum directed against glycoprotein antigens isolated from rat embryonic visceral yolk-sac microvilli (VYS-MV). The glycoprotein antigens were isolated by extracting the VYS-MV with detergent Nonidet P-40 followed by gel filtration in Sephacryl S-300 and finally by lectin affinity chromatography with Ricinus communis agglutinin I. In vitro immunofluorescent localization studies demonstrated that the nephritogenic antibodies were localized along the apical region of the visceral yolk-sac endodermal cells and the brush border of the proximal tubular cells of the kidney. Rats injected with a single dose of the antiserum manifested proteinuria. Indirect immunofluorescent studies showed that the injected rabbit IgG was localized in vivo along the capillary walls of the glomerulus in a granular fashion. Electron microscopic examination of the same kidney glomeruli revealed numerous electron-dense deposits along the lamina rara externa of the glomerular basement membrane. Fusion of the epithelial foot processes was also present. These findings represent the typical immunopathological characteristics of Heymann nephritis. Furthermore, with the aid of Ouchterlony analysis, the antiserum against the isolated VYS antigens exhibited an immunoprecipitin band which was in common with that formed by the antiserum against the homogeneous nephritogenic antigen (gp330) of renal brush border origin. Thus, the nephritogenic antigens which have been found to be associated with the brush border of the renal proximal tubules may also be present or cross-reacted in the microvilli of the rat embryonic visceral yolk-sac.     

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics

Summay Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology. This paper was resented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by grants GM 07145, The Johns Hopkins Center for Alternatives to Animal Testing, and a Sigma Xi Grants-in-Aid of Research Award.     

Isolation of membrane-bound renal kallikrein and kininase.

Fractions highly enriched in plasma membrane, endoplasmic reticulum or brush border were prepared from homogenized rat kidney cortex. Kallikrein was concentrated in the plasma-membrane fraction, but not in the brush border of the proximal tubules. Kininase II or angiotensin I-converting enzyme was localized in the brush-border membrane. It is suggested that kallikrein in the urine may originate from the plasma membrane of the distal tubules and the conversion of angiotensin I and the inactivation of bradykinin may occur on the lumen membrane of the proximal tubular cells.     

Changes in expression and subcellular localization of annexin IV in rabbit kidney proximal tubule cells during primary culture

In the present study, we investigated the polarized expression of annexin IV at various stages in the growth of rabbit kidney proximal tubule cells (PTC) in primary cultures. The results of immunoblotting analysis and indirect immunofluorescence studies using a specific anti-annexin IV monoclonal antibody, indicated that annexin IV is expressed in proximal tubule cultured cells, although it was not detected in the proximal tubules present in frozen sections of kidney cortex and freshly isolated proximal tubule cells. In either non-confluent or confluent cells which remained attached to the collagen-coated support, annexin IV was mainly concentrated around the nucleus, whereas in PTC forming the monolayer of domes, it was restricted to the basolateral membrane domain. This basolateral localization was identical to that observed in other polarized epithelial cell types such as enterocytes. When the domes burst, the cells returned to the collagen-coated support and the annexin IV was again localized around the nuclei. The fact that the change of localization was very rapid suggested the existence of a considerable difference between the differentiation states of dome forming and adherent confluent cells. Moreover, a transient association of annexin IV with the basal body of apically located cilia also seemed to be correlated with a particular polarization state and/or differentiation states of adherent cultured cells, corresponding to the beginning of the polarized expression of aminopeptidase N, a hydrolase located in the apical brush border membrane, and to the falling of cells onto the support, subsequent to the bursting of the domes. In conclusion, these results provide evidence that annexin IV may constitute a new marker of the basolateral membrane domain of polarized epithelial renal cells in primary cultures. © 1995 Wiley-Liss, Inc.     

Antigenic expression of aminopeptidase M,dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Summary Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured, to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occured during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occured only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Antigenic expression of aminopeptidase M, dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

THE COLUMNAR CELLS OCCURRING IN THE PARIETAL LAYER OF BOWMAN''S CAPSULE : Cellular Fine Structure and Protein Transport

The renal corpuscles of adult, C3H Swiss, male mice contain testosterone-sensitive, columnar cells in the parietal layer of Bowman's capsule. A study of the normal fine structure of these cells reveals several distinctive characteristics: a microvillous brush border; apical tubular invaginations and apical tubules; an elaborate infolding of the basal surface membrane forming cellular compartments, which contain numerous mitochondria; and a complex group of membrane-limited cytoplasmic inclusions. This appearance is remarkably similar to the fine structure of cells in the proximal convoluted tubule. 1 hr after an in vivo injection of horseradish peroxidase, numerous protein-absorption droplets occur in the columnar cell cytoplasm. The speed and cytomorphology of protein transport by these capsular cells closely resemble the handling of peroxidase by the proximal convoluted tubule. Origins for these testosterone-sensitive cells are discussed briefly. Morphological evidence is presented for the differentiation of squamous cells in Bowman's parietal capsule into columnar cells, which appear structurally and functionally identical with proximal convoluted tubular epithelium.     

