Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity.

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.

Enhancement of nitric oxide production by pulmonary cells following silica exposure.

In vivo exposure of rat lungs to crystalline silica either by intratracheal instillation or by inhalation results in an increase in mRNA levels for inducible nitric oxide synthase (iNOS) in bronchoalveolar lavage cells (BALC), elevated nitric oxide (.NO) production by BALC, and an increase in .NO-dependent chemiluminescence (CL) from alveolar macrophages (AM). Induction of iNOS message occurs in both AM and polymorphonuclear leukocytes (PMN) harvested from silica-exposed lungs but is not significantly elevated in lavaged lung tissue. In vitro exposure of AM to silica does not stimulate .NO production or enhance iNOS message. However, treatment of naive AM with conditioned media from BALC harvested from silica-exposed rats does increase iNOS message and .NO production by these AM. The potency of this conditioned medium is dependent on interaction between AM and PMN. In the rat model, a relationship exists between the ability of various dusts to cause PMN recruitment or protein leakage into the alveolar space and the induction of iNOS message in BALC, i.e., silica > coal mine dust > carbonyl iron > titanium dioxide. Similarly, a comparison of BALC from a healthy volunteer, a silica-exposed coal miner with a normal chest radiograph, and a silica-exposed coal miner with an abnormal chest radiograph shows a correlation between pathology and both the level of iNOS message in BALC and the magnitude of .NO-dependent CL from AM. These data suggest that .NO may play a role in silicosis and that human pulmonary phagocytes exhibit enhanced .NO production in response to an inflammatory insult.

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.

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.

Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age.

Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn. The growth medium utilized was a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 supplemented with selenium (5 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained. Confluency was reached within 3 to 6 days. A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates. That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained. By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity. However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or "micro-domes" that would not be visible by light microscopy. Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy. However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions. It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.

Induction of metallothionein mRNA and protein following exposure of cultured human proximal tubule cells to cadmium.

Humans 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, in contrast to most animals which have one or two functional MT genes. In the present study, human proximal tubule (HPT) cells were exposed to cadmium (Cd) to determine if these cultures might serve as a model system to study MT gene expression in the renal proximal tubule. Three independent isolates of HPT cells were shown to repeatably induce MT protein when exposed continually to a non-toxic dose of 1 microgram/ml of Cd administered as CdCl2. Accumulation of MT protein was noted within 3 h and persisted over the 16-day time course. The expression of mRNA for the MT-IIA, MT-IA, B, E, F and G genes was also assessed through 16 days of exposure to 1 microgram/ml of Cd versus control media. Of these, the mRNA for the MT-IIA, MT-IE, MT-IF and MT-IG genes were detected in the cells exposed to 1 microgram/ml of Cd. Overall, the results were supportive that the HPT cells can provide a valuable model system to study the regulation of MT gene expression as it applies to the human renal proximal tubule.

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.

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.

Variation in the electrical properties of cultured human proximal tubule cells.

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (RT). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apical-negative PD of 1.5 +/- 0.1 mV, an Isc of 2.7 +/- 0.2 microA/cm2, and an RT of 480 +/- 19 ohms x cm2. Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na+,K(+)-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na+,K(+)-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.

Variation in sorbitol accumulation and polyol-pathway activity in cultured human proximal tubule cells.

The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

Aminoglycoside antibiotics alter the electrogenic transport properties of cultured human proximal tubule cells.

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD) and short-circuit current (Isc). These electrical parameters were used in the present study to determine if aminoglycoside exposure altered electrogenic sodium transport by HPT cells. The results of this determination demonstrated that exposure to gentamicin, at levels below that producing cell necrosis, caused a marked reduction in Isc and that this reduction followed the known in vivo nephrotoxicities of the aminoglycosides streptomycin, gentamicin, and neomycin. It was concluded through a similar analysis on a total of 14 isolates of HPT cells that the aminoglycosides repeatably reduced the electrogenic sodium transport of HPT cells. It was further determined that this alteration in electrogenic transport by gentamicin was mediated through exposure of the drug to the basolateral cell surface and that apical exposure had little effect. Evidence was obtained against the involvement of Na+, K(+)-ATPase, adenosine 3',5'-cyclic monophosphate, and sodium-coupled substrate transport in this alteration in electrogenic transport by the aminoglycosides. The basolaterally located Na+: CO3(-2):HCO3(-1) symporter is a possible site for aminoglycoside-induced nephrotoxicity.

Elicitation of sorbitol accumulation in cultured human proximal tubule cells by elevated glucose concentrations.

The human proximal tubule (HPT) is the characteristic site within the kidney that mediates absorption of glucose. This study was designed to determine whether cultured HPT cells would respond to a hyperglycemic environment through activation of the polyol pathway. The results of this study clearly indicate that exposure of the HPT cells to an extracellular glucose concentration greater than or equal to 11 mM results in substantial intracellular accumulation of sorbitol. This accumulation is inhibited by approximately 70% by treatment with 100 microM sorbinil. When cells growing 24 h on 27.5 mM glucose were changed to medium containing 5.5 mM glucose, sorbitol concentration returned to the control level within 12 h. The activity of aldose reductase was increased by a factor of 1.6 by exposure to elevated glucose concentrations, and the relative reactivity of the enzyme with glucose as substrate was approximately 0.1 compared with that of glyceraldehyde as substrate. Together, these results indicate that cultured cells derived from the HPT undergo activation of the polyol pathway when exposed to a hyperglycemic environment.

Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice.

Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo. Both relaxin B and relaxin C produced an early (3-hr) elevation in in vitro protein synthesis and a later (6-hr) increase in collagen incorporation of proline at the time when the uterotrophic effect was maximal. In vivo effects of relaxin C on the uterus were in some cases greater than relaxin B in contrast to the complete inactivity of the former upon the pubic ligament of the mouse. These findings suggest a high degree of tissue specificity for relaxin stimulation, a variability in responsiveness among tissues in the same animal, and perhaps a primary role of relaxin in uterine function with pelvic relaxation representing a secondary function which has developed in certain species.