Normal Brush Border Research Articles

Biochemical and morphological evaluation of the effects of propolis on cisplatin induced kidney damage in rats

ABSTRACTCisplatin (CP) is a chemotherapeutic agent used to treat various types of cancer; nephrotoxicity is the most common adverse effect of the drug. We investigated the protective effects of propolis against CP induced kidney injury. Thirty-six male rats were divided into six equal groups: untreated control group, 50 mg/kg/day propolis group, 100 mg/kg/day propolis group, single-dose 7 mg/kg CP group, 7 mg/kg CP + 50 mg/kg/day propolis and 7 mg/kg CP + 100 mg/kg propolis. Rats were sacrificed after 14 days and kidneys were removed for histopathological and biochemical analyses. We used hematoxylin & eosin and periodic acid-Schiff staining to evaluate kidney histopathology and we used the TUNEL technique to assess apoptosis. We also measured total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), ischemia-modified albumin (IMA) and malondialdehyde (MDA) levels in tissue and blood specimens. Normal morphology was observed in the control, 50 mg/kg/day propolis and 100 mg/kg/day propolis groups by light microscopy. Degeneration of tubule cells, edema and tubule dilation were increased in the CP group compared to the control group. Degeneration of tubule cells and dilation of Bowman’s spaces were decreased in the CP + 50 mg/kg/day propolis and CP + 100 mg/kg/day propolis groups compared to the CP group. Tubule dilation decreased significantly in the CP + 100 mg/kg propolis group compared to the CP group. Also, the 7 mg/kg CP group exhibited altered proximal tubule epithelial cells, loss of brush border and thickening of the parietal layer of Bowman’s capsule in glomeruli and basal laminae of tubules. A normal brush border was observed in the CP + 50 mg/kg/day propolis and CP + 100 mg/kg/day groups. Serum OSI and MDA levels were increased in the CP group compared to the control group. Serum MDA levels decreased significantly in the CP + 50 mg/kg/day propolis and 100 mg/kg CP + propolis groups compared to the CP group. CP caused significant damage to kidney tissue; propolis exhibited dose-dependent prevention of tissue damage.

Antioxidant Effect of Clorella vulgaris on Wistar Rat Kidney Induced by CCl4: A Histopathological Review

Kidney is very susceptible to damage by toxicological compounds such as carbon tetrachloride (CCl­4). CCl4 produce free radicals, which cause lipid peroxidation and kidney damage and free radical release, which can be prevented by the administration of exogenous antioxidants, such as Chlorella vulgaris.The aim of study was to determine an effect of antioxidant of C. vulgaris on the histopathological features of Wistar rat kidney which is induced by CCl4. Experimental study with completely randomized design. The variable was histopathology features of the kidneys. The doses of C. vulgaris extract were 3 mg, 4 mg, and 5 mg per 100 grams of rat body weight (BW). The administration of C. vulgaris extract was performed within 30 days, while the CCl4 induction (0.25 ml / 100 g BW) was administered orally on the day 9, 12, 16, 19, 23, and 26. Parameters were histopathology features of renal damage, proportion of tubular cell damage, and Bowman's space diameter. The results showed the administration of C. vulgaris extract was able to reduce the impact of damage caused by CCl4 (p<0.05). This was supported by histologic observations, which was showing a decrease of picnotic and vacuolated cells, normal brush border, and decrease of Bowman's space. In conclusion, 5 mg / 100 g BW of C. vulgaris extract can effectively protect the kidney from damage caused by CCl4. The results of this study strongly support further research on immunostimulant content test C. vulgaris and determine the efficient dose for representative animals mammals also in humans.

