Nephrol Dial Transplant (2000) 15: 16–22
Nephrology Dialysis Transplantation
Original Article
Acute glomerular upregulation of ornithine decarboxylase is not essential for mesangial cell proliferation and matrix expansion in anti-Thy-1-nephritis Markus Ketteler, Ralf Westenfeld, Alexander Gawlik, Emile de Heer1 and Armin Distler Department of Endocrinology and Nephrology, University Hospital Benjamin Franklin, Free University of Berlin, Germany and 1Department of Pathology, University of Leiden, The Netherlands
Abstract Background. Pathways of -arginine metabolism including nitric oxide, agmatine and polyamine synthesis are upregulated during glomerular inflammation in experimental glomerulonephritis. In anti-Thy1-glomerulonephritis -arginine-deficient diets ameliorate the disease course in this model. However, it is unclear which metabolic pathway is affected by this substrate depletion. Since polyamines are important proproliferative molecules, we studied the effect of specific polyamine synthesis blockade in vivo on mesangial cell proliferation and glomerular fibrosis in this model. Methods. Anti-Thy-1-glomerulonephritis was induced in male Sprague–Dawley rats by single-bolus injection of monoclonal ER4-antibodies. Rats were treated with difluoromethylornithine (0.5–2% in the drinking water), a selective inhibitor of the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC ). Mesangial cell proliferation and matrix expansion were evaluated in PAS-stained kidney tissues. Glomerular TGF-b and biglycan-mRNA-expression were determined by Northern blot analysis and albuminuria was measured using a competitive ELISA. Data were compared to untreated controls. Results. Though complete inhibition of ODC activity was achieved at any time point, difluoromethlornithine treatment had no significant effect on albuminuria, glomerular matrix protein expression and mesangial cell count in this model. Conclusions. The acute upregulation of glomerular ODC activity above baseline in anti-Thy1-glomerulonephritis is not pathophysiologically important for disease development however, biological effects of available polyamine pools cannot be excluded by our study. Key words: arginine metabolism; glomerulonephritis; Correspondence and offprint requests to: Markus Ketteler, MD, Department of Endocrinology and Nephrology, University Hospital Benjamin Franklin, Hindenburgdamm 30, D-12203 Berlin, Germany. E-mail:
[email protected].
mesangial proliferation; nitric oxide; ornithine decarboxylase; polyamines
Introduction Since the discovery of nitric oxide (NO) as a multifunctional effector molecule, -arginine metabolism has attracted much attention as a central mechanism in the control of haemodynamics, neurotransmission, inflammation and tissue repair [1–4]. The arginine/NO-system is known to be involved in the regulation of glomerular perfusion pressure, in tubuloglomerular feedback regulation and in the control of renin release [5]. In models of renal disease, de novo expression of the inducible nitric oxide synthase (iNOS ) has been observed and may play a role as a mediator of inflammatory processes with both deleterious and beneficial effects dependent on the experimental conditions [6–9]. However, upregulation of other -arginine metabolizing enzymes including arginase, arginine decarboxylase (ADC ) and ornithine decarboxylase (ODC ) was also observed in some models of renal disease and may have pathophysiologically relevant impacts on the course of kidney diseases [3,4,10,11]. Arginase produces -ornithine from -arginine and, by competing with nitric oxide synthases for their common substrate, may locally alter the magnitude of NO-synthesis [3,4,8,10]. -ornithine is the substrate for polyamine synthesis (putrescine, spermidine, spermine). Polyamines are small cationic molecules and essential for tissue growth in most living organisms [12,13]. ODC is the rate-limiting enzyme of polyamine synthesis and shares expression characteristics with some immediate early genes, while the proto-oncogene c-myc is involved in the control of ODC transcription [12–14]. Polyamines were shown to play a major role in the induction and regulation of cell differentiation and proliferation in a large number of cells including mesangial cells in vitro [12,13,15–17]. Studies per-
