Structural adaptation to altered electrolyte metabolism by cortical distal segments

The four different renal cell types in the cortical segments beyond the macula densa--distal convoluted tubule (DCT) cell, the connecting tubule (CNT) cell, the principal (P) cell, and the intercalated (I) cell--each respond to prolonged specific stimuli with changes in cell membrane area. Increases in basolateral cell membrane area, reflecting the transport capacity of the cell, are associated in DCT cells with luminal, chronically high sodium load, in CNT cells with mineralocorticoids and tubular solute load, and in P cells with mineralocorticoids. In I cells the luminal cell membrane area seems to be influenced by the local luminal environment of the cells as well as by the peritubular environment. These structural findings indicate that tubular fluid composition as well as peritubular factors (e.g., mineralocorticoid levels) may have a role in regulating the transport capacity of distal cell types in the kidney.     

Structural adaptation of intercalated cells in rat renal cortex to acute metabolic acidosis and alkalosis

The structural responses of cells in the distal convoluted, connecting, and collecting tubule to acute acid/base changes were investigated by electron microscopy. Acute metabolic acidosis was induced by administration of ammonium chloride, and acute metabolic alkalosis by potassium or sodium bicarbonate. Morphometric analyses were performed on micrographs of randomly selected distal nephron cells. No structural responses were found in distal convoluted tubule cells, connecting tubule cells, or principal cells but prominent changes were observed in intercalated cells (I cells). Thus, the surface density of the luminal membrane in I cells was significantly higher in acidotic animals and lower in KHCO3 alkalotic animals than in controls. On the contrary, the surface density of the membrane that bounds apical vesicles was higher in KHCO3 alkalotic and lower in acidotic animals than in controls. These results suggest that the luminal membrane is internalized during alkalosis and that the membrane that bounds apical vesicles is transferred to the luminal membrane during acidosis. Since a proton translocating ATPase may be present in the luminal membrane the observations are consistent with the possibility that cortical I cells participate in the maintenance of acid/base homeostasis.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells, segment of the nephron preceding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. The close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Ultrastructure of distal nephron cells in rat renal cortex

Distal nephron segments in the rat renal cortex contain distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells), and principal cells (P cells). The present study was carried out to expand present knowledge on the ultrastructure of these cells. The cells were sampled from superficial cortex and analyzed by electron microscopy. Several morphometric parameters were determined and statistical comparison between cell types was performed. Significant structural differences between the cell types were demonstrated. DCT cells showed the highest volume density of mitochondria whereas the amplification of basolateral membranes was higher in CNT cells than in I and P cells. The surface density of the membrane that bounds intermediate vesicles in the apical cytoplasm was twofold higher in I cells than in the other cell types. The morphological differentiation found in the present study adds to available evidence indicating a functional differentiation between the cell types and provides a reference for structure-function correlations in these cells.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

Summary The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells segment of the nephron preceeding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. the close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Microheterogeneity of the collecting duct system in rabbit kidney as revealed by monoclonal antibodies

Summary This report describes the immunolocalization of three monoclonal antibodies along the collecting duct system in rabbit kidney. The antibodies were raised against antigens derived from a membrane fraction of homogenized papillary tissue. Western Blot analysis demonstrated that each of the antibodies recognized a single band of about 190000 (P_CD1), 210000 (P_CD2) and 50000 (P_CD3) daltons. In renal tissue, the antibodies bound specifically to the epithelia of the connecting tubule (CNT), the collecting duct (CD) and the papillary surface epithelium. Differences in the binding patterns of the antisera were limited to the cortex. p_CD1 labeled only a few scattered cells in the CNT, and exhibited a heterogeneous binding along the cortical collecting duct (CCD). P_CD2 and P_CD3 binding patterns were similar. In the CNT, these antibodies bound to the intercalated cells (IC-cells) but not to the CNT-cells proper. In the CCD, both IC-cells and principal cells were labeled. The binding to the medullary collecting duct by all three antisera was identical. The ureter was labeled only by P_CD2 and P_CD3, and none of the antisera bound to the bladder epithelium.The antibody binding patterns provide information concerning tubular axial heterogeneity and embryogenetic aspects of the CNT and the CCD. These antibodies may be used as differentiation markers in studies of the developing kidney and of renal tissue culture systems.These studies were supported by Deutsche Forschungsgemeinschaft, Forschergruppe Niere, Kr 546/5-1     

Immunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium

We examined the localization of fodrin in epithelial cells of rat uriniferous and collecting tubules by immunofluorescence and immunoelectron microscopy of frozen sections. In the uriniferous tubule, fodrin was found along the cell membrane and in the well-developed terminal web, as previously reported in other epithelial cells: in the terminal web and along the basolateral cell membrane in the proximal tubule; all around the cell surface in the thin limb of Henle; along the basolateral surface in the thick limb of Henle's thick segment and the distal tubule. In the intercalated cells of the collecting tubule, fodrin was found not only along the basolateral cell membrane but also in the apical cytoplasm. The most peculiar labeling was obtained in the principal cells of the collecting tubule. In addition to labeling in the basolateral cell membrane, fodrin was found diffusely in the cytoplasmic matrix. Association of fodrin with any particular structure could not be identified, but the Golgi area was apparently free of labeling. Cytoplasmic labeling was more conspicuous in the principal cells of the medulla than in those of the cortex. The present results show that fodrin need not always exist in association with the cell membrane or the cytoskeleton but can occur in the cytoplasmic matrix, at least in epithelial cells. We discuss the possible physiological significance of the latter distribution.     

Ultrastructural organization of the transition from the distal nephron to the collecting duct in the desert rodent Psammomys obesus

Summary The transition from the nephron to the collecting duct is formed by three tubular segments (convoluted part of the distal tubule, connecting tubule, cortical collecting duct), which in the desert rodent, Psammomys obesus, transform gradually from one segment to the next, due to intermingling of their different cell types.The convoluted part of the distal tubule (DTC) starts abruptly, shortly beyond the macula densa and initially is homogeneously composed of characteristic DTC-cells. Subsequently, the DTC-cells intermingle with intercalated cells. The first appearance of the connecting-tubule cell, which gradually replaces the DTC-cell, is regarded as the beginning of the connecting tubule. The major portion of the connecting tubule is lined by connecting-tubule cells and intercalated cells. The first appearance of the principal cell between them defines the beginning of the cortical collecting duct, which in the medullary ray is lined by principal and intercalated cells only.Each cell type is described in detail and discussed in relation to the assumed function of the tubular segments.Interspecies differences in the cellular composition of the transitional zone from the nephron to the collecting duct are discussed in relation to the different organization of the collecting duct system.     

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.     

Role of adrenal hormones in regulating distal nephron structure and ion transport

Mineralocorticoid levels are an important determinant of membrane area and ion transport in the renal initial (ICT) and cortical (CCT) collecting tubules. Adrenalectomy leads to a dramatic and specific decrease in basolateral membrane area of principal (P) cells and depresses sodium reabsorption and potassium secretion. Although aldosterone replacement for 10 days restores basolateral membrane area and ATPase activity to control levels and dramatically elevates ion transport, glucocorticoids have no effect on basolateral membrane area, ion transport, or ATPase. It is suggested that the aldosterone-induced amplification of membrane area occurs as a mechanism whereby cells increase the number of ATPase pumps in the basolateral membrane. An acute (of 2-3 h) increase in aldosterone, but not dexamethasone, also stimulates potassium transport by the ICT. Future studies will have to establish whether the acute stimulation of transport by aldosterone involves a change in basolateral membrane area. It is concluded that mineralocorticoids, but not glucocorticoids, regulate sodium and potassium transport by P cells of the ICT and CCT, in part, by determining the number of ATPase pumps available for transport.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

Summary The ultrastructure of the distal nephron, the collecting duct and the Wolffian duct was studied in a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) by transmission and scanning electron microscopy (TEM, SEM). The distal tubule (DT) is made up of one type of cell that has a well-developed membrane labyrinth established both by interdigitating processes and by interlocking ramifications. The processes contain large mitochondria, the ramifications do not. The tight junction is shallow and elongated by a meandering course. The connecting tubule (CNT) is composed of CNT cells proper and intercalated cells, both of which are cuboidal in shape. The CNT cells are characterized by many lateral interlocking folds. The intercalated cells have a dark cytoplasm densely filled with mitochondria. Their apical cell membrane is typically amplified by microplicae beneath which a layer of globular particles (studs) is found. The collecting duct (CD) is composed of principal cells and intercalated cells, again both cuboidal in shape. The CD epithelium is characterized by dilated intercellular spaces, which are often filled with lateral microfolds projecting from adjacent principal cells. The apical membrane is covered by a prominent glycocalyx. The intercalated cells in the CD are similar to those in the CNT. The Wolffian duct (WD) has a tall pseudostratified epithelium established by WD cells proper, intercalated cells and basal cells. The WD cells contain irregular-shaped dense granules located beneath the apical cell membrane. The intercalated cells of the WD have a dark cytoplasm with many mitochondria; their nuclei display a dense chromatin pattern.Research fellow of the Alexander von Humboldt Foundation     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600 X g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum. Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.     

