Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

Ultrastructural location of albumin and fibrinogen in primary cultures of new-born rat liver tissue

In primary cultures of new-born rat liver tissue, albumin and frbrinogen, two proteins normally synthesized by the liver and secreted into plasma were demonstrated by specific antibodies labelled with peroxidase in about 50 and 70% of the hepatocytes; these proteins were not demonstrated in the other types of cells, in particular fibroblasts, present in primary cultures. These two proteins were detected on the ribosomes of the rough endoplasmic reticulum and were also present in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus. It is concluded that

1. 1. In primary cultures of liver tissue, only the hepatocytes synthesize albumin and fibrinogen.

2. 2. Proliferating cultured hepatocytes are able to synthesize albumin and fibrinogen.

3. 3. The presence of detectable albumin and fibrinogen in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in hepatocytes of primary cultures and their absence in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in the hepatocytes of adult rat liver might indicate an alteration in the translocation of albumin and fibrinogen through these organelles in cultured hepatocytes.

     

Glycocalyx heterogeneity of rat kidney urinary tubule: demonstration with a lectin-gold technique specific for sialic acid

The distribution of sialic acid residues in rat kidney urinary tubule was investigated by light and electron microscopy with a lectin-gold technique. The application of the sialic acid specific Limax flavus lectin resulted in intense plasma membrane labeling of the epithelium of the entire proximal tubule and thin limbs of loop of Henle. In contrast, the plasma membrane of the epithelium lining the medullary portion of the thick ascending limb of Henle was not labeled. In the cortical portion, however, microvilli-bearing positive and smooth-surfaced negative cells were present. Moreover, all cells of the convoluted distal tubules were labeled along their plasma membrane. These data demonstrate the existence of a gross difference in glycocalyx composition between proximal tubules and thin limbs of loop of Henle on one hand and thick ascending limbs on the other. In addition, fine heterogeneity in glycocalyx composition between medullary and cortical portion of thick ascending limb exists. It is concluded that the differences in sialic acid content of the glycocalyx may be related to the functional diversity exhibited by these tubular regions.     

Selenite metabolism in rat and human blood

The binding of selenite, the form of selenium used in the treatment of Keshan disease, to plasma proteins and the role of erythrocytes in this process have been studied. The experiments were carried out by incubating⁷⁵Se as selenite with plasma and whole bloodin vitro (human and rat) andin vivo (rat) and subsequent fractionation by Sephadex G-150 gel filtration. Human and rat plasma proteins were unable to incorporate selenium from selenite, as shown by the negligible amount present in proteins after incubation of plasma with selenite. The incorporation can be carried out after internalization of selenite by the erythrocytes in a fast, temperature dependent process. Hemoglobin, being the major binding protein for the newly reduced selenium in the erythrocyte, might have a role in the uptake of selenite by erythrocytes. The greater affinity of plasma proteins for the final selenium compound resulting from reduction could be the cause of the efflux. However, this is minimal in the absence of plasma, as is evident from the results obtained from blood reconstituted with saline solution instead of plasma. At least two proteins, one albumin like, probably albumin itself and the other of molecular weight close to or greater than 200,000 in the plasma, are involved in the binding and efflux processes.     

Immunohistochemical analyses on albumin and immunoglobulin in acute hypertensive mouse kidneys by "in vivo cryotechnique"

The purpose of this study is to visualize topographical changes of serum proteins, albumin and immunoglobulin, passing through mouse glomerular capillary loops and their reabsorption in renal proximal tubules by immunohistochemistry in combination with our "in vivo cryotechnique". The "in vivo cryotechnique" was performed on left mouse kidneys under normotensive, experimentally acute hypertensive and heart-arrest conditions. The cryofixed tissues by the technique were routinely processed for freeze-substitution. Serial deparaffinized sections were stained with hematoxylin-eosine and immunostained with anti-mouse albumin, immunoglobulin G (IgG), kappa or lambda light chain and IgG1 heavy chain antibodies. Under the normotensive and heart-arrest conditions, albumin and IgG were clearly immunolocalized in blood vessels and slightly in apical cytoplasmic parts of some proximal tubules. Under the acute hypertensive condition, the albumin and kappa or lambda light chains, but not IgG1 heavy chain, were strongly immunolocalized in the apical cytoplasm of almost all proximal tubules. This study is the first in vivo visualization for glomerular passage of serum proteins and their transtubular absorption. Thus, the "in vivo cryotechnique" with freeze-substitution can be used for clarifying not only the functional morphology of living animal cells, but also in situ immunohistochemical localization of their components.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation

Summary Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation.

Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

The role of the kidney in lipid metabolism

PURPOSE OF REVIEW: Cellular uptake of plasma lipids is to a large extent mediated by specific membrane-associated proteins that recognize lipid-protein complexes. In the kidney, the apical surface of proximal tubules has a high capacity for receptor-mediated uptake of filtered lipid-binding plasma proteins. We describe the renal receptor system and its role in lipid metabolism in health and disease, and discuss the general effect of the diseased kidney on lipid metabolism. RECENT FINDINGS: Megalin and cubilin are receptors in the proximal tubules. An accumulating number of lipid-binding and regulating proteins (e.g. albumin, apolipoprotein A-I and leptin) have been identified as ligands, suggesting that their receptors may directly take up lipids in the proximal tubules and indirectly affect plasma and tissue lipid metabolism. Recently, the amnionless protein was shown to be essential for the membrane association and trafficking of cubilin. SUMMARY: The kidney has a high capacity for uptake of lipid-binding proteins and lipid-regulating hormones via the megalin and cubilin/amnionless protein receptors. Although the glomerular filtration barrier prevents access of the large lipoprotein particles to the proximal tubules, the receptors may be exposed to lipids bound to filtered lipid-binding proteins not associated to lipoprotein particles. Renal filtration and receptor-mediated uptake of lipid-binding and lipid-regulating proteins may therefore influence overall lipid metabolism. The pathological mechanisms causing the pronounced atherosclerosis-promoting effect of uremia may involve impairment of this clearance pathway.     

