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Protocol no. 5
ISOLATED RAT GLOMERULI AND PROXIMAL TUBULES

Specific cell types are isolated from the kidney and the cytotoxic effect of chemicals assessed by examining cell glucose and/or fatty acid oxidation and de novo protein synthesis.

CONTACT

Dr. P.H. Bach
Head of the School of Science
Polytechnic of East London, Romford Rd, London E15 4LZ. UK

RATIONALE

Many nephrotoxic chemicals selectively attack one or more of the discrete cell types of the kidney, leaving adjacent, morphologically different, cells unaffected. The basis of this procedure is that two specific cell type preparations may be isolated, exposed separately to various compounds over a range of concentrations, and the cytotoxicity of these determined. Parameters deemed indicative of a cytotoxic effect include a reduction in de novo protein synthesis and decreased glucose and fatty acid metabolism. A cytotoxic effect may indicate that a chemical is likely to be nephrotoxic in vivo. A differential effect upon the two cell types under study would provide information as to the potential site of injury and, furthermore, may give an insight into the interaction between the toxic compound and the target cell.

BASIC PROCEDURE

Kidneys are dissected from rats, the cortical tissue removed, chopped up, and suspended in solution. The tissue suspension is then passed through metal sieves and mechanically broken down to yield proximal tubule fragments and glomeruli. These cell fractions are immediately incubated in the absence and presence of test compounds and the rate of protein synthesis, glucose metabolism and fatty acid metabolism determined. De novo protein synthesis Cell suspensions are incubated in the presence of tritiated amino acids, the protein fraction precipitated and the incorporation of radioactivity determined by liquid scintillation counting. Glucose or fatty acid oxidation Cell suspensions are incubated with radiolabelled linolenic acid (for fatty acid oxidation) or glucose (glucose oxidation) and the 14CO2 liberated as a result of oxidation is collected and measured by liquid scintillation counting. All results are compared to the control situation and are expressed as percentage values, thus providing an indication of the inhibitory effect of a test compound.

CRITICAL ASSESSMENT

One animal provides several preparations (» 10 samples of both cell fractions). One of the main advantages of dispersing cells by mechanical shearing is the ability to isolate relatively homogeneous populations of glomeruli and proximal tubular fragments from the same kidney. One compound can thus be tested on different renal cell types. Unlike enzymatic dispersion, mechanical shearing does not damage proximal tubular basement membrane. The selectivity of different chemicals for particular cells in the kidney has complicated the assessment of nephrotoxicity in vivo. A variety of techniques are available to study nephrotoxicity in vitro, but each is subject to technical difficulties and the data generated must be interpreted in relation to the strengths and weaknesses of the method. Ideally, it is better to use several different, complementary techniques to provide a firmer basis upon which to predict nephrotoxicity in vivo. This technique, however, does enable a comparison of the toxic effect of chemicals upon two kidney cell types. Should the potency be the same on both the indication may be that the compound is generally cytotoxic or nephrotoxic. On the other hand, a differential degree of toxicity may identify the preferred cell target and, perhaps, suggest a mechanism of action in a nephrotoxic response. There is evidence that many nephrotoxins exhibit target cell-specific toxicity and that this feature is retained in the in vitro situation and is demonstrable in this system. For example, differences have been found in the response of glomeruli and proximal tubular fragments to heavy metals (Wilks et al., 1990; Wilks et al., in press), and also, in some species of rat, in the metabolic response of cortical and juxtamedullary glomeruli to adriamycin (Kastner et al., in press). The procedure described in this protocol yields glomeruli and proximal tubular fragments which are more than 90% pure as assessed by phase contrast microscopy (Wilks et al, 1990). GSH requirements For particular classes of compounds (e.g. heavy metals) GSH plays an important role in nephrotoxicity. GSH is found predominantly in the proximal convoluted tubules, although the glomerulus itself has considerable levels of GSH (» 50% of that found in proximal tubules). GSH contributes to metal accumulation in the proximal tubule by trapping the ion with its SH moiety, but it also represents an important defense mechanism against metal-induced injuries. It was found (Wilks et al., 1990) that pre-exposure to GSH reduced certain metal-induced inhibition (e.g. by Hg) of proline incorporation. Freshly isolated proximal tubular fragments retain a number of amino acid transport processes, and there is evidence that perfused kidneys have a Na+-coupled transport system for tubular uptake of intact GSH. This may account for the protective effect of GSH in pre-incubation experiments. Other in vitro assays A variety of cell systems are used to test for potential nephrotoxicity including the following: clonal growth inhibition of Chinese hamster ovary cells; inhibition of metabolic functions and growth assays using Chinese hamster kidney cell cultures; viability of 3T3 fibroblasts. These in vitro cytotoxicity assays, based on lines, have only a limited predictive potential with respect to target organ toxicity. This is possibly due to the cells not being derived from the target organ, or to the loss of some of their original functional characteristics on account of dedifferentiation in culture. Comparison to in vivo events A good agreement has been found between the relative toxicity of heavy metals to isolated glomeruli and acute nephrotoxic potential in vivo (Wilks et al., 1990). When aminoglycosides were tested in this system (Kwizera et al., 1990), their ability to inhibit incorporation of amino acids did not relate to their in vivo nephrotoxicity. Thus, streptomycin, the least nephrotoxic of the compounds studied, was the most potent inhibitor of amino acid uptake in vitro. Conversely, neomycin was the second least potent inhibitor in vitro and the most potent nephrotoxin in vivo. The effects seen in vitro probably mirror early events in the action of aminoglycosides on renal proximal tubules. However, of potentially greater significance in vivo is the rate of clearance from the renal cortex (t½ = 5 hours for streptomycin, t½ = over 60 hours for the other aminoglycosides). The degree of nephrotoxicity in vivo will be influenced by the longer period of renal cortical exposure to high concentrations of accumulated compound. Therefore, while this system may elucidate possible mechanisms of early events in the toxic process, it does not necessarily predict in vivo nephrotoxic potential. Choice of endpoint A pronounced degree of cell selective toxicity has been documented for several established in vivo nephrotoxins using de novo protein synthesis to measure cytotoxicity. The choice of amino acid may increase or decrease the sensitivity of the assay, e.g. the incorporation of radio-labelled histidine, lysine and leucine into proximal tubular fragments is inhibited by streptomycin (up to 1mM) and by neomycin, gentamicin and amikacin (up to 10mM), but only amikacin inhibits the incorporation of glycine. Thus, inhibition of amino acid incorporation may not be a suitable criterion for all chemical classes if other factors are not taken into consideration. The incorporation of proline is the most frequently used. Two assays are used routinely - protein synthesis and oxidative metabolism, either fatty acid (Wilks et al., 1990; Kwizera et al., 1990) or glucose. The use of assays with two different reactions and end points provides for better extrapolation. Linolenic acid is the substrate of choice for fatty acid oxidation, because it gives rise to a higher rate of CO2 formation in proximal tubules than occurs with oleic, palmitic and stearic acids. Glucose oxidation, as an endpoint, may be inappropriate for proximal tubular fractions, since in vivo the energy they used is derived primarily from fatty acid metabolism.