Electron microscopic study of the innervation of the renal tubules and urinary bladder epithelium in Rana catesbeiana and Necturus maculosus

The fine structure of the kidney and the bladder of the bullfrog (Rana catesbeiana), the bullfrog tadpole, and the mudpuppy (Necturus maculosus) were studied with special attention to the innervation of renal tubule cells and bladder epithelial cells. In the bullfrog kidney, nerve terminals and varicosities were frequently associated with the tubule cells, apparently in an increasing order from the proximal tubule to the connecting tubule. Although these terminals and varicosities did not directly contact the tubular cell membrane, an aggregation of synaptic vesicles on the side facing the tubule was considered as morphological evidence that neurotransmitter can be released here and can affect the transport activity of the tubule cells. The association of nerve varicosities with canaliculi cells in the connecting tubule was also demonstrated. In the bullfrog tadpoles, renal tubule cells were occasionally innervated. In the mudpuppy, renal tubule cells were only poorly innervated. The epithelium of the bullfrog bladder was commonly innervated. Nerve terminals with synaptic vesicles were located very near basal cells and even contacted them directly on rare occasions. In the mudpuppy, the innervation of the bladder epithelium was observed infrequently. The bullfrog tadpoles did not possess an apparent bladder. In all materials studied, renal arterioles and bladder smooth muscle cells were innervated.     

L-glutamate transport in renal plasma membrane vesicles

Summary This review describes the uptake of L-glutamate by well-characterized preparations of renal brush border (luminal) and baso-lateral membrane vesicles derived from the plasma membrane of the polar proximal tubular cell. L-glutamate is taken up against its concentration gradient, from both sides, by co-transport systems in which the movement of the amino acid into the cell is coupled to the influx of Na⁺ and efflux of K⁺ down their respective electrochemical gradients. The presence of these ion gradient-energized systems, specific for L-glutamate, may account for the exceedingly high intracellular concentration of this metabolically important amino acid in the renal tubule.     

Differential effect of 1,25-dihydroxycholecalciferol on phosphoinositide turnover in the antipodal plasma membranes of colonic epithelial cells.

1,25-dihydroxycholecalciferol stimulates membrane phosphoinositide turnover in colonic epithelial and other cells, but the effects of this hormone on phosphoinositide metabolism in specific antipodal plasma membranes has not been examined. In the present studies, addition of 10(-8)M 1,25-dihydroxycholecalciferol to rat colonic crypts for 90 seconds decreased the phosphatidylinositol-4,5-bisphosphate content and increased the diacylglycerol content of the baso-lateral, but not the brush border plasma membrane. Using Caco-2 cells grown as tight polarized monolayers, 1,25-dihydroxycholecalciferol reduced cellular phosphatidylinositol-4,5-bisphosphate and increased cellular inositol-1,4,5-triphosphate and diacylglycerol when added to the buffer bathing the baso-lateral, but not the brush border membrane surface. These data indicate, therefore, that 1,25-dihydroxycholecalciferol activates the phosphoinositol signal transduction cascade specifically in the baso-lateral cell membrane of colonic cells.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Summary Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600×g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum.Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.     

Quantitative immunogold localization of Na,K-ATPase along rat nephron

Summary Ultrastructural localization of Na, K-ATPase -subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against -subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per m of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6–3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Interaction of mammalian kidney membrane and mitochondria in vitro

1. In many parts of the kidney tubule, there exists a close physical association of membrane and mitochondria apparently related to salt transport. The present study examines the influence of adding rat kidney membrane fragments on mitochondrial respiration in vitro. The results are compared with previous findings on the effect of rat kidney membrane fragments on cytoplasmic glycolysis.

2. The membrane fraction stimulates mitochondrial respiration. The stimulus decreases with higher levels of ADP and is abolished by dinitrophenol. Stimulus of glycolysis by kidney membranes is related to a specific coupling of membrane ATPase activity and phosphoglycerate kinase. The stimulus of respiration appears to be due solely to the enhanced availability of ADP in presence of an ATPase system.