Biosafety testing of genetically modified ryegrass (Lolium perenne) using a model for the optimum selection of test invertebrates.

Selection of test species for use in biosafety evaluation of genetically modified plants is challenging but important, as regulators in many jurisdictions require tests to determine the potential for adverse environmental impacts before the release of plants into the environment. This contribution provides an example of an evidence-based process whereby species from the receiving environment can be ranked in order of susceptibility to potential impact, and guide test species selection. The case study used for this example was ryegrass, a forage plant, which had been modified to produce elevated levels of the lipid triacylglyceride. The previously described priority ranking of nontarget invertebrates model (PRONTI), designed to rank invertebrates for biosafety testing, has been adapted for use with these plants, which could, potentially, be beneficial to invertebrate populations, and applied to data on 246 known pasture invertebrate species. The output from the model for the top 20 ranked pasture invertebrate species is discussed, the attributes of these are considered along with the level of uncertainty in the information used. Consideration is given to how the model output can be interpreted and used in a biosafety risk assessment. While some subjectivity is involved in establishing the scores, all invertebrate species are subjected to the same analysis, and treated equally. In this way, regulators have a method of a risk assessment that is evidence-based, and transparent in its assumptions thereby avoiding potential for bias.

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.

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.

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+).

Metallothionein isoform 1 and 2 gene expression in the human prostate: downregulation of MT-1X in advanced prostate cancer.

BACKGROUND: Studies have shown an association of metallothionein (MT) overexpression with tumor type and grade. However, a family of genes underlies the expression of these proteins. The goals of this study were to define the expression of MT genes and protein in normal human prostate and to provide evidence that the expression of the MT isoforms is altered in prostate cancer. METHODS: Immunohistochemistry was used to localize MT protein, reverse transcription-polymerase chain reaction (RT-PCR) to determine the MT isoform-specific mRNAs, and immunoblot analysis to determine MT protein levels. RESULTS: The localization of MT in the prostate was further defined using the E9 antibody. Using normal prostate tissue dissected from glands removed for prostate cancer, it was demonstrated that MT protein expression in the normal prostate is supported by mRNA from the MT-1A, MT-1E, MT-1X, and MT-2A genes. No expression of the MT-1X gene was demonstrated in cases of advanced prostate cancer. The expression of MT-1 and MT-2 isoform-specific mRNA varied among three commonly utilized prostate cancer cell lines. CONCLUSIONS: MT protein in the normal human prostate is supported by transcription of mRNA from the MT-1A, MT-1E, MT-1X, and MT-2A genes. Expression of MT-1X mRNA is downregulated in advanced prostate cancer. Variable expression of MT mRNA in prostate cell lines provides evidence that MT gene expression may be altered among individual prostate cancers.

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.

Tissue culture of human renal epithelial cells using a defined serum-free growth formulation.

BACKGROUND: Development of the culture of renal epithelial cells in a serum-free growth medium was driven by the need to examine the effects of hormones and other effector molecules on differentiated cell function without interference from the complex mixture of substances in serum. The present report details this laboratory's cumulative experience in the use of a defined growth medium for the propagation of epithelial cells from adult, fetal, and malignant human renal tissue. METHODS: Routine cell culture technology was used to determine the capability of a defined growth medium to support the growth of renal epithelial cells isolated by collagenase dissociation of tissue from adult and fetal kidneys, renal cell carcinoma, and Wilms' tumors. RESULTS: The defined growth medium formulation consistently allows the isolation and growth of transporting renal epithelial cells from both normal adult and fetal kidneys. This growth medium only rarely supports the growth of epithelial cells from renal cell carcinomas and Wilms' tumors. CONCLUSIONS: The method developed for the culture of human proximal tubule cells requires minimal cell culture expertise and equipment, and results in the repeatable isolation of transporting epithelial cell cultures that retain features of differentiated proximal tubule cells.

