Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Cytoskeletal proteins of the rat kidney proximal tubule brush border

Cytoskeletal components backing the brush border of the rat kidney proximal tubule cell were identified and compared with those of the well characterized intestinal brush border by immuneoverlay and immunocytochemistry. Antibodies reactive against the intestinal microvillus core components, villin and fimbrin, as well as against the terminal web components, spectrin (fodrin) and myosin, were used. Proteins of similar molecular weight to these intestinal brush border cytoskeletal components were identified in isolated kidney brush borders by immuneoverlay. Spectrin, a major component of the terminal web region of both cell types, was more concentrated in the kidney brush border relative to both actin and myosin. By immunofluorescence, villin and fimbrin were localized in the microvilli, and spectrin and myosin were localized to the terminal web region of the brush border. In addition, spectrin was found along the basolateral membranes of the proximal tubule cell, and myosin was detected in a punctate staining pattern throughout its cytoplasm. By immunoelectron microscopy using immunogold labeling procedures, fimbrin and villin were localized in the terminal web as well as in microvilli, and spectrin and myosin were localized to fibrils in the terminal web. A key difference between the epithelia of the two organs is the extensive network of clathrin coated pits found in the terminal web region of the kidney but not the intestinal brush border. The clathrin-rich terminal web region of the kidney, like the intestinal brush border, proved to be quite stable and resistant to disruption by non-ionic detergents and harsh mechanical treatment.     

Ba2+-sensitive potassium permeability of the apical membrane in newt kidney proximal tubule

Summary The apical membrane K⁺ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V _a change) during alteration of luminal K⁺ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V _a change/decade for K⁺ was 50 mV at K⁺ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K⁺ concentration was increased from 2.5 to 25mm. Ba²⁺ (1mm in the lumen) reducedV _a change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba²⁺-sensitive K⁺ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K⁺ activity measured by K⁺-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K⁺ across both cell membranes. Thus, it is concluded that cell K⁺ passively diffuses, at least in part, through the K⁺ conductive pathway of the apical membrane.     

Sex different cytochrome-c uptake in the proximal tubule of the rat kidney

The present study deals with the dose- and time-dependent uptake of cytochrome c (CYT c) in the proximal tubule of the rat kidney, and shows that there are segment and sex differences in the reabsorption of CYT c. Rats of both sexes were intravenously injected with different doses of CYT c (0.75-9.0 mg per 100 g body weight), and the kidneys were investigated by light and electron microscopy at different times (3 min, 10 min, and 2 h) after the injection. After 3 and 10 min, CYT c was demonstrated in apical vacuoles of different sizes and in some lysosomes of the S1 and S2 segments, whereas after 2 h, CYT c was found only in lysosomes of all three segments of the proximal tubule. At these times, the S1 segment contained more CYT c than the S2 and S3 segments. However, 2 h after the injection of 6 or 9 mg CYT c, the differences between the S1 and S2 segments disappeared almost completely, due to a strong lysosomal accumulation of CYT c in the S2 segment. At all studied times and CYT-c doses, the S3 segment contained less CYT c than the S1 and S2 segments. On the whole, different levels of CYT-c reabsorption were found in the different segments of the proximal tubule, which was saturable with increasing CYT-c doses, i.e. firstly in the proximal and then in the distal parts of the proximal tubule. Two hours after the injection of CYT c, a difference between males and females was observed, with the lysosomes of the S1 and S2 segments of females containing more CYT c than those of males. Thus, more CYT c was reabsorbed in the proximal tubule of females than in that of males.     

Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.     

Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury

Summary Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated γ-glutamyltranspeptidase in >95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectinArachis hypogaea (rat proximal tubule) and negatively forLotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, γ-glutamyltranspeptidase and leucine aminopeptidase, and Na⁺-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium. This project was supported by grant 2-R01-DK15440-16A1 from the National Institutes of Health, Bethesda, MD, and by grant N0001 4-88-K-0427 from the Department of the Navy, Washington, DC.     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Ultrastructural observations on the pars descendens of the proximal tubule in the kidney of the male rat

Summary The pars descendens (pars recta) of the proximal tubule in the male rat kidney, consisting of the terminal part of the second proximal segment (P₂) and of the third proximal segment (P₃), was studied with the electron microscope. A technique of tissue orientation and trimming was used which permitted precise topographic definition of the tubules studied in the electron microscope. The terminal descending part of the P₂ showed some minor differences from the convoluted part of this segment, and ultrastructure also changed along the course of the P₃. In the beginning of the latter segment numerous, shallow interdigitations were observed between adjacent cells; along the course of the segment they decreased in number or disappeared. In the initial part of the P₃ mitochondria were more abundant than in the terminal portion of the segment and at least as numerous as in the straight part of the P₂. Also, the dense, acid phosphatase-positive cytoplasmic bodies decreased somewhat in size along the course of the P₃. The smooth surfaced endoplasmic reticulum reached a higher development in the P₃ than anywhere else in the proximal tubules.Investigation supported by grants from: Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — The authors are indebted to Mrs. J. Barslund and Mrs. M. Jacobsen for excellent technical assistance.     