© 2000 European Renal Association–European Dialysis and Transplant Association
ODC inhibition in experimental glomerulonephritis
formed in cultured glomerular mesangial cells conclusively demonstrated that ODC activity is important for mesangial cell proliferation and may be controlled by an array of growth factors including platelet-derived growth factor (PDGF ) [15,16 ]. Recently, a potential role for ODC in the induction of apoptosis was also described [18,19]. Upregulation of the expression and activity of ODC associated with increases of tissue polyamine levels was previously described in a number of models of tissue injury and repair including some models of kidney diseases [20–24]. Biphasic upregulation of ODC in the early phase of anti-Thy-1-glomerulonephritis (antiThy-1 GN ) was also observed in vivo with the second peak coinciding with the onset of mesangial cell proliferation and growth-factor overexpression [10]. Further, substrate depletion by feeding -argininedeficient diets to nephritic rats ameliorated the disease course in this model while it remained unclear which pathway of -arginine metabolism was affected by this treatment modality [9]. Mesangial cell proliferation and apoptosis are key factors in determining the disease development of mesangioproliferative anti-Thy-1 GN. Since increased polyamine synthesis was suggested to play a significant role in this context, difluoromethlornithine (DFMO)induced inhibition of ODC activity seemed an appropriate tool to directly evaluate the pathophysiological role of acute polyamine synthesis in the outcome of this model. The influence of this treatment approach on albuminuria, mesangial cell proliferation and glomerular matrix expansion was subsequently determined in a time course experiment and compared with untreated controls. Effects of DFMO-treatment in rat models of experimental glomerulonephritis were previously not reported.
Methods
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lected in metabolic cages on days 1, 3 and 7. Rats were sacrificed under ether anaesthesia at 6 h, on day 3 and on day 7 following disease induction. After renal perfusion with 25 ml ice-cold phosphate-buffered saline (PBS ), kidneys were excised. Cortex slices of the left kidneys were formalin-fixed or snap-frozen in ODC buffer, respectively. The remainder of the kidneys were subjected to glomerular isolation using graded sieving as previously described [9,10].
Morphology and mesangial cell count Formalin-fixed kidney tissue sections were stained with periodic acid Schiff (PAS ). Glomerular matrix expansion was scored in a blinded fashion by two investigators as previously published (matrix score: 1=normal glomerular morphology–4=diffuse and complete glomerulosclerosis) [10]. Mesangial cell count was determined by counting cell nuclei in 35 glomerular cross-sections per animal [10]. Swollen nuclei at the 6-h-time point were regarded as severely damaged cells and excluded from counting.
Albuminuria measurements Rat albuminuria was determined by using a modified competitive ELISA technique [26 ]. Ninety-six-well-plates (NuncImmuno Maxisorp, Denmark) were coated with rat albumin (solution: 0.2 mg/l in 0.1 M sodium bicarbonate) by incubation at 37°C for 3 h and at 4°C for an additional 12 h using 100 ml per well. The plates were subsequently washed three times with buffer A (20 mM diethylmalonic acid, 150 mM sodium chloride, 0.1 mM EDTA, 1 ml Tween 20, 0.5% gelatine; pH 7.4). Twenty-four-hour urines were diluted with buffer A at the following concentrations: 1550, 15500, 155000 and 1520 000. Aliquots of 50 ml of these diluted samples were pipetted into the wells and 50 ml of antibody conjugate (GAR a/ALB/PO) (BIOGENZIA/Nordic Immunologie, Bochum, Germany) diluted 155000 in buffer A were added. After additional washings with buffer A, 200 ml of 0.5 mM TMB were pipetted into each well. The plates were then put on a shaker and incubated for 1 h at room temperature. Extinction was determined every 10 min for 90 min in an automated plate reader at 650 nm. Values were compared to rat albumin standards.
