Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells.

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.

Heat shock protein 27 expression in human proximal tubule cells exposed to lethal and sublethal concentrations of CdCl2.

The expression of hsp 27 mRNA and protein was determined in cultured human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd2+ under both acute and extended conditions. Initial procedures demonstrated that HPT cells display the classic stress response following physical and chemical stress. Heat stress (42.5 degrees C for 1 hr) caused an increase in both hsp 27 mRNA and protein as well as a shift in the protein to a more phosphorylated state. Results were similar when the cells were subjected to chemical stress (exposure to 100 microM sodium arsenite for 4 hr). Acute exposure to 53 microM CdCl2 for 4 hr also resulted in an increase in hsp 27 mRNA and protein and a shift to the more phosphorylated protein isoform. Extended Cd2+ exposure involved continuous treatment with Cd2+ at both lethal and sublethal levels over a 16-day time course. The results of this treatment showed that chronic exposure to Cd2+ failed to increase either hsp 27 mRNA or protein expression in HPT cells, even at lethal Cd2+ concentrations. In fact, hsp 27 protein levels decreased as compared to controls at both lethal and sub-lethal exposure to Cd2+. These findings imply that hsp 27 expression in human proximal tubule cells may have two distinct modes depending on the nature (acute vs. chronic) of the stress.

Expression of the constitutive and inducible forms of heat shock protein 70 in human proximal tubule cells exposed to heat, sodium arsenite, and CdCl(2).

We determined the expression of the constitutive (hsc 70) and inducible (hsp 70) forms of heat shock protein 70 mRNA and protein in human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd(+2) under both acute and extended conditions of exposure. The HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite); hsc 70 mRNA and protein levels were constant or slightly increased, whereas hsp 70 mRNA and protein were greatly elevated. Acute exposure to 53.4 microM CdCl(2) for 4 hr failed to increase either hsc 70 mRNA or protein, a finding similar to that observed under classic conditions of stress. However, under identical conditions of acute exposure to Cd(2+), the expected increase in hsp 70 protein level was suppressed as compared to that found under classic conditions of physical or chemical stress. The decrease in hsp 70 protein level correlated to the reduced expression of mRNA from the hsp 70B gene. The expression of mRNA from the hsp 70A and hsp 70C genes was similar to that found when the cells were treated with heat shock or sodium arsenite. We modeled an extended exposure to Cd(2+) by treating the cells continuously with Cd(2+) at both lethal and sublethal levels over a 16-day time course. Chronic exposure to Cd(2+) failed to increase either hsc 70 mRNA or protein levels in the HPT cells at a nonlethal dosage level and decreased hsc 70 mRNA and protein levels late in the time course of lethal exposure. Under identical conditions, the expression of hsp 70 protein remained at basal levels that were only marginally detectable throughout the time course. Hsp 70A and hsp 70C mRNA levels were unaltered by extended exposure to Cd(2+), and hsp 70B mRNA was not detected during the 16-day time course. Cd(2+) is a poor inducer of hsc 70 and hsp 70 in the proximal tubule under both acute and long-term exposure. These results reinforce the fact that the expression of hsp 70 protein does not result from the transcription of a single gene, but is derived from what may be a complex interplay of several underlying genes.

Differential expression of human metallothionein isoform I mRNA in human proximal tubule cells exposed to metals.

In contrast to the single metallothionein (MT)-1 gene of the mouse, the human MT-1 gene family is composed of seven active genes and six pseudogenes. In this study, the expression of mRNA representing the seven active human MT-1 genes was determined in cultured human proximal tubule (HPT) cells under basal conditions and after exposure to the metals Cd2+, Zn2+, Cu2+, Hg2+, Ag2+, and Pb2+. Basal expression of MT-1X and MT-1E mRNA in HPT cells was similar to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. In contrast, mRNAs representing the basal expression of MT-1A and MT-1F were a minor transcript in HPT cells. Treatment of HPT cells with Cd2+, Zn2+, or Cu2+ increased the levels of MT-1E and MT-1A mRNA, but not the levels of MT-1X or MT-1F mRNA. The increase in MT-1E mRNA appeared to be influenced mainly by exposure to the various metals, whereas the increase in MT-1A mRNA was influenced more by exposure to a metal concentration eliciting a loss of cell viability. Treatment of HPT cells with the metals Hg2+, Ag2+, and Pb2+ was found to have no effect on the level of MT-1 mRNA at either sublethal or lethal concentrations. Using HPT cells as a model, these results suggest that new features of MT gene expression have been acquired in the human due to the duplication of the MT-1 gene.

