Dimethylarginine metabolism during acute and chronic rejection of rat renal allografts

Abstract

Background: Dimethylarginines are inhibitors of NO synthesis and are involved in the pathogenesis of vascular diseases. In this study, we ask the question if asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) levels change during fatal and reversible acute rejection, and contribute to the pathogenesis of chronic vasculopathy.

Methods: The Dark Agouti to Lewis rat strain combination was used to investigate fatal acute rejection. Fischer 344 kidneys were transplanted to Lewis rats to study reversible acute rejection episode and the process of chronic rejection. Isograft recipients and untreated Lewis rats were used as controls. l-arginine derivatives were determined by HPLC, and ADMA-metabolizing enzymes were studied by quantitative RT-PCR and western blotting.

Results: Renal transplantation transiently increased dimethylarginine levels independent of acute rejection. ADMA plasma levels did not importantly differ between recipients undergoing fatal or reversible acute rejection, whereas SDMA was even lower in recipients of Fisher 344 grafts. In comparison to isograft recipients, ADMA and SDMA levels were slightly elevated during reversible, but not during the process of chronic rejection. Increased dimethylarginine levels, however, did not block NO synthesis. Interestingly, protein methylation, but not ADMA degradation, was increased in allografts.

Conclusions: Our data do not support the concept that renal allografts are protected from fatal rejection by dimethylarginines. Dimethylarginines may play a role in triggering chronic rejection, but a contribution to vascular remodelling itself is improbable. In contrast, differential arginine methylation of yet unknown proteins by PRMT1 may be involved in the pathogenesis of acute and chronic rejection.

Fig. 1

ADMA, SDMA and L-arg plasma concentration. ADMA (A), SDMA (B) and L-arg (C) were quantified by HPLC in the blood plasma from healthy control rats (C, white), isograft recipients (iso, dotted), and allograft recipients of DA and F344 donors (allo, grey). Graft recipients were investigated on Day (d) 4, 9 and 42 post-transplantation. Data are displayed as median (bar), 25–75 percentiles (box), and the highest and lowest data point (whiskers); circles indicate data beyond 3 × standard deviation; P-values ≤0.05 and number of animals (n) used in the study are indicated.

Fig. 2

DDAH mRNA expression and activity in renal grafts. DDAH1 (A) and DDAH2 (B) mRNA expression were analysed by qRT–PCR. Data are expressed as the ΔCt of PBGD as a house-keeping gene, and DDAH1 or DDAH2. Healthy control kidneys (C, white), isografts (iso, dotted), and allografts of DA and F344 donors (allo, grey) were investigated on Day (d) 4, 9 and 42 post-transplantation. (C, D) For the measurement of DDAH activity, excess amounts of ADMA and SDMA were added to protein extracts form control kidneys, isografts and allografts, and incubated for 2 h at 37°C. Thereafter, ADMA and SDMA were measured by HPLC. (C) HPLC chromatograms of a typical experiment: the upper panel shows a sample before, and the lower panel after incubation at 37°C. Note that in contrast to ADMA, SDMA is not degraded. (D) Quantification of ADMA degradation; circles indicate data beyond 3 × standard deviation; P-values ≤0.05 and number of animals (n) used in the study are indicated. (A, B, D) Data are displayed as median (bar), 25–75 percentiles (box), and the highest and lowest data point (whiskers).

Fig. 3

PRMT1 expression in renal tissue. Homogenates of healthy control kidneys (C, white), isografts (iso, dotted), and allografts of DA and F344 donors (allo, grey) were separated by SDS–polyacrylamide (12%) gel electrophoresis. PRMT1 expression was (A, C, E) analysed by western blotting on Day (d) 4, 9 and 42 post-transplantation. The intensity of the resulting bands (B, D, F) was quantified by densitometry and divided by the values obtained for the house-keeping protein GAPDH. The mean of the PRMT1/GAPDH ratio of the controls was set to 1, and each individual value including the control values was calculated accordingly. Data are displayed as median (bar), 25–75 percentiles (box), and the highest and lowest data point (whiskers); P-values ≤0.05 and number of animals (n) used in the study are indicated.

Fig. 4

Localization of PRMT1 in renal isografts and allografts. Immunohistochemistry using antibodies to PRMT1, a CD68-like antigen (macrophages), and the β-chain of the α/β T-cell receptor (T lymphocytes) was performed on paraffin sections of renal isografts (iso), DA allografts (allo) on Day (d) 4 post-transplantation. Immunopositive structures were stained in brown, and the sections were lightly counter-stained with hemalum. Arrows are pointing to small arteries. Note the strongly PRMT1-immunoreactive infiltrate consisting of macrophages and T lymphocytes in the perivascular region as well as in the interstitium of renal allografts. The micrograph is representative for at least three independent experiments.

Fig. 5

NOS protein expression. Homogenates of healthy control kidneys (C, white), isografts (iso, dotted), and allografts of DA and F344 donors (allo, grey) were separated by SDS–polyacrylamide (8%) gel electrophoresis. Grafts were investigated on Day (d) 4 (A, D), 9 (B, F) and 42 (C, H) post-transplantation. Western blots were performed using specific antibodies to iNOS (A–C) and eNOS (D–H). The positive control for iNOS expression is a Day 4 DA allograft (B, C). The intensity of the resulting bands was quantified by densitometry and divided by the values obtained for the house-keeping protein GAPDH (E, G, I). The mean of the eNOS/GAPDH ratio of the controls was set to 1, and each individual value including the control values was calculated accordingly (E, G, I). Data are displayed as median (bar), 25–75 percentiles (box), and the highest and lowest data point (whiskers); P-values ≤0.05 and number of animals (n) used in the study are indicated.

Fig. 6

NO_x concentration in plasma and renal tissue. Rat plasma (A) or renal tissue extracts (B) were subjected to Griess reaction. Untreated healthy control rats (C, white), isograft recipients (iso, dotted) and allograft recipients of DA and F344 kidneys (allo, grey) were investigated. Plasma and grafts were investigated on Day (d) 4, 9 and 42 post-transplantation. Data are displayed as median (bar), 25–75 percentiles (box), and the highest and lowest data point (whiskers); circles indicate data beyond 3 × standard deviation; P-values ≤0.05 and number of animals (n) used in the study are indicated.