Association of immunosuppressant-induced protein changes in the rat kidney with changes in urine metabolite patterns: a proteo-metabonomic study

Abstract

The basic mechanisms underlying calcineurin inhibitor (CI) nephrotoxicity and its enhancement by sirolimus are still largely unknown. We investigated the effects of CIs alone and in combination with sirolimus on the renal proteome and correlated these effects with urine metabolite pattern changes. Thirty-six male Wistar rats were assigned to six treatment groups (n = 4/group for proteome analysis and n = 6/group for urine (1)H NMR metabolite pattern analysis): vehicle controls, sirolimus 1 mg/kg/day, cyclosporine 10 mg/kg/day, cyclosporine 10 mg/kg/day + sirolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day + sirolimus 1 mg/kg/day. After 28 days, 24 h-urine was collected for (1)H NMR-based metabolic analysis and kidneys were harvested for 2D-gel electrophoresis and histology. Cyclosporine affected the following groups of proteins: calcium homeostasis (regucalcin, calbindin), cytoskeleton (vimentin, caldesmon), response to hypoxia and mitochondrial function (prolyl 4-hydroxylase, proteasome, NADH dehydrogenase), and cell metabolism (kidney aminoacylase, pyruvate dehydrogenase, fructose-1,6-bis phosphate). Several of the changes in protein expression, confirmed by Western blot, were associated with and explained changes in metabolite concentrations in urine. Representative examples are an increase in kidney aminoacylase expression (decrease of hippurate concentrations in urine), up regulation of pyruvate dehydrogenase and fructose-1,6-bisphosphatase, (increased glucose metabolism), and down regulation of arginine/glycine-amidino transferase (most likely due to an increase in creatinine concentrations). Protein changes explained and qualified immunosuppressant-induced metabolite pattern changes in urine.

Figure 1. Representative histology (HE stain) of kidney tissues from the different treatment groups

The total number of tissue samples evaluated was n=6/ treatment group. All figures: ×440 magnification, except for B: 7220 magnification to better illustrate the overall appearance of tubular dilation. None of the groups showed structural changes of either glomerular or vascular aspects and changes caused by the immunosuppressants alone and in combination at the doses tested were mostly focused on the tubuli. A) kidney sample from vehicle-treated control B) 1 mg/kg/d sirolimus: minor tubular epithelial atrophy and luminal dilation, C) 1 mg/kg/d tacrolimus: no differences compared to vehicle-treated controls, D) 1 mg/kg/d tacrolimus+ 1 mg/kg/day sirolimus: tubular dilation similar to B), in addition dilated tubular lumen filled with cytoplasmic vesicles (blebs) leading to luminar narrowing in some parts. E) 10 mg/kg/d cyclosporine: macro- and microvesicular intracellular vacuolization of the tubular epithelial cells combined with marked shrinking (atrophy) and luminal dilation of the proximal and distal tubular cytoplasm, F) 10 mg/kg/d cyclosporine+ 1 mg/kg/day sirolimus: same effects as described for E) but more progressed damage. Areas adjacent to the tubuli appeared normal leading to an overall inhomogeneous appearance.

Figure 2. Changes in urinary metabolite patterns after 28 days as assessed using ¹HNMR spectroscopy

Metabolites known to be associated with proximal tubulus injury (21) are shown. Significance level: * p<0.05 (Tukey test pairwise comparison to controls). All values are normalized based on the total integral to compensate for differences in urine concentrations and are presented at means + standard deviations (n=6). The arrows show the expected direction of changes indicating tubular damage (21,30). Abbreviations: CsA10: 10 mg/kg/d cyclosporine, CsA10+Srl1: 10 mg/kg/d cyclosporine+ 1 mg/kg/d sirolimus, Srl1: 1 mg/kg/d sirolimus, Tac1: 1 mg/kg/d tacrolimus, Tac1+Srl1: 1 mg/kg/d tacrolimus + 1 mg/kg/d sirolimus.