Metallothionein isoform 3 as a potential biomarker for human bladder cancer.

The goal of the present study was to determine if the expression of metallothionein isoform 3 (MT-3) might serve as a biomarker for human bladder cancer. To accomplish this goal, we defined the localization and expression of MT-3 protein and mRNA using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders. We used immunohistochemistry, immunoblot, and RT-PCR analysis to define the localization and expression of MT-3 protein and mRNA. Immunohistochemical analysis disclosed no immunoreactivity for MT-3 in normal bladder cells. The absence of MT-3 expression in the normal bladder was further confirmed by demonstrating that MT-3 mRNA could not be detected using reverse transcriptase-polymerase chain reaction (RT-PCR) or MT-3 protein using immunoblot. Immunohistochemistry also disclosed no immunoreactivity for MT-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders. Immunohistochemical analysis demonstrated that MT-3 was expressed in carcinoma in situ (CIS), high-grade bladder cancer, low-grade bladder cancer, and dysplastic lesions. MT-3 immunostaining was intense in both CIS and high-grade bladder cancer, and low to moderate in low-grade bladder cancer and dysplastic lesions. We determined MT-3 mRNA expression in a subset of these bladder cancer specimens; expression was elevated as compared to that of the housekeeping gene, ss-actin. The cDNA from the RT-PCR reaction primed for MT-3 contained a FokI restriction site, a site unique for MT-3 as compared to other MT family members. In conclusion, this study demonstrates that MT-3 is up-regulated in human bladder cancer and that this up-regulation increases with increasing tumor grade. The finding that MT-3 expression is minimal in normal bladder suggests that MT-3 might be developed into an effective biomarker for bladder cancer.

The immortalized UROtsa cell line as a potential cell culture model of human urothelium.

The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

Electrical and freeze-fracture analysis of the effects of ionic cadmium on cell membranes of human proximal tubule cells.

We previously reported that cell cultures of human proximal tubule (HPT) cells respond to ionic cadmium in a manner consistent with well-defined Cd(2+)-elicited responses reported for in vivo systems. However, one unique finding was that the transepithelial electrical resistance and tight junction sealing strands were altered as a result of Cd2+ exposure at micromolar concentrations. These alterations are reexamined in detail in the present report to determine whether the Cd(2+)-induced alterations are specific alterations in the tight junction structure or reflect a general alteration in the cell membrane. Exhaustive analysis of tight junction sealing strands demonstrated no significant alterations due to Cd2+ exposure, even at the concentration that elicited a significant reduction in transepithelial resistance. Further analysis of intramembrane particle distribution demonstrated a significant increase in apical intramembrane particles, indicating that Cd2+ exposure altered the characteristics of the apical cell membrane. Overall, the results were consistent with evidence of Cd(2+)-induced alteration in the apical cell membrane of the HPT cell.

Walker-Warburg syndrome: report of three affected sibs.

Walker-Warburg syndrome (WWS) is a lethal, autosomal recessive disorder characterized by Type II lissencephaly, retinal malformation, cerebellar malformation, and congenital muscular dystrophy. We report on 3 sibs with WWS born to a consanguineous couple. The fetal hydrocephalus associated with this syndrome, while not consistent or necessary for diagnosis, is the key manifestation for its prenatal detection. These sibs illustrate the importance of a careful search for associated malformation(s) in a fetus or newborn infant with hydrocephalus and the potential pitfalls of accurate genetic risk estimation in families of such propositi.

Cytologic manifestations of ballistic injury.