A New Genetic Mutation in a Patient With Syndromic Diarrhea and Hepatoblastoma

A small-for-gestational-age preterm infant presented with prominent forehead and cheeks, broad nasal root, hypertelorism, and woolly and brittle hair. Intractable diarrhea began at 1 month. Upper endoscopy showed no abnormality, and histology showed mild and nonspecific villous atrophy with a normal brush border (Fig. 1). Microscopic analysis of the hair shaft revealed trichorrhexis nodosa (twisted hair, longitudinal breaks, cuticolar cell disruption) (Fig. 2). She died after 12 months from hepatic failure. Autopsy revealed a multinodular liver, with fibrosis and a macrotrabecluar-type hepatoblastoma (an area of immature hepatic cells arranged in trabecular structures and ribbons with intraluminar bilious thrombi) (Fig. 3).FIGURE 1: Microscopic findings. Semithin slide under optical microscope shows a short intestinal villus.FIGURE 2: Hair findings. Microscopic analysis of a hair shaft reveals abnormalities, including twisted hair (pili torti), longitudinal breaks, and cuticolar cell disruptions leading to the separation and fragmentation of the cortical fibers with the formation of nodes. These features are pathognomonic of trichorrhexis nodosa.FIGURE 3: Postmortem liver histology. A small hepatic nodule showing macrotrabecular structure, resembling a degeneration area of hepatoblastoma and cirrhosis.We suspected trichohepatoenteric syndrome (THES), a congenital entheropathy causing diarrhea in infants born small for gestational age. The syndrome is associated with nonspecific villous atrophy, facial dysmorphism, immune disorders, liver disease, and trichorrhexis nodosa (1). Hartley et al (2,3) identified the THES locus mapped to 5q14.3–5q21.2. A new homozygous mutation of the THES locus was identified in our patient (C2921-2G>A, skipping of the exon 29). This mutation, together with the occurrence of hepatoblastoma, has not been previously reported. Acknowledgments We thank Dr Antonello Villa of Milano-Bicocca University for the electronic microscopy, Prof ssa Margherita Silengo of Turin University for the evaluation of the hair shaft, Dr ssa Francesca Crosti and the genetic laboratory of San Gerardo Hospital for genetic supervision, Prof Jane Hartley of Birmingham Children's Hospital for the genetical analysis, and the medical and nursing staff of San Gerardo's NICU for the attention and the care paid to this patient.

176 Loss of Functional MYO5B in CaCo2-BBe Cells and Navajo MVID Patient Enterocytes Leads to Loss of Polarity and Deficits in Maintenance of Microvilli

Myosin Vb (MYO5B) is an actin based motor that localizes specific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains in polarized epithelial cells. Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apical trafficking process, because it arises as a result of inactivating mutations in MYO5B that leads to the loss of microvilli in intestinal enterocytes and chronic unremitting diarrhea in the affected newborns. Understanding how mutations in MYO5B lead to aberrant enterocyte phenotype will provide novel insights into the fundamental mechanisms governing apical recycling system trafficking, microvilli formation, and MVID. We hypothesize that MYO5B plays a crucial role in apical trafficking and polarization in enterocytes, and that defective MYO5B in MVID patients mislocalizes Rab small GTPases leading to disruption in apical transport and loss of polarity in enterocytes. To test this hypothesis we have established cellular models of aberrant enterocyte apical trafficking by stably knocking down MYO5B in CaCo2-BBE cells. The cells grown on permeable filters for 15 days were analyzed using immunostaining, Scanning EM, and Transmission EM to examine markers of apical and basolateral polarity, intracellular trafficking, and the establishment of apical microvilli. MYO5B KD knockdown (KD) caused a loss of microvilli and dispersal of Rab8a and Rab11acontaining vesicles from their usual subapical distribution to diffusely cytoplasmic in the cells. MYO5B KD elicited an increase in claudin-2 and a decrease in claudin-1 expression as well as a decrease in lateral membrane staining for p120-catenin (p120). MYO5B KD also caused a loss of apical active GTP-bound cdc42 in the cells. Rescue of theMYO5B KDwith synthetic MYO5B wild type elicited recovery of normal microvilli. However, re-expression of MYO5B(P660L), the Navajo MVID mutation, failed to rescue and in addition caused the formation of microvillus inclusions. Navajo MVID MYO5B(P660L) patient samples demonstrated a loss of p120 and a pseudostratified columnar epithelium at the villus tips where ezrin-staining microvillus inclusions were most predominant. Patient biopsies also showed aberrant localization of MYO5B, Rab8a, Rab11a, Rab11-FIP2, DPPIV, and Ezrin. Rab11a and MYO5B localized around microvillus inclusions, while Rab8a staining was diffuse. Importantly, while a severe loss of brush border was observed in cells at the tips of microvilli, the cells in the mid-villus appeared to have a preserved normal brush border. Our results suggest that MYO5B regulates the polarity of intestinal epithelial cells, thereby maintaining the functional brush border. The normal brush border seen in the proximal villi in the Navajo MVID patients suggests that the pathogenesis of MVID lies in a loss of polarity in enterocytes at the tips of intestinal villi.

