Toxicity of Holotransferrin but Not Albumin in Proximal Tubule Cells in Primary Culture
Total Page：16
File Type：pdf, Size：1020Kb
Toxicity of Holotransferrin But Not Albumin in Proximal Tubule Cells in Primary Culture LIGUANG CHEN,* ROSS A. BOADLE,t and DAVID C. H. HARRIS* *Department of Renal Medicine and Electron Microscopy Unit, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia. Abstract. Proteinuria has been invoked as a cause of tubuloin- 0.25 versus 0.46 ± 0.23 nmol/dish, P < 0.01) were increased terstitial injury in chronic renal disease, and in vivo studies compared with values at pH 7.4. In contrast, pH 6.0 did not have suggested indirectly the particular nephrotoxicity of one increase iron uptake from FeCl3. Lysine (100 mM) inhibited urinary protein hobotransferrin (Tf-Fe). However, to date there Tf-Fe uptake, decreased intracellular iron concentration, and has been no direct evidence for the nephrotoxicity of Tf-Fe. To attenuated Tf-Fe-induced cytotoxicity. The iron chelator des- examine the potential cytotoxicity of Tf-Fe and the mechanism ferrioxamine (200 j.tM) and hydroxyl radical scavenger dim- involved, and to compare this to another urinary protein albu- ethylpyrroline N-oxide (32 mM) abolished lactate dehydroge- mm, rat proximal tubule cells were studied in primary culture. nase leakage induced by Tf-Fe at pH 6.0. Lipid peroxidation, Tf-Fe at pH 6.0 caused functional and ultrastructural injury, but as assessed by production of mabondialdehyde, preceded lac- no cytotoxicity was seen with cells exposed to albumin, apo- tate dehydrogenase leakage. In summary, holotransferrmn, but transferrin (transferrin), or Tf-Fe at pH 7.4. The influence of not albumin, is toxic to rat proximal tubule cells, a pH-depen- pH on Tf-Fe-induced cytotoxicity was not due to pH per se, but dent effect involving its uptake into tubule cells, its iron could be explained by an effect on Tf-Fe uptake. At pH 6.0, moiety, and its lipid peroxidation. (J Am Soc Nephrol 9: uptake of ‘251-Tf-Fe (3.55 ± 0.05 versus 1 .25 ± 0. 10 fmol/ 77-84, 1998) dish, P < 0.01) and intracellular iron concentration (1.14 ± In humans with chronic glomerular disease associated with (8). Alternatively. lysosomes might be overwhelmed by exces- renal insufficiency, tubulointerstitial changes are uniformly sive reabsorption of proteins, with consequent leakage of dam- present (1-3). Moreover, patients with nephrotic-range protein- aging lysosomal enzymes into the tubular cytoplasm (9), or uria are more likely to have chronic interstitial disease than fatty acids bound to reabsorbed protein could be tubulotoxic those with minor proteinuria (4-5). In animal experiments, rats (10,1 1). However, no ultrastructural evidence of lysosomal with proteinuria induced by the intraperitoneal administration damage in proteinuric humans or several animal models of of heterologous serum albumin develop tubulointerstitial ne- proteinuric renal disease has been found, nor was there any phritis (6). In humans and animals alike, tubulointerstitial functional evidence of lysosomal fragility in the rat remnant damage correlates better with prognosis than does gbomerular kidney (12-18). pathology (7). In vivo studies by other investigators ( 19 -2 1) and from our Because of the close association between proteinuria and own laboratory (1 2-1 8) have suggested that the toxicity of tubulointerstitial disease, it has been suggested that urinary nonselective proteinuria resides in the iron moiety of the fib- protein may play a direct role in the pathogenesis of tubuloin- tered protein holotransferrin (Tf-Fe). In in vivo studies, we terstitial injury that develops in association with chronic gb- have demonstrated that iron accumulates within proximal tu- merulonephritis. However, the actual mechanisms by which bule lysosomes in humans with a variety of renal diseases proteinuria induces tubulointerstitial disease have not been (12,14) and in rats with partial nephrectomy, streptozotocin- delineated. Several hypotheses have been proposed to explain induced diabetic nephropathy, Adriamycin nephropathy, and how proteinuria might damage the tubular epithelium. For puromycin nephrosis (12-18). The accumulation of iron in example, it has been suggested that obstructing intrabuminal proximal tubular bysosomes correlated with proteinuria and proteinaceous casts might cause damage due to pressure effects was associated with evidence of oxidative stress and functional and structural injury (12-18). Received April 2. 1997. Accepted June 25. 