The adherent residue from 60 projectiles in 38 consecutive gunshot wound deaths was analyzed by cytologic technique to determine whether a bullet, while passing through the body or intermediate target, retains tissue and other trace evidence. The projectiles, which were recovered from both the body and shooting area, contained microscopically recognizable cellular and inert material in all cases. Direct ballistic trauma could be documented in several tissue types, most notably in muscular tissue. Progressive damage to skeletal and cardiac muscle was seen in multiple preparations. This ranged from partial separation of the fascicles to cytoplasmic homogenization and nuclear rupture. Except in cases of severe ballistic trauma, skeletal and cardiac muscle could be distinguished on the preparations. In addition to neural tissue, projectiles traversing the central nervous system (CNS) contained elongated fragments of intact microvascular structures, sheets of cerebral covering cells, and connective tissue from the scalp. The vascular structures present in CNS preparations may clarify some of the clinical findings in victims of gunshot wounds and elucidate possible pathophysiologic mechanisms in craniocerebral projectile injuries.

Fatal Streptobacillus moniliformis infection in a two-month-old infant.

Streptobacillus moniliformis is an uncommon human pathogen contracted from exposure to rodents. It usually produces a mild, protracted illness (rat-bite fever, Haverhill fever, erythema arthriticum epidemicum) that has either a favorable response to antibiotic therapy or spontaneously resolves. This report describes a fatal case of Streptobacillus moniliformis in an infant bitten by a wild rat. The autopsy findings included an interstitial pneumonia, fibrinous endocarditis, mild mononuclear meningitis, hepatosplenomegaly and lymphadenopathy, erythrophagocytosis, and sinusoidal mononuclear cell infiltrates in regional lymph nodes and the liver. To the authors' knowledge, this is the first report of the autopsy pathology findings of this agent.

Implications of the immunohistochemical localization of the carbonic anhydrase isozymes for their function in normal and pathologic cells.

Histochemical knowledge of the distribution of CA and the two isozymes CA I and CA II has been reviewed here. An abundance of CA occurs most commonly in epithelial cells specializing in transport of ions and water. A mechanism is favored whereby the polarity of efflux of CA-generated protons and bicarbonate across the apical versus the basolateral plasmalemma depends not on the location of CA, which is probably in the cytosol in most sites, but rather on the transport properties of the luminal compared with the serosal region of the plasma membrane in each epithelial cell type. CA exists also in some protein-secreting, merocrine cells including serous cells of salivary and tracheobronchial glands. Available evidence supports the possibility that CA stored as a secretory product in the cytoplasmic granules is released from these cells and, thus, implies extracellular biologic activity for CA in these sites. CA exists also in abundance in various nonepithelial cells performing different and not fully defined biologic functions in these cells. Prevalence of one isozyme over another varies in different cell types. A question remains whether the significance of this variability depends on work load or other undetermined factors.

In situ freeze-fracture of monolayer cell cultures grown on a permeable support.

The growth of cultured epithelial cells on permeable supports allows increased cell differentiation and the assessment of a variety of transcellular and paracellular transport processes. The need to assess the corresponding ultrastructural characteristics of these cells under identical conditions prompted this laboratory to develop a reliable method for producing freeze-fracture replicas of these cultures. Sections of filter inserts with the cell-side facing up are placed between layers of polyvinyl alcohol with a strip of mylar positioned on the layer of polyvinyl alcohol. Following freezing, the monolayer is fractured by lifting the mylar strip from the assembly. The result is a consistent fracture of the apical membrane sufficient for analysis of tight junction sealing strands, microvilli distribution, and intramembranous particle (IMP) distribution between apical and lateral membrane domains. This method utilizes standard equipment and readily available materials and, most importantly, allows the freeze-fracture and replication of an undisturbed cell monolayer.

Expression of heat shock protein 60 in human proximal tubule cells exposed to heat, sodium arsenite and CdCl(2).

The expression of hsp 60 mRNA and protein were determined in human proximal tubule cells (HPT) exposed to lethal and sub-lethal concentrations of Cd(2+) under both acute and extended conditions of exposure. It was demonstrated that HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite). Heat stress, elevated temperature at 42.5 degrees C for 1 h, caused an increase in both hsp 60 mRNA and protein following removal of the stress. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 microM sodium arsenite for 4 h. Acute exposure of HPT cells to 53.4 microM CdCl(2) for 4 h also resulted in an increase in hsp 60 mRNA and protein following removal of the metal. An extended exposure to Cd(2+) was modeled by treating the cells continuously with Cd(2+) at both lethal and sub-lethal levels over a 16-day time course. It was demonstrated that chronic exposure to Cd(2+) failed to increase either hsp 60 mRNA or protein expression in HPT cells, even at concentrations of Cd(2+) that were lethal to the cells during the time course. In fact, hsp 60 protein levels were decreased compared to controls at lethal levels of Cd(2+) exposure. These findings suggest that hsp 60 expression may have two distinct roles when the human proximal tubule cell is exposed to Cd(2+). A protective role through hsp 60 induction when the proximal tubule cell is acutely exposed to Cd(2+) and a deleterious role when hsp 60 protein is down-regulated during extended exposure to Cd(2+).