An MBoC Favorite: Myosin-1a is critical for normal brush border structure and composition

An MBoC Favorite: Myosin-1a is critical for normal brush border structure and composition

Light Microscopic Diagnosis of Microvillus Inclusion Disease on Colorectal Specimens Using CD10

Microvillus inclusion disease (MID) is a rare neonatal enteropathy that is typically diagnosed using electron microscopy to show characteristic inclusions in conjunction with light microscopy and periodic acid-Schiff staining to show lack of the normal brush border on biopsies obtained endoscopically from the small bowel. MID has also been diagnosed using CD10 immunoreactivity that shows abnormal intense cytoplasmic staining in absorptive small bowel enterocytes. As ultrastructural studies also show abnormal microvillus inclusions in absorptive colonocytes in these patients, we investigated the use of CD10 immunoreactivity on colon specimens. We studied the CD10 staining pattern in 4 colon specimens from patients with MID and in colon biopsy specimens from pediatric control patients with and without histopathologic abnormalities. All MID cases had cytoplasmic CD10 staining in absorptive colonocytes in contrast to the control patients who did not show any epithelial CD10 staining. We conclude that abnormal cytoplasmic CD10 staining of absorptive colonocytes can aid in the diagnosis of MID, which may be invaluable in the situations where only colon biopsy specimens are available for examination.

Disaccharidase Activity in Infants and Comparison Based on Symptoms and Histological Changes

There is uncertainty regarding disaccharidase activity (DA) in infants. In this study, values for DA in infants were established and compared with symptoms and intestinal mucosal histological appearance. Disaccharidase activity and histological appearance of endoscopically obtained intestinal mucosal biopsy specimens from 131 infants (75 males; mean age 180 days; range 20-364 days) obtained during an 8-year period were reviewed. Patients were divided into 2 groups on the basis of absence (group 1; n = 56) or presence (group 2; n = 75) of failure to thrive (FTT) and/or diarrhea. These groups were subdivided into 3 subgroups on the basis of histological findings: normal histological appearance (A), mild histological abnormalities (B), and moderate to severe histological abnormalities (C). The DA from patients in group 1A represent values in infants who were free of diarrhea/FTT and who had normal intestinal mucosal histological appearance. The geometric means (95% CI) in units of DA were as follows: lactase 33.7 (normal range 29.0-39.1), sucrase 48.9 (normal range 44.2-54.1), maltase 160.5 (normal range 144.4-178.3), and palatinase 11.2 (normal range 9.7-12.9). Differences in DA were not related to symptoms, in the absence of histological abnormalities (1A vs 2A), but rather on the presence of histological abnormalities even in the absence of symptoms (1A vs 1B). Differences were also found when patients with FTT and/or diarrhea with abnormal histological appearance (2B and 2C) were compared with patients with no FTT and/or diarrhea with a normal brush border (1A). We outline DA values in a large cohort of infants. DA in infants, as in children, relates to intestinal mucosal histological appearance rather than to symptoms.