TEST STATUS

Interlaboratory
ORGANISATIONS USING THE TEST
In some German laboratories.

CHEMICALS TESTED

Anticancer drugs/antibiotics:
adriamycin
streptomycin
PAN
Neomycin
Kanamycin
Gentamicin
Amikacin
Heavy metals :
mercury
cadmium
chromium
arsenic
nickel
selenium

REFERENCES

  1. Bach, P.H. (1989) The detection of chemically induced renal injury, the cascade of degenerative morphological and functional changes that follow the primary nephrotoxic insult and the evaluation of these changes by in vitro methods. Toxicology Letters, 46, 237-250.
  2. Bach P.H., & Kwizera E.N. (1988) Nephrotoxicity: a rational approach to target cell injury in vitro in the kidney. Xenobiotica, 16 (6), 685-698
  3. Kastner S., Wilks M.F., Soose M., Bach P.H., & Stolte, H. (1991) Metabolic studies on isolated rat glomeruli; A valuable tool to investigate glomerular damage In: Nephrotoxicity : Mechanisms, early diagnosis and therapeutic management (eds. P.H. Bach, N.J. Gregg, M.W. Wilks and L. Delacruz) New York; Marcel Dekker, 1991, pp. 467-474.
  4. Kastner S., Wilks M.F., Gwinner W., Soose M., Bach P.H., & Stolte H. (1990) Metabolic heterogeneity of isolated cortical and juxtamedullary glomeruli in adriamycin nephrotoxicity. Renal Physiology and Biochemistry 14: 48-54.
  5. Kwizera E.N., Wilks M.F., & Bach P.H. (1990) Effects of aminoglycosides on the incorporation of amino acids and on fatty acid oxidation in freshly isolated rat renal proximal tubules. In Vitro Toxicology, 3(3), 243-253.
  6. Wilks M.F., Kwizera E.N., & Bach P.H. (1990) Assessment of heavy metal nephrotoxicity in vitro using isolated rat glomeruli and proximal tubular fragments. Renal Physiology and Biochemistry, 13, 275-284.
  7. Wilks M.F., Kwizera E.N., & Bach P.H. (1991) Effects of heavy metals on metabolic functions in isolated glomeruli and proximal tubule fragments. In: Nephrotoxicity : Mechanisms, early diagnosis and therapeutic management (eds. P.H. Bach, N.J. Gregg, M.W. Wilks and L. Delacruz) New York; Marcel Dekker, 1991, pp. 363-366.

IP-5 © June 1991