The long range goal of this research is to demonstrate that cultured human proximal tubule (HPT) cells are a valid, and possibly singular, model system for the study of human metal- and agent-induced nephropathies that invoke induction of the metallothionein stress response. It is well- established that the metallothioneins (MTs) are important in the renal response to toxiC insult, especially as it applies to heavy metal exposure and the resulting proximal tubular necrosis. However, it is not as well- known that the gene organization of MT between humans and rodents is quite different. Whereas the rodent has two coordinately regulated MT genes, MT1 and MT2, the human possesses 7 functional genes for MT-1 and one for MT-2; none of which appear to be coordinately regulated. It is not known if this increase in gene number is important in the human renal response to toxic agents or simply represents a needless duplication of function. As such, the initial experimental aim in this validation process is to determine the expression of the individual MT genes in cultured human proximal tubule cells (HPT) under basal conditions and following exposure to agents known to induce renal-toxicity. This response will then be compared to the MT response known to occur in animal model systems. The agents to be employed are copper, silver, cadmium, zinc, mercury and lead (control) . The second specific aim of this validation process will be to determine which MT genes are intimately involved in attenuating or enhancing human agent-induced renal toxicity. This will be determined for expressed MT genes by selectively inhibiting expression using antisense technology and for unexpressed MT genes by eliciting expression by transient transfection of the unexpressed gene into the HPT cells. Together, the above studies should validate the HPT cell system for use in experimental studies defining the mechanism of heavy metal toxicity in humans and, at the very least, allow replacement of primary cultures of similar cells isolated from animal species.
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