33 Disaccharidase Activities in Infants: Normal Values and Comparison Based on Symptoms and Histological Changes.

Background: There is an uncertainty and paucity of data regarding normal levels of disaccharidase activities (DA) in infants. Aim: To establish normal values for DA in infants. To determine the relationship between symptoms, intestinal mucosal histology and DA. Methods: Histology and DA of intestinal mucosal specimens from 131 infants (75 males; mean age 180 d; age range, 20-364 d) obtained endoscopically over an 8 year period were reviewed. Patients were divided into 2 groups based on absence (Group 1; n=63) or presence (Group 2; n=68) of failure to thrive (FTT) and/or diarrhea. These groups were further subdivided into 3 subgroups based on histological findings: (normal: A, mild abnormalities: B, and moderate/severe changes: C). Results: DA from group 1A represent normal values as these infants were free of FTT/diarrhea, and had normal intestinal mucosal histology (Table). Differences in DA were not dependent on symptoms, in absence of histological abnormalities (groups 1A and 2A), but rather on presence of histological changes even in the absence of symptoms (groups 1A vs 1B). Also, differences were found when patients with FTT and/or diarrhea with abnormal histology were compared to patients with no FTT and/or diarrhea with a normal brush border (groups 2B and 2C vs 1A). Conclusions: We describe normal levels of DA in infants. These may serve as reference values for clinical practice and laboratory analyses. Additionally, DA were related to mucosal histology and not certain symptoms.

Myosin-1a Is Critical for Normal Brush Border Structure and Composition

To develop our understanding of myosin-1a function in vivo, we have created a mouse line null for the myosin-1a gene. Myosin-1a knockout mice demonstrate no overt phenotypes at the whole animal level but exhibit significant perturbations and signs of stress at the cellular level. Among these are defects in microvillar membrane morphology, distinct changes in brush-border organization, loss of numerous cytoskeletal and membrane components from the brush border, and redistribution of intermediate filament proteins into the brush border. We also observed significant ectopic recruitment of another short-tailed class I motor, myosin-1c, into the brush border of knockout enterocytes. This latter finding, a clear demonstration of functional redundancy among vertebrate myosins-I, may account for the lack of a whole animal phenotype. Nevertheless, these results indicate that myosin-1a is a critical multifunctional component of the enterocyte, required for maintaining the normal composition and highly ordered structure of the brush border.

Receptor-associated protein is important for normal processing of megalin in kidney proximal tubules.

The receptor-associated protein (RAP) has been identified as a chaperone regulating the expression and processing of the LDL receptor-related protein. RAP also binds to the related 600-kD multiligand endocytic receptor megalin expressed in many absorptive epithelia including renal proximal tubule. The present study examines the effect of RAP gene disruption on megalin expression and subcellular distribution in the proximal tubule as well as the effect on tubular protein reabsorption. It is shown that RAP is important for the normal expression and function of megalin. Megalin expression was reduced to approximately 23% estimated by immunoblotting and supported by immunocytochemistry and by the amount of megalin recovered by RAP affinity chromatography. Light- and electron microscope immunocytochemistry as well as analyses on separated membrane fractions showed significant changes in the subcellular distribution of megalin. A significant reduction in the normal brush border labeling was observed in association with increased labeling of rough endoplasmic reticulum and the smooth paramembranous endoplasmic reticulum along the basolateral membranes. RAP deficiency was associated with changes in urinary protein composition, enabling the identification of alpha-amylase as a new ligand for megalin. In addition, an increased excretion of vitamin D-binding protein, a recently identified ligand to megalin, was observed supporting changes in tubular protein reabsorption. The present data show that RAP is of crucial importance for normal processing and function of megalin, suggesting a chaperone-like function of this protein in the kidney proximal tubule.

Lenten cell: ultrastructure, absorptive properties, and enzyme expression of a novel type of cell in the newborn and suckling pig intestinal epithelium.