Animal model and study design Ornithine decarboxylase activity assay Eight-week-old male Sprague–Dawley rats were obtained from the Breeding Center Scho¨nwalde, Germany. DFMO was kindly provided by Dr Peter McCann, Hoechst Marion Russell (formerly: Marion Merrell Dow), Madison, WI, US. All chemicals were obtained from the Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany, unless otherwise indicated. Rats (n=4 per group) were intravenously injected into the tail vein with a monoclonal anti-Thy-1-antibody (ER4-hybridoma, 1 mg/kg body weight). DFMO was administered in the drinking water in the following concentrations: $ $ $ $ $
0.5% pretreatment: day −3–day −1; 1% pretreatment: day −1–induction of anti-Thy 1 GN; 1% treatment: day 0–day 1; 1.5% treatment: day 1–day 3; 2% treatment: day 3–day 7.
Increasing doses of DFMO were administered to ensure effective inhibition of polyamine synthesis, since upregulation of ODC gene transcription was previously reported as a counter-regulatory feedback mechanism in the presence of enzyme inhibition [24]. Twenty-four-hour urines were col-
ODC activity was determined by measuring the conversion of -[1-14C ]ornithine (Amersham International, Little Chalfont, UK ) to 14CO as previously described [10]. Briefly, 2 cortex slices were homogenized in assay buffer (50 mmol/l Na HPO , 0.1 mmol/l EDTA, 5 mmol/l NaF, 0.2 mmol/l 2 4 pyridoxalphosphate, 2.5 mmol/l DTT, 2 mmol/l PMSF; pH 7.2) on ice and subsequently centrifuged at 14 000×g for 25 min. Aliquots of 200 ml of the supernatant were subjected to a 15-ml conical centrifuge tube, 0.25 mCi of radiolabelled -ornithine and cold -ornithine (final concentration: 0.33 mmol/l ) were added. After incubation for 1 h at 37°C in a water bath, 200 ml of 2 M citric acid was injected into the tube to release all CO . 14CO was trapped on a filter 2 2 paper soaked with 20 ml NaOH, which was mounted in the top of the tube. After an additional incubation of 45 min at 37°C, filter papers were placed in 5 ml scintillation fluid and subjected to scintillation counting. Protein content of samples was measured by the Bradford method. Linearity of the assay was determined by a standard curve employing bovine ODC (0.1, 0.5, 1 and 2 mU, respectively) under the described
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assay conditions. ODC activity was calculated as mU per g protein. Interassay background activity ranged between 50 and 250 mU/g protein due to condensation of radiolabelled -ornithine on the filter paper. Therefore, ODC activity values below 250 mU/g protein were considered as completely suppressed activity.
Northern blot hybridization Northern blot analysis was performed as previously described [9,10]. Isolated, pooled glomeruli from four rats per group were lysed in 4 M guanidine isothyanate and subsequently ultracentrifuged on a 5.7 M cesium chloride cushion. Following electrophoresis on a 2.2 M formaldehyde/0.9% agarose gel and transfer to a nylon membrane, prehybridization of membranes (5×Denhardt’s solution, 5×SSC, 50 mmol/l sodium phosphate (pH 6.5), 0.1 mg/ml herring sperm, 0.1% SDS, 50% formamide) was performed at 42°C for 3 h and membranes were subsequently hybridized overnight using the following cDNA probes after random primer labelling with [32P]CTP (Boehringer Mannheim, Germany): (i) Rat ODC-cDNA (Dr Jaenne, University of Helsinki, Finland ) [27]; (ii) Murine TGF-b-cDNA (Dr Derynck, San Francisco, CA) [28]; (iii) Human Biglycan-cDNA (Dr Fisher, NIH, Bethesda, MD) [29]; and (iv) Rat GAPDHcDNA (Dr Kondaiah, Dr Sporn, NIH, Bethesda, MD) [30] as a control house-keeping gene. After hybridization, membranes were washed three times in 2×SSC, 0.1% SDS for 5 min at room temperature, and once in 0.1×SSC, 0.1% SDS for 15 min at 50°C. Expression of ODC-, TGF-b- and biglycan-mRNA was quantitated by phophoimaging technique (FUJI Biolmaging Analyzer (Type BAS-IIIs), Raytest, Berlin, Germany) and by comparison to GAPDH-mRNAexpression. Northern blot experiments were performed twice, and increases of mRNA-expression of >50% vs controls and untreated animals at the same time point respectively, were regarded as significant.