1997. Thus, there is indirect evidence that Tf-Fe may cause dam- Part of this work was presented in abstract form at the 27th annual meeting of age to the proximal tubular epithelium. However, it is difficult the American Society of Nephrobogy held October 26 to 29, 1994. in Orlando, to localize changes to the proximal tubule cell and differentiate FL. the effects of individual proteins by in vivo studies. Cell culture Correspondence to David C. H. Harris. Department of Renal Medicine, West- can overcome these problems and can be used to study cyto- mead Hospital. Westmead, NSW 2145, Australia. toxicity under defined conditions, without the confounding 1046-6673/0901 -0077$03.00/0 Journal of the American Society of Nephrology background of humoral and nervous factors present in intact Copyright 0 1998 by the American Society of Nephrology animals. Thus, the present studies have used proximal tubule 78 Journal of the American Society of Nephrology cell culture to examine the differential toxicity of individual with the enzyme assay or decreased the total enzyme content. Results proteins and the mechanisms of injury of one of these proteins, are expressed as the percentage of LDH leakage. Tf-Fe. Malondialdehyde Assay Materials and Methods Mabondialdehyde (MDA), a lipid peroxidation product, was mea- Animals sured by the thiobarbituric acid reaction, as described previously (25), This study was approved by the Animal Care and Ethics Commit- but with some modification. Cells were washed three times with 0.01 tee of the Western Sydney Area Health Service, and experiments M phosphate buffer and then transferred into 1-mi Eppendorf tubes. conformed to standards of the National Health and Medical Research After centrifugation. cells were resuspended in 80 pA of distilled water Council. Male Wistar rats weighing 200 to 250 g were housed under and lysed using an ultrasonic homogenizer. Aliquots were taken for conditions of constant temperature (22#{176}C)and humidity on a 12-h MDA measurement (40 ,.d) and for protein measurement (40 l). light/dark cycle (light on 6 am. to 6 p.m.) with free access to Samples (40 l) were added to an 80-Ml mixture of 8.1% sodium commercial rat pellets (Allied Foods, Sydney, Australia) and water. dodecyl sulfate, 0.8% thiobarbituric acid reaction, and 20% acetic acid (2: 15: 15, vol/vol/vol), and then heated in boiling water for 70 mm. After cooling with tap water, 20 jtl of distilled water and 100 pA of a Isolation and Priman’ Culture of Proximal Tubule mixture of n-butanol and pyridine (I 5: 1 , vol/vol) were added and Cells vortexed. Samples were centrifuged at 4000 rpm for 10 mm, the Rat renal proximal tubule segments were isolated according to the organic layer was separated. and its absorbance at 532 nm was method of Vinay et a/. (22). Briefly. rats were anesthetized with measured (Beckman DU-68 Spectrophotometer, Fullerton, CA) using ketamine (40 mg/kg, intraperitoneally) and xylazine (4 mg/kg, intra- MDA bis (dimethyl acetal) as the standard (Aldrich, Milwaukee, WI). peritoneally), and kidneys were perfused via the aorta with Krebs- Protein content was determined using Lowry’s method (26). MDA Henseleit buffer. After removal, kidneys were cut in half, the medulla values are expressed as picomoles per microgram of protein. was carefully dissected out, and the cortex was cut into small pieces and digested in Krebs-Henseleit solution with 0. 1% collagenase and 5% bovine serum albumin (BSA) at 37#{176}Cfor20 mm. After digestion, Iron Measurement the cell suspension was filtered through a 50 mesh and centrifuged at Iron content in culture medium and cells was determined by flame- 2000 rpm for 2 mm in 32% Percoll (Sigma-Aldrich, NSW, Australia). less atomic absorption spectroscopy. using a Perkin-Elmer Spectro- This procedure yielded a preparation consisting primarily of proximal photometer (PE 3030) (27). After treatment, medium was transferred tubule fragments (>95%) with approximately 90% viability as as- to iron-free tubes. Cells were rinsed twice with 0.01 M phosphate sessed by exclusion of the vital dye trypan blue. Tubule fragments buffer and harvested, and resuspended cells (in I ml of iron-free were suspended in the mixture of Dulbecco’s modified Eagle’s me- water) were transferred to an iron-free tube and lysed using an dium (DMEM) and nutrient F- I 2 Ham ( 1 : 1 ) medium supplemented ultrasonic homogenizer. The results were expressed as nanomoles per with 10% fetal calf serum and plated onto plastic 30-mm culture dish. dishes coated with rat tail collagen. Twenty-four hours later, unat- tached tubules were washed away and new medium was added with- out fetal calf serum but with 10 ng/ml epidermal growth factor, 5 Uptake of Transferrin mg/mI insulin, 5 mg/ml transferrmn, and 5 X l0 M hydrocortisone. The uptake oftransferrmn by proximal tubule cells was measured by Thereafter, culture medium was changed every other day. Proximal replacing the culture medium with DMEM and F-l2 Ham medium tubule cells became confluent after 4 d of culture. The proximal origin containing ‘ 25I-transferrmn-Fe, according to the method of Trinder of cultured cells was supported by their expression of proximal (28). ‘25I-transferrin-Fe (0.54 pmol) was added to the cell culture brush-border enzymes, ultrastructure, and formation of domes. Con- medium in pH 7.4 or pH 6.0 and incubated for 1 h at 37#{176}C,and fluent monolayers were assayed in situ for the brush-border enzyme internalized radioactivity was measured.