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The expression of heat shock protein (hsp) 90alpha and beta mRNA and protein were determined in the human kidney and in human proximal tubule (HPT) cells exposed to lethal and sub-lethal concentrations of Cd(+2) under both acute and extended conditions of exposure. Using immunohistochemical analysis, it was demonstrated that hsp 90 was widely distributed in the human adult and fetal kidney. Moderate to strong staining was observed in the straight portions of the distal and proximal tubules, the distal convoluted tubule, the collecting ducts and the parietal epithelium of Bowmans capsule in the glomerulus. Moderate staining was observed in the proximal convoluted tubule of the cortex and the thick loops of Henle within the medulla. In addition, the fetal kidney demonstrated strong staining of the blastema, the 'S-shaped' bodies, and the developing glomeruli. Analysis of hsp 90alpha and beta mRNA expression in total RNA isolated from in situ microdissected proximal tubules or HPT cells demonstrated similar expression levels of both the alpha and beta isoforms in this tubule segment. It was demonstrated that HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (NaAsO(2)). Heat stress, elevated temperature at 42.5 degrees C for 1 h, caused a modest increase in both hsp 90alpha and beta mRNA and protein. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 microM NaAsO(2) for 4 h. In contrast, acute exposure of HPT cells to 53.4 microM CdCl(2) for 4 h resulted in no consistent increase in hsp 90alpha and beta mRNA or protein. Chronic exposure to Cd(+2) likewise failed to increase either hsp 90 mRNA or protein expression, even at concentrations of Cd(+2) that were lethal to the cells during the time course. This study shows that the HPT has a high basal expression of hsp 90, which is not induced by Cd(+2) exposure.

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+).

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.

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.

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.

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.

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.

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.