The alpha macroglobulins of rat serum.

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..     

Apical trehalase expression associated with cell patterning after inducer treatment of LLC-PK1 monolayers

Trehalase, a differentiation-specific marker of renal proximal tubule brush border membrane, is expressed in confluent long-term cultures of the renal epithelial cell line LLC-PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membrane-associated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64-73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC-PK1 cultures after HMBA treatment is localized in cells forming a three-dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100-110 kD both in LLC-PK1 cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'-dimethylformamide (DMF) and equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound-repair of the monolayer. In addition to the membrane-bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers. These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.     

Localization of dehydropeptidase-I, an enzyme processing glutathione, in the rat kidney

An antibody prepared against dehydropeptidase-I, one of the glutathione processing enzymes, from the renal membrane fraction of rats was employed to localize at the light microscopic level the enzyme in the kidney using the avidin/biotin-peroxidase complex method. Dehydropeptidase-I was found to be present on both the brush border and the basolateral membranes of proximal tubular cells. Furthermore, the enzyme activity in the isolated brush border and basolateral membrane fractions was also determined. Distribution profiles of the enzyme activity showed good agreement with the results of the immunohistochemical observations.     

Expression of sodium-linked nucleoside transport activity in monolayer cultures of IEC-6 intestinal epithelial cells

Mature, confluent monolayer cultures of IEC-6 rat intestinal epithelial cells in conventional growth media express both Na(+)-linked, concentrative nucleoside transport (NT) activity and equilibrative, inhibitor-sensitive NT activity, but do not show morphologic differentiation. Na(+)-dependent fluxes of Ado and formycin B were minor in early subconfluent IEC-6 monolayers, but increased severalfold to become the major component of influx of these agents in confluent monolayers grown in medium containing Nu-Serum, a commercial medium supplement with a low serum content. In monolayers cultured in medium with fetal bovine serum, cell proliferation rates were similar to those in medium supplemented with Nu-Serum, but expression of Na(+)-linked NT activity was 6-8-fold lower than in monolayers grown in the latter medium. Inclusion of hydrocortisone in growth medium with Nu-Serum caused a 2-fold increase in the expression of Na(+)-linked NT activity. Experiments in which components of medium supplementation were withheld showed that insulin and epidermal growth factor were important in expression of the Na(+)-linked NT activity. Because the Na(+)-linked NT system has a brush border location in fresh intestinal epithelium, it is concluded that the regulated expression of this activity in the IEC-6 monolayers is a differentiative change.     

Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration

We investigated in vivo catabolism of apolipoprotein A-II (apo A-II), a major determinant of plasma HDL levels. Like apoA-I, murine apoA-II (mapoA-II) and human apoA-II (hapoA-II) were reabsorbed in the first segment of kidney proximal tubules of control and hapoA-II-transgenic mice, respectively. ApoA-II colocalized in brush border membranes with cubilin and megalin (the apoA-I receptor and coreceptor, respectively), with mapoA-I in intracellular vesicles of tubular epithelial cells, and was targeted to lysosomes, suggestive of degradation. By use of three transgenic lines with plasma hapoA-II concentrations ranging from normal to three times higher, we established an association between plasma concentration and renal catabolism of hapoA-II. HapoA-II was rapidly internalized in yolk sac epithelial cells expressing high levels of cubilin and megalin, colocalized with cubilin and megalin on the cell surface, and effectively competed with apoA-I for uptake, which was inhibitable by anti-cubilin antibodies. Kidney cortical cells that only express megalin internalized LDL but not apoA-II, apoA-I, or HDL, suggesting that megalin is not an apoA-II receptor. We show that apoA-II is efficiently reabsorbed in kidney proximal tubules in relation to its plasma concentration.     

Activity of calcium/phospholipid dependent protein kinase during rat kidney development

The activity of the calcium sensitive phospholipid dependent protein kinase C (PKC) was studied in cytosol and in the proximal tubular luminal membrane of rats during growth. Cytosolic activity was elevated at 14 and 21 days of age and fell to adult levels by day 60. Luminal brush border membrane activity on the other hand was low on day 14 but reached adult levels by day 21. Changes in brush border membrane PKC activity may have important consequences for the development of electrolyte transport in proximal tubular cells.