Isoform-specific expression of metallothionein mRNA in the developing and adult human kidney.

The organization of the metallothionein (MT) gene family has been demonstrated to be much more complex in humans than in the mouse, and possibly rodents in general. For humans, the MTs are encoded by a family of genes located at 16q13 representing 10 functional and 7 non-functional MT isoforms. In the present study, the 5' and 3' untranslated region sequences of the highly conserved, functional MT genes were utilized to generate primer pairs for the analysis of isoform-specific MT mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR). Human kidneys from 13 weeks gestation through adulthood were examined for the expression of MT protein and mRNA. Immunohistochemical analysis demonstrated MT immunoreactivity to be confined exclusively to the proximal tubules of the adult and developing kidney. For all MT-positive cells, MT was localized in the cytoplasm and nuclear localization was variable. There was no correlation between nuclear staining and stage of development. Of the 10 MT genes examined (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, MT-3, and MT-4), mRNAs representing the MT-1E, MT-1F, MT-1X, and MT-2A genes were consistently expressed in all samples regardless of gestational age. There was no indication of a 'fetal form' of MT analogous to that noted to occur in human liver. Messenger RNA for the MT-1A gene was detected in 2 of 6 renal samples without correlation to gestational age. In no instance was mRNA for the MT-1B, MT-1G, MT-1H, MT-3 or MT-4 genes detected. These studies detail the initial determination of MT gene expression in the human renal system and provide the PCR primers for testing and determination of MT gene expression in other organ systems.

Expression of heat shock protein 27 in developing and adult human kidney.

The expression of heat shock protein (hsp) 27 was examined in the developing and adult human kidney. Immunolocalization using a monoclonal antibody against human hsp 27 demonstrated immunoreactivity in both the developing and adult kidney. Low to moderate immunoreactivity for hsp 27 was observed in the fetal and adult proximal tubule, distal tubule, and mesangial cells of the glomeruli. Intense immunoreactivity for hsp 27 was localized to the cortical and medullary collecting ducts in both the adult and fetal kidney, with the most intense staining in the medullary regions. The loop of Henle demonstrated no immunoreactivity for hsp 27. The blastemal element of the developing kidney showed no hsp immunostaining and the ureteric bud demonstrated moderate staining. Western, northern, and reverse transcription-polymerase chain reaction (RT-PCR) analyses disclosed no significant differences in hsp 27 mRNA or protein level as a function of gestational age. An analysis of the phosphorylation state of hsp 27 showed the majority of hsp 27 to be present in the unphosphorylated isoform for both adult and fetal samples. These studies are the first to demonstrate the presence of hsp 27 in the human kidney. It is suggested that this pool of hsp 27 is constitutive as it appears in an inactivated state; localized to the cytoplasm and in an unphosphorylated state.

Exposure of human proximal tubule cells to cytotoxic levels of CdCl2 induces the additional expression of metallothionein 1A mRNA.

Humans, in contrast to animals, have a complex expression of metallothionein (MT) genes which involves many MT isoforms encoded by a family of genes containing an upper limit of 12 possible functional genes. It is unknown if these human isoforms of MT have distinct functions or if they simply represent a non-essential duplication of gene function. In the present study, MT protein and mRNA for the MT-2A, MT-1A, B, E, F, and G genes was determined for 3 isolates of human proximal tubule (HPT) cells having distinct sensitivities to cadmium. For all 3 HPT isolates, the expression of MT protein and mRNA for the MT-2A, MT-1E, MT-1F and MT-1G isoforms was similar among the isolates and demonstrated no correlation to lethality. However, each isoform mRNA was expressed at different levels when compared to one another. In contrast, the expression of MT-1A mRNA differed in expression and correlated with the differing lethalities displayed by each isolate. The finding of different profiles of mRNA expression provides evidence that the MT isoforms may have unique functions and that mRNA for the MT-1A gene could be a potential marker for heavy metal exposure and/or toxicity.