The small intestinal epithelium is made up of columnar absorptive enterocytes and a smaller number of specialized non-absorptive cells, including goblet cells, enteroendocrine cells, M cells, cup cells, and tuft cells. During a study on milk protein absorption in newborn pigs, we identified an enterocyte that showed no uptake of milk proteins and that could be found only in the jejunum and ileum of pigs during the first 2 weeks of life. We call this previously undescribed enterocyte the lenten cell. We used light microscopy and transmission electron microscopy in conjunction with immunolabelling and cytochemical techniques to determine the occurrence, ultrastructure, absorptive properties, and brush border hydrolase expression of lenten cells. Lenten cells constituted approximately 1-2% of the villous epithelium. They were seen in newborn and suckling pigs 1-9 days of age, but were not found in weaned pigs. Morphologically, lenten cells were spindle- or wineglass-shaped, with a ventrally sited nucleus and an electron-dense cytoplasm with numerous cytokeratin filaments. Lenten cells had a normal brush border with microvilli that were slightly thicker than those of absorptive enterocytes, but they did not express the brush border hydrolases lactase, aminopeptidase N, and alkaline phosphatase. Lenten cells did not endocytose milk proteins or horseradish peroxidase, but contained some endocytic or secretory vacuoles and a few dense granules. No role for lenten cells has been identified in this study, but presence of these cells during the neonatal period, when growth and differentiation of the gastrointestinal tract is at a peak, clearly suggests that lenten cells may play a role in this process.

Lenten cell: Ultrastructure, absorptive properties, and enzyme expression of a novel type of cell in the newborn and suckling pig intestinal epithelium

Background The small intestinal epithelium is made up of columnar absorptive enterocytes and a smaller number of specialized non-absorptive cells, including goblet cells, enteroendocrine cells, M cells, cup cells, and tuft cells. During a study on milk protein absorption in newborn pigs, we identified an enterocyte that showed no uptake of milk proteins and that could be found only in the jejunum and ileum of pigs during the first 2 weeks of life. We call this previously undescribed enterocyte the lenten cell. Methods We used light microscopy and transmission electron microscopy in conjunction with immunolabelling and cytochemical techniques to determine the occurrence, ultrastructure, absorptive properties, and brush border hydrolase expression of lenten cells. Results Lenten cells constituted ∼1–2% of the villous epithelium. They were seen in newborn and suckling pigs 1–9 days of age, but were not found in weaned pigs. Morphologically, lenten cells were spindle- or wineglass-shaped, with a ventrally sited nucleus and an electron-dense cytoplasm with numerous cytokeratin filaments. Lenten cells had a normal brush border with microvilli that were slightly thicker than those of absorptive enterocytes, but they did not express the brush border hydrolases lactase, aminopeptidase N, and alkaline phosphatase. Lenten cells did not endocytose milk proteins or horseradish peroxidase, but contained some endocytic or secretory vacuoles and a few dense granules. Conclusions No role for lenten cells has been identified in this study, but presence of these cells during the neonatal period, when growth and differentiation of the gastrointestinal tract is at a peak, clearly suggests that lenten cells may play a role in this process. © 1996 Wiley-Liss, Inc.

Wheat-sensitive enteropathy in Irish setter dogs: possible age-related brush border abnormalities

The pathogenesis of a wheat-sensitive enteropathy was explored in a litter bred from two Irish setters with a naturally occurring enteropathy. Jejunal biopsies from all eight progeny exhibited morphological changes comparable to those in the parents, while biochemical abnormalities appeared to be related to age. In biopsies obtained from the first group of four dogs at eight months, the activities of alkaline phosphatase and of leucyl-2-naphthylamidase were almost undetectable while disaccharidases were unaltered. In contrast, analytical subcellular fractionation of biopsies obtained from the second group of four dogs at nine months showed that specific activities now reflected a major deficiency of brush border alkaline phosphatase, and normal brush border leucyl-2-naphthylamidase accompanied by elevated soluble activity. Further studies are indicated to determine whether these findings represent an age-related abnormality affecting specific microvillus membrane proteins.

Further characterization of intestinal lactase/phlorizin hydrolase

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca 2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electropboresis the same size of constituent polypeptides and the same catalytic and immunological properties as normal brush border lactase/phlorizin hydrolase.

Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.