Metallothionein isoform 3 expression in the human prostate and cancer-derived cell lines.

BACKGROUND: Expression of metallothionein isoform 3 (MT-3) was initially reported to be confined to neural tissues. However, it was recently demonstrated that MT-3 is expressed in epithelial cells of the human kidney. This motivated the current examination of the expression of MT-3 in the human prostate. METHODS: Immunohistochemistry (IHC) was used to localize the expression of MT-3, RT-PCR to determine the expression of MT-3 mRNA, and Western blot analysis to determine the level of MT-3 protein. RESULTS: Selected epithelial and stromal cells of the normal human prostate were shown to have low levels of MT-3 expression. MT-3 was increased in prostatic intraepithelial neoplasia (PIN) lesions and further increased in a highly variable fashion in prostatic adenocarcinoma. In some adenocarcinomas, MT-3 expression exceeded that of nerve. Three cell culture models of prostate cancer were also shown to variably express MT-3. Restriction enzyme analysis confirmed the expression of MT-3 in the cells and tissues. CONCLUSIONS: MT-3 is expressed in the normal human prostate, and expression is enhanced and highly variable in PIN lesions and primary prostate cancer cells. The variable nature of MT-3 expression was also noted in commonly utilized prostate cancer cell lines.

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 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.

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.

Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA.

The organization of the human metallothionein (MT) gene family is more complex than the commonly used mouse and rat models. The human MTs are encoded by a family of genes consisting of 10 functional and 7 nonfunctional MT isoforms. One objective of this study was to determine if the accumulation of MT protein in cultures of human proximal tubule (HPT) cells exposed to metals is similar to that expected from the knowledge base obtained from rodent models. To accomplish this objective, HPT cells were exposed to both lethal and sublethal concentrations of Cd2+, Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ and MT protein levels were determined. The results were in general agreement with animal model studies, although there were some exceptions, mainly in areas where the animal model database was limited. In clear agreement with animal models, Cd2+, Zn2+, and Cu2+ were demonstrated to be potent inducers of MT protein accumulation. In contrast to the similarity in MT protein expression, we obtained evidence that the human renal MT-2 gene has a unique pattern of regulation compared to both animal models and human-derived cell cultures. In the present study, we determined that MT-2A mRNA was not induced by exposure of HPT cells to Cd2+ or the other metals, a finding in contrast to studies in both animal models and other human cell culture systems in which a high level of MT-2 mRNA induction occurs upon exposure to Cd2+ or Zn2+. While MT protein expression may be similar between humans and animal models, this finding provides initial evidence that regulation of the genes underlying MT protein expression may be divergent between species.

Differential expression of the MT-1E gene in estrogen-receptor-positive and -negative human breast cancer cell lines.

The goal of this study was to determine which of the 10 functional metallothionein (MT) genes are expressed in four human breast cancer cell lines and whether expression varies among the cell lines. Using reverse transcription polymerase chain reaction (RT-PCR) technology, it was shown that there was no expression of mRNA for the MT-1A, MT-1B, MT-1F, MT-1G, MT-1H, MT-3, and MT-4 genes in any of the four cell lines. All four cell lines were shown to express mRNA for the MT-2A and MT-1X genes. The expression level of mRNA for the MT-2A gene demonstrated modest differences among the cell lines, whereas expression of the MT-1X gene was consistent. In contrast, mRNA for the MT-1E gene was expressed in only two of the four cell lines and expression correlated to the estrogen receptor status of the cell lines. The two estrogen-receptor-positive cell lines showed no mRNA expression for the MT-1E gene. In the two estrogen-receptor-negative cell lines, mRNA expression for the MT-1E gene was elevated with expression levels similar to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The cellular content of MT protein was also shown to be elevated in the estrogen-receptor-negative cell lines that express MT-1E mRNA. These results suggest a possible relationship between estrogen receptor status and MT-1E gene expression in human breast cancer.

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.

Expression of hsc 70, but not hsp 70, in human third molar dental pulp.