Hemoglobin radiolabeling: In vitro and in vivo comparison of iodine labeling with iodogen and a new method for technetium labeling

Radiolabeled hemoglobin may be a useful tool in the study of the body distribution of hemoglobin solutions developed as plasma expanders with oxygen-transporting capacity. The present investigation compares the suitability of two radiolabeling techniques for hemoglobin. ¹²⁵I labeling of hemoglobin with Iodogen® as iodinating agent caused major changes in the chromatographic behaviour and an accelerated plasma clearance of the labeled hemoglobin in rats. A recently developed two-step procedure for ^99mTc labeling gave better results. The label had only minimal influence on the chromatographic behaviour of hemoglobin. In vivo, no free label occurred in the circulation and no transfer of the label to other plasma proteins took place. The plasma clearance of ^99mTc-labeled hemoglobin in rats was slowed. However, this could be explained entirely by diminishing glomerular filtration, probably by inhibition of the dissociation of the hemoglobin molecule into dimers. The plasma clearance of hemoglobin modified by intramolecular cross-linking, which prevents dissociation of the molecule into dimers and thus excretion by the kidney, was not influenced by the label.We conclude that the ^99mTc labeling procedure is suitable for in vivo distribution studies of hemoglobin when it is taken into account that the urinary excretion is underestimated. For cross-linked hemoglobin, which is more promising as plasma expander, no such restriction exists.     

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments. proximal tubule cell     

T-system formation in cultured rat skeletal tissue

By using a lanthanum-staining technique which enhances the visualization of the plasma membrane and its derivatives we have studied the formation of the T system in rat muscle cells differentiating in vitro. We have found that: (1) T-system formation normally occurs after myoblast fusion and is especially extensive in mature myotubes; myoblasts grown in calcium-deficient medium to prevent fusion show increased number of sarcolemmal caveolae but rare, short T tubules. (2) T-system formation in vitro differs from that displayed by rat muscle cells in vivo in that it precedes and is independent of junctional SR differentiation; the uncoordinated development of T tubules and junctional SR in vitro leads to the formation of ‘inverted’ triads and labyrinthine T-system networks. (3) Coated vesicles are frequently found either free in the cytoplasm or associated with growing T tubules in rat muscle cells differentiating in vitro. A role of coated vesicles in T-system formation is proposed.     

Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

Immunocytochemical localization of cathepsin H in rat kidney. Light and electron microscopic study

Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For light microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

FETAL RAT INTESTINAL ABSORPTION OF HORSERADISH PEROXIDASE FROM SWALLOWED AMNIOTIC FLUID

Horseradish peroxidase (HRP) injected into amniotic fluid is swallowed by rat fetuses and within 3–6 h reaches the gut lumen. This macromolecular protein is then absorbed by the columnar lining cells via a system of apical cytoplasmic tubules formed by invaginations of the plasma membrane. From cytoplasm subjacent to the brush border HRP is transported, within vacuoles, to the supranuclear region, where some is retained for at least 18 h, and to interepithelial spaces. Extracellular enzyme is then found throughout the epithelial basement membrane and between connective tissue cells of the mucosal and submucosal layers Finally, HRP can be detected within lumina of blood and lymphatic capillaries, strongly suggesting that it is transported from the intestine to the circulation.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.     

Immunohistochemical analyses on serum proteins in nephrons of protein-overload mice by "in vivo cryotechnique"

Immunohistochemical analyses on local distributions of serum proteins in living mouse kidneys are usually difficult to examine with conventional preparation methods. By using our "in vivo cryotechnique" combined with freeze-substitution, we have checked immunolocalizations of the serum proteins in nephrons of bovine serum albumin (BSA)-overload mice, and compared them with those obtained by the conventional preparation methods. In two days of daily BSA-injected mice, the immunolocalization of BSA could be observed in Bowman's space and urinary tubules with their overt proteinuria, where another endogenous mouse albumin was similarly immunolocalized. The leakage of BSA and mouse albumin in Bowman's space and their reabsorption into proximal tubules were detected in 55% of nephrons, where no leakage of immunoglobulin G1 (IgG1) was detected. However, the leakage of IgG1, in addition to BSA and mouse albumin, was detected in the other nephrons. By carefully examining immunolocalizations of BSA and IgG1, they were obviously different from those obtained by the conventional preparation methods without normal blood circulation into the kidneys. The immunolocalizations of both BSA and mouse serum proteins could be directly analyzed with the "in vivo cryotechnique", suggesting that functional damage to glomerular filtration barriers are different at early stages of the BSA-overload mouse model, depending on each nephron of living mice.     

The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer

In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.