Expression of MT-3 mRNA in human kidney, proximal tubule cell cultures, and renal cell carcinoma.

The human metallothionein 3 (MT-3) gene has recently been identified and characterized as a brain-specific MT having growth inhibitory activity for neuronal cells. One objective of the present study was to determine if MT-3 is brain-specific or also present in the renal system, a site for chronic toxicity due to heavy metal exposure. Using RT-PCR methodology, MT-3 mRNA was shown to be expressed in the human renal system at levels below mRNA for the beta-actin gene. MT-3 mRNA was shown to be expressed in all samples obtained from both the developing and adult renal systems, from 20 weeks of fetal age to 72 years. Cultures of human proximal tubule (HPT) cells were used to determine if MT-3 mRNA expression is influenced by metal exposure. Exposure of HPT cells to either Zn2+ or Cd2+ resulted in an early (within 24 h), but unsustained increase in MT-3 mRNA. The demonstration of MT-3 mRNA expression in the kidney indicates that MT-3 may play an important early role in the response of the cell to metal exposure. MT-3 mRNA expression was also examined in tissues and cells from three cases of renal cell carcinoma. MT-3 was found to be expressed in all three cases at levels similar to those found for normal kidney, providing evidence that MT-3 mRNA expression is not altered in this cancer.

Heterogeneity in the amount of ionic cadmium necessary to elicit cell death in independent cultures of human proximal tubule cells.

Eleven separate isolates of human renal proximal tubule cells (HPT) were analyzed for toxic response to ionic cadmium exposure over a 16-day period in vitro. This study demonstrates a heterogeneous response to Cd2+ exposure in isolates from different individuals with some individuals nearly 3-times more sensitive to ionic cadmium exposure than others. There was no apparent correlation to the race, sex or age of the individuals in the response to cadmium. In addition, readily identifiable culture artifacts, i.e., culture age, passage number, had no influence on the response to Cd2+ exposure and the different isolates had homogeneous baseline HPT properties and morphology. This difference in response to Cd2+ may reflect a heterogeneous response within the human population to cadmium exposure.

Selective exposure of human proximal tubule cells to gentamicin provides evidence for a basolateral component of toxicity.

The goal of the present study was to determine if cultured human proximal tubule (HPT) cells could provide evidence for a basolateral component of gentamicin toxicity. Six isolates of HPT cells were grown on Millicell filters and exposed to gentamicin either apically, basolaterally, or both apically and basolaterally. Toxicity was determined by the release of lactate dehydrogenase into the growth media. The results clearly demonstrated that basolateral exposure and combined apical and basolateral exposure to gentamicin resulted in significant levels of cell toxicity. In contrast, apical exposure to gentamicin elicited only marginal toxicity. The transepithelial flux of gentamicin was shown to be the same in either the apical to basolateral or the basolateral to apical direction. A two-step mechanism of gentamicin toxicity is proposed in order to integrate basolateral toxicity with known features of aminoglycoside nephrotoxicity.

Expression of MT-3 protein in the human kidney.

The objective of the present study was to determine the expression of MT-3 in the human kidney. To accomplish this, an antibody was generated against a unique 8 amino acid sequence present in MT-3 that is not shared by any other MT family member. Western analysis demonstrated that the resulting antibody reacted with a protein band of approximately 6 kDa, corresponding to the known molecular weight of MT-3. Immunohistochemical staining using this antibody demonstrated reactivity with several epithelial components of the nephron. In the glomerulus, moderate intensity was demonstrated in parietal epithelial cells of Bowman's capsule and in visceral epithelial cells of the glomerular tuft. Proximal convoluted tubule cells exhibited moderate cytoplasmic MT-3 reactivity. Distal tubules showed strong cytoplasmic staining for MT-3, particularly in the medullary rays. In the medulla, MT-3 staining was the most variable, with weak to moderate staining in the medullary collecting ducts and a general absence of staining in the thin loops of Henle and in the transitional epithelium of the renal pelvis. The finding that MT-3 is constitutively expressed in several glomerular and tubular epithelial elements of the human kidney warrants consideration of an expanded role for this protein family in maintaining renal homeostasis.