The influence of cortisol on intestinal calcium transport was studied in isolated duodenal loops and brush border membrane (BBM) vesicles of vitamin D-deficient or replete chickens. Four- to five-week-old vitamin D-deficient cockerels were dosed intraperitoneally with 1 microgram of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] alone 15 hr before sacrifice or in combination with 1, 3, or 5 mg of cortisol 24 and 48 hr before sacrifice. After a 1-microgram dose of 1,25-)OH)2D3 the in situ intestinal ligated loop technique revealed a 60% increase in calcium absorption compared to control birds (P less than or equal to 0.001). However, the administration of cortisol in various doses (3 and 5 mg) to chickens given 1,25-(OH)2D3 resulted in significant decreases in intestinal calcium transport in vivo (P less than or equal to 0.05; P less than or equal to 0.05). When intestinal BBM vesicles were prepared from birds treated in a manner identical with that described above, there was no observable difference between calcium uptake in BBM vesicles of the 1,25-(OH)2D3-treated birds and that of the cortisol plus 1,25-(OH)2D3-treated birds. 1,25-(OH)2D3-treated and 1,25-(OH)2D3 plus cortisol-treated chicks had intestinal BBM vesicle uptakes that were significantly greater than those of vitamin D-deficient controls (P less than or equal to 0.02; P less than or equal to 0.025). These data show that in vivo intestinal calcium transport may be markedly reduced in the presence of normal intestinal BBM vesicle calcium uptake. This suggest that factors other than BBM calcium uptake (e.g., protein synthesis or contraluminal membrane events) play an important role in the movement of calcium from the intestinal lumen into the bloodstream and extracellular fluid of the organism.

Ultrastructural localization of concanavalin a surface receptors on brush-border enterocytes in normal children and during coeliac disease.

Concanavalin A surface receptors were detected on brush border enterocytes from child intestinal biopsies by the concanavalin A-peroxidase method. On electron microscopy, these receptors appeared as electron-dense deposits, located on a band running along the innermost part of the brush border membrane glycocalyx. In the five control subjects tested, brush border length was 1.06 +/- 0.18 micrometer, and band thickness, 19 +/- 4 nm. Deposits were regular in shape and evenly spaced out along the entire membrane, reflecting homogeneous glycosylation of the normal brush border. In seven patients with villous atrophy induced by gluten, the brush border was damaged and its length was 1.04 +/- 0.39 micrometer. The thickness of the electron-dense deposit band was 19 +/- 6 nm; deposit shape was irregular and the band ran discontinuously along the membrane. The degree of this abnormality seemed to correspond to the degree of brush border damage. In six treated coeliac patients with normalized mucosa, the brush border was structurally normal but it was significantly longer (1.25 +/- 0.32 micrometer) than in the controls (p less than 0.01). The electron-dense band was significantly thinner (16 +/- 7 nm) than in the controls (p less than 0.01). The distribution of the electron-dense deposits was sporadic in some parts of the band and regular in others. These results suggest abnormal glycosylation of the brush border membrane in coeliac disease, and might be due to the presence of abnormal glycoconjugates. It remains to be established if these changes are induced by gluten toxicity or are the consequences of nonspecific intestinal disorders.

Para-aminohippurate and tetraethylammonium transport in fragments of rat renal cortex.

SummaryTo develop a simple in vitro procedure which, compared to slices, yields less variation in organic anion and cation transport, we used fragments of renal cortex. Each fragment contained 11–15 glomeruli and, after 90 min of incubation, showed normal brush border and well-preserved epithelium. With this technique, there was less variation in transport studies within a given experiment, and fragments responded in the same manner as slices to addition of acetate and serum to the medium.

Folate deficiency and the pill.

A 21-year-old woman who had been taking oral contraceptives (OCs) for 13 months presented with severe megaloblastic anemia. oc therapy was discontinued and 5 mg folic acid 3 times/day was administered. 20% reticulocytosis resulted in 5 days. 2 weeks after beginning therapy a small intestinal biopsy revealed partial villous atrophy with a convolut ed pattern and a dense inflammatory infiltration of plasma cells and lymphocytes in the lamina propria and the surface cells of the villi were low columnar with a poor brush border. The woman later became pregnant and remained well on a normal diet. A second small intestinal biopsy revealed a mixed pattern of leaves and ridges. The villi revealed a normal tall columnar surface epithelium with a normal brush border and the lamina propria contained scanty plasma cells and some lymphocytes.