John H. Todd

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 Bowmans 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.

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.

Metallothionein isoform 1 and 2 gene expression in the human prostate: Downregulation of MT‐1X in advanced prostate cancer

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.

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

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.

Enzymatic Isolation and Serum-Free Culture of Human Renal 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.

Expression of heat shock protein 60 in human proximal tubule cells exposed to heat, sodium arsenite and CdCl2

Abstract 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°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 μM sodium arsenite for 4 h. Acute exposure of HPT cells to 53.4 μM 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+ .

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.

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.

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.

Aminoglycoside Antibiotics Alter the Paracellular Transport Properties of Cultured Human Proximal Tubule Cells

Cell cultures retaining properties of the human proximal tubule were utilized to determine whether or not the aminoglycoside antibiotics alter paracellular transport. The transepithelial resistance (RT) of the human proximal tubule (HPT) cell monolayers was determined by Ussing chamber analysis of cells grown on permeable supports. This analysis revealed that RT was reduced as a result of aminoglycoside exposure and that the reduction corresponded to the known clinical nephrotoxicities of the aminoglycosides. Variation in the aminoglycoside concentration necessary to elicit this response was documented using 14 individual cell isolates. Ultrastructural analysis provided evidence indicating that the alterations in RT were not associated with general damage to the HPT cells. An examination of the structure of the tight junctions by freeze-fracture analysis demonstrated only minimal alteration of the sealing strands as a result of aminoglycoside exposure. Consequently, the reductions in RT were not directly associated with discernible tight junction structural alterations. Alteration in the paracellular route of transport, as indicated by altered RT values, was clearly documented as a result of aminoglycoside exposure. In addition, this alteration was accompanied by an increased density of intramembrane particles within the apical cell membrane.