Statistics Differences between DFMO-treated and untreated animals at given time points were analysed by unpaired Student’s ttest. Open bars indicate standard deviations in the figures.
Results As shown in Figure 1, complete inhibition of cortical ODC activity could be achieved by our DFMO treatment schedule at any time point under investigation. ODC-mRNA expression was higher in DFMO-treated vs untreated rats in controls and at 6 h, but enzyme activity was totally suppressed at these time points (Figure 2). Though there was a tendency of increased albuminuria in DFMO-treated rats on day 1 and day 7, this result was not statistically significant (Figure 3). Maximal albuminuria on day 3 was not different between DFMO-treated and untreated rats in this model. Mesangial cell count and matrix expansion were evaluated in PAS-stained kidney tissue sections. As shown in Figure 4, the observed reduction in glomerular cell nuclei at 6 h and on day 3 following disease
M. Ketteler et al.
induction was similar in DFMO-treated and untreated rats. Thus, DFMO treatment did not affect anti-Thy1-antibody-induced mesangial cell lysis. Mesangial cell proliferation as expressed by an increased mesangial cell count on day 7 was also not different between the two groups. Further, increased glomerular matrix expansion on day 7 in this model developed to the same degree in both DFMO-treated and untreated animals (Figure 5). Glomerular TGF-b- and biglycan-mRNA-expression were determined as a relevant fibrogenic growth factor and matrix protein, respectively, in the pathophysiology of anti-Thy1-GN (Figure 2). Glomerular TGF-b-mRNA-expression was characteristically increased on day 3 and on day 7 without a difference in magnitude between the two groups. Biglycan-mRNA expectedly decreased during mesangial cell lysis, since mesangial cells are the major source of the proteoglycan biglycan, and significantly increased on day 7 without being affected by DFMO treatment.
Discussion We had previously shown that ODC-mRNA-expression and ODC activity were upregulated in anti-Thy-1 GN and that dietary -arginine restriction, which should have lowered substrate supply for ODC, ameliorated the disease course of this model [9,10]. In this study, we evaluated the role of acute upregulation of glomerular polyamine synthesis in the disease course of experimental mesangioproliferative glomerulonephritis by blocking increased ODC activity in vivo using the enzyme inhibitor DFMO. DFMO treatment successfully blocked cortical ODC activity in our study, however, there were no significant differences in the degree of albuminuria, mesangial cell proliferation, glomerular matrix expansion and in the glomerular expression of TGF-b and biglycan between DFMOtreated and untreated animals. Our treatment approach was started with a 3-day DFMO-pretreatment period, and DFMO concentrations were stepwise increased from 0.5 to 2% in the drinking water. Pretreatment was performed to achieve a near maximal effect by reaching a relative glomerular polyamine depletion prior to the induction of disease. The DFMO dose was gradually increased, because it had been previously suggested that ODC inhibition by DFMO can be counteracted by a dose-dependent increase in ODC gene transcription [25]. Indeed, in pretreated controls and at 6 h after disease induction ODC-mRNA was higher in DFMO-treated rats potentially due to this mechanism, but ODC activity was efficiently blocked despite increased gene transcription. DFMO had been employed as an effective antiproliferative substance in vitro and in some animal models of cancer [12,31]. DFMO has also been previously used in a number of models of kidney diseases [22–24,32–34]. Early kidney hypertrophy in streptozotocin-induced diabetes mellitus could be prevented by DFMO, but later changes in the development of
ODC inhibition in experimental glomerulonephritis
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Fig. 1. Cortical ODC-activity is shown in DFMO-treated and untreated rats in control animals and at 6 h, on day 3 and on day 7 following induction of anti-Thy1-GN (n=4 per time point). In untreated rats, increased ODC-activity is observed at 6 h and on day 7 in this model. In DFMO-treated rats, ODC-activity is completely suppressed at any time point. Data are given as means±standard deviation (open bars); *P0.05; open bars represent standard deviations). Albuminuria in control animals was