Figure 3. Representative ¹H-NMR spectra of urine samples

The total number of urine samples evaluated for each group was n=6. Signal assignments: 1 valine leucine isoleucine 2 sirolimus drug vehicle compounds, 3 lactate, 4 alanine, 5 acetate, 6 succinate, 7 2-oxoglutarate, 7 citrate, 8 dimethylamine, 10 trimethylamine, 11 dimethyl glycine, 12 creatine, 13 creatinine, 14 trimethylamine oxide, 15 taurine, 16 hippurate, 17 glucose, 18 α-glucose. Abbreviations: CsA10: 10 mg/kg/d cyclosporine, CsA10/Srl1: 10 mg/kg/d cyclosporine+ 1 mg/kg/d sirolimus, Srl1: 1 mg/kg/d sirolimus, Tac1: 1 mg/kg/d tacrolimus, Tac1/Srl1: 1 mg/kg/d tacrolimus + 1 mg/kg/d sirolimus.

Figure 4. Changes in concentration of proteins that are involved in structural integrity and contractile processes based on the analysis of 2D-gels

Significance levels: *: p<0.05 (Tukey test pairwise comparison to controls). All values are means± standard deviations (n=4). Abbreviations: CsA10: 10 mg/kg/d cyclosporine, CsA10+Srl1: 10 mg/kg/d cyclosporine+ 1 mg/kg/d sirolimus, Srl1: 1 mg/kg/d sirolimus, Tac1: 1 mg/kg/d tacrolimus, Tac1+Srl1: 1 mg/kg/d tacrolimus + 1 mg/kg/d sirolimus.

Figure 5. Immunosuppressant-induced changes of regucalcin compared to vehicle controls after 28 days of treatment

The two spots associated with regucalcin as displayed in the IMAGE-Master software are shown. The pI and molecular weight as detected on the gel matched the theoretical values of regucalcin (Figure 2). The spot at pH 4.35 and 33kDa (left spot) is markedly reduced after treatment with sirolimus, cyclosporine and cyclosporine + sirolimus. This is associated with the increase of volume of a corresponding spot (right) at the same molecular mass but at a different pH (4.25). Both spots were independently cut from the gel, digested and identified by HPLC/MS analysis. The right and left spots showed amino acid coverage of 41% and 38% with 10 and 9 distinct amino acids, respectively. Abbreviations: con: vehicle controls, CsA: 10 mg/kg/d cyclosporine, CsA/Srl: 10 mg/kg/d cyclosporine+ 1 mg/kg/d sirolimus, Srl: 1 mg/kg/d sirolimus, Tac: 1 mg/kg/d tacrolimus, Tac/Srl: 1 mg/kg/d tacrolimus + 1 mg/kg/d sirolimus.

Figure 6. Comparison of calbinding-D₂₈ among treatment groups using Western blot analysis

Analysis of variance: p= 0.00015. Significance levels *: *:p<0.05; **: p<0.001; (Tukey method). β-Actin was used as control and was expected not to change. Abbreviations: CsA10: 10 mg/kg/d cyclosporine, CsA10+Srl1: 10 mg/kg/d cyclosporine+ 1 mg/kg/d sirolimus, Srl1: 1 mg/kg/d sirolimus, Tac1: 1 mg/kg/d tacrolimus, Tac1+Srl1: 1 mg/kg/d tacrolimus + 1 mg/kg/d sirolimus.

Figure 7. Comparison of the effects of treatment with immunosuppressants alone and in combination at the doses tested on NADH-ubiquinone oxidoreductase subunit 10 expression in the kidney

Top: Representative 2D-gel regions. The region at 38kDa and pH 6.1 corresponded to NADH-ubiquinone oxidoreductase subunit 10. Bottom: Western blot analysis of the Complex I of the respiratory chain, NADH-ubiquinone oxidoreductase subunit 10. Three different modifications of the subunit 10 were observed. Treatment with different immunosuppressants and their combination resulted in an alteration of the pattern as compared to the control group. The band at 38 kDa in the Western blots (bottom) is labeled with an arrow. The modifications have not yet been identified. The protein changes observed in the Western blots matched those after 2D-gel electrophoresis.