Alterations in intermediate filament proteins in rat kidney proximal tubule epithelial cells

Changes in the intermediate filament composition of rat kidney proximal tubule cells in culture have been investigated. The data suggest that differentiated tubular epithelial cells do not express vimentin, but vimentin expression is induced when the cells begin to proliferate in culture. The cultured cells are positive for both cytokeratins and vimentin by immunofluorescence microscopy. The data support the concept that the intermediate filament composition of proximal tubule epithelial cells can be altered during proliferation induced by nephrotoxic chemicals or by neoplastic transformation.     

Conductive properties of the proximal tubule in Necturus kidney

The electrical properties of the proximal tubule of the in vivo Necturus kidney were investigated by injecting current (as rectangular waves) into the lumen or into the epithelium of single tubules and by studying the resulting changes of transepithelial (VL) and/or cell membrane potential (VC) at various distances from the source. In some experiments paired measurements of VL and VC were performed at two abscissas x and x'. The luminal length constant of about 1,030 micrometer was shown to provide a good estimate of the transepithelial resistance, specific resistance (RTE = 420 omega.cm2) and/or per unit length (rTE = 1.3 x 10(4) omega.cm). The apparent intraepithelial length constant was subject to distortions arising from concomitant current spread in the lumen. The resistances of luminal membrane (rL), basolateral membrane (rB), and shunt pathway (rS) were estimated by two independent methods at 3.5 x 10(4), 1.2 x 10(4), and 1.7 x 10(4) omega.cm, respectively. The corresponding specific resistances were close to 1,200, 600, and 600 omega.cm2. There are two main conclusions of this study. (a) The resistances of cell membranes and shunt pathway are of the same order of magnitude. The figure of the shunt resistance is at variance with the notion that the proximal tubule of Necturus is a leaky epithelium. (b) A rigorous assessment of the conductive properties of concentric cylindrical double cables (such as renal tubules) requires that electrical interactions arising from one cable to another be taken into account. Appropriate equations were developed to deal with this problem.     

The proximal tubule in the pathophysiology of the diabetic kidney

Diabetic nephropathy is a leading cause of end-stage renal disease. A better understanding of the molecular mechanism involved in the early changes of the diabetic kidney may permit the development of new strategies to prevent diabetic nephropathy. This review focuses on the proximal tubule in the early diabetic kidney, particularly on its exposure and response to high glucose levels, albuminuria, and other factors in the diabetic glomerular filtrate, the hyperreabsorption of glucose, the unique molecular signature of the tubular growth phenotype, including aspects of senescence, and the resulting cellular and functional consequences. The latter includes the local release of proinflammatory chemokines and changes in proximal tubular salt and fluid reabsorption, which form the basis for the strong tubular control of glomerular filtration in the early diabetic kidney, including glomerular hyperfiltration and odd responses like the salt paradox. Importantly, these early proximal tubular changes can set the stage for oxidative stress, inflammation, hypoxia, and tubulointerstitial fibrosis, and thereby for the progression of diabetic renal disease.     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Summary Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluores-ceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence mciroscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.In honour of Prof. P. van DuijnSupported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluoresceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence microscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S1/S2 segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 microns. Maximal DAP II activities were demonstrated at pH 5.5. The Km of DAP II was about 2.3 mM. In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S1/S2 segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

Summary The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S₁/S₂ segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 m. Maximal DAP II activities were demonstrated at pH 5.5. The K _m of DAP II was about 2.3 mM. — In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S₁/S₂ segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. T.H. Schiebler, Chairman of the Institute of Anatomy of the University of Würzburg, on the occasion of his 60th birthday     

Separation of proximal tubule cells from suspensions of rat kidney cells by free-flow electrophoresis

Suspensions of rat kidney cells obtained by disaggregation of the kidney with 0.25% trypsin were separated by electrophoresis. Previously, we found a correlation between cells with histochemically demonstrable alkaline phosphatase (HDAP) and cells with brush borders which established that HDAP is a useful marker for rat proximal tubule cells (Kreisberg et al., '77). The starting suspension of cells for electrophoresis consisted of 38.4 +/- 5.7% nucleated cells with HDAP, 39.8 +/- 5.7% nucleated cells without HDAP, and 20.8 +/- 9.2% red blood cells. After electrophoresis, the purest fraction contained 85.8 +/- 3.5% nucleated cells with HDAP, 8.4 +/- 2.2% nucleated cells lacking HDAP, and 5.8 +/- 2.8% red blood cells; 91.9 +/- 2.4% of the nucleated cells in the purest fractions had HDAP.     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.