The constitutive (hsc 70) and inducible (hsp 70) isoforms of heat shock protein 70 are important members of the superfamily of stress related proteins that protect and promote the recovery of cells from physiological and pathologic stress. The goal of this study was to define the baseline expression of hsc 70 and hsp 70 in disease-free, minimally stressed human dental pulp of the adult 3rd molar. Immunolocalization demonstrated moderate to heavy staining intensity for hsc 70 in both the cytoplasm and nucleus of odontoblasts and fibroblasts comprising the human pulp. Endothelial and smooth muscle cells displayed weak to moderate immunoreactivity for hsc 70 in both the cytoplasm and nucleus. Schwann cells demonstrated only weak nuclear staining for hsc 70. No immunoreactivity for hsp 70 was observed in any cell type in human pulp. Western, northern, and RT-PCR analysis of pulp preparations confirmed the expression of hsc 70 mRNA and protein within components of the pulp. These results demonstrate that cells of the human pulp express, under conditions of minimal stimulation, a key component of the stress response protein superfamily. The expression of hsc 70 under conditions of minimal stress may provide pulp components an advantage in resisting cell injury when stress occurs.

Poly-l-aspartic acid protects cultured human proximal tubule cells against aminoglycoside-induced electrophysiological alterations.

Cultured human proximal tubule cell monolayers maintained on permeable supports were treated simultaneously with the aminoglycoside antibiotic, gentamicin, and poly-L-aspartic acid (PAA), an inhibitor of aminoglycoside nephrotoxicity. Following 4 days of exposure, cell monolayers were placed into Ussing chambers to allow monitoring of transepithelial electrical properties. For each of the three cell isolates examined, aminoglycoside-induced alterations in electrogenic transport, reflected by changes in short-circuit current (Isc), as well as alterations in paracellular properties, indicated by changes in transepithelial electrical resistance (RT), were diminished in the presence of PAA. Alterations resulting from selective basolateral exposure to gentamicin were unchanged in the case of apically applied PAA and attenuated only when PAA acid was added basolaterally. This is the first demonstration of PAA inhibition of aminoglycoside-induced cellular alterations involving human cells.

Expression of heat shock protein 27 in adult human third molar dental pulp.

This study is the first to define the expression of hsp 27 in the pulp of the adult human third molar. Using a monoclonal antibody against human hsp 27, immunoreactivity was demonstrated in the odontoblasts, odontoblast processes, pulp fibroblasts, and smooth muscle and endothelial cells of vessel walls. Nerves were negative. Pulp fibroblasts were characterized by cytoplasmic staining and variable nuclear staining. Odontoblasts also displayed consistent cytoplasmic staining and variable nuclear staining. Western, Northern, and RT-PCR analysis confirmed the expression of hsp 27 mRNA and protein. Hsp 27 was also shown to be present in both the unphosphorylated and phosphorylated isoforms. In general, nuclear localization and phosphorylation of hsp 27 has been correlated with cells responding to stress or other stimuli. This study demonstrates that pulp from a single human third molar provides sufficient material to support a detailed molecular analysis of gene expression.

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.

Enzymatic isolation and serum-free culture of human renal cells : retaining properties of proximal tubule cells.

Procedures for the culture of the human renal proximal tubule (HPT) cellutilizing explanted tissue have been previously reported by this laboratory (1). Several other investigators have also reported the isolation and culture of human renal tubule cells (2, and references therein). Although explantation of tissue fragments remains an effective way to initiate cell cultures, cell outgrowth and the attainment of confluent cultures may take several weeks. The cell-culture methodology described in this chapter results in a high yield of confluent primary cultures in 7-10 d. The technique involves the digestion of minced cortical tissue with collagenase, followed by a filtering step to remove tissue fragments. The filtrate is centrifuged, and the cell pellet is resuspended in serum-free growth medium and dispensed onto prepared growth surfaces.