As in human disease, macrophages (MØ) are central players in the development and progression of experimental atherosclerosis. In this study we have evaluated the phenotype of MØ associated with progression of atherosclerosis in the apolipoprotein E (ApoE) knockout (KO) mouse model.We found that bone marrow-derived MØ submitted to M1 and M2 polarization specifically expressed arginase (Arg) II and Arg I, respectively. This distinct arginase expression was used to evaluate the frequency and distribution of M1 and M2 MØ in cross-sections of atherosclerotic plaques of ApoE KO mice. Early lesions were infiltrated by Arg I+ (M2) MØ. This type of MØ favored the proliferation of smooth muscle cells, in vitro. Arg II+ (M1) MØ appeared and prevailed in lesions of aged ApoE KO mice and lesion progression was correlated with the dominance of M1 over the M2 MØ phenotype. In order to address whether the M2->M1 switch could be due to a phenotypic switch of the infiltrated cells, we performed in vitro repolarization experiments. We found that fully polarized MØ retained their plasticity since they could revert their phenotype. The analysis of the distribution of Arg I- and Arg II-expressing MØ also argued against a recent recruitment of M1 MØ in the lesion. The combined data therefore suggest that the M2->M1 switch observed in vivo is due to a conversion of cells already present in the lesion. Our study suggests that interventional tools able to revert the MØ infiltrate towards the M2 phenotype may exert an atheroprotective action.

Arginase II (AII) has been almost exclusively studied in male mammalian kidneys. Our investigations were conducted to localize AII gene expression in the female mouse kidney, and to analyze the differential expression of AII gene at the transcriptional and translational levels in the kidneys of female and male mice. Total RNAs and soluble proteins extracted from renal zones and whole kidneys were analyzed by Northern and Western blots, respectively. Mitochondrial and cytosolic proteins were analyzed by Western blot. L-[guanidino-14C]arginine hydrolysis by AII was detected in microdissected tubules and the 14CO2 released from [14C]urea hydrolysis was quantified. The results of these experiments showed that: (1) both AII mRNA and protein were highly expressed in the deep cortex and the outer stripe of the outer medulla, (2) urea was produced mainly in the proximal straight tubules (PST), (3) the 38-kDa AII protein was more abundant in the mitochondria than the cytosol, and (4) the renal content of AII mRNA and protein was about three-fold higher in female than in male mice. In conclusion, in both genders, AII gene expression is restricted to the PST and localized into mitochondria. AII gene is differentially expressed in the kidney of female and male mice since higher levels of AII mRNA, protein and activity were observed in the kidneys of the former than those of the latter. Renal AII gene expression was gender-dependent in mice but not in rats. Finally, in the PST of females, L-arginine-derived ornithine may be a precursor for the renal production of L -glutamate and L-glutamine because high levels of AII, ornithine aminotransferase and glutamine synthetase are expressed in this nephron segment.

Arginine production was measured in isolated rat nephron segments. Segments were incubated with 0.3 mM aspartate and 0.1 mM L-[ureido-14C]-citrulline in a sealed chamber. Arginase and urease were added to the medium to hydrolyze arginine and to release 14CO2, which was trapped in KOH and counted. Arginine synthesis was found only in the proximal tubule, with decreasing intensity from proximal convoluted (PCT) to proximal straight tubule (PST). Results were as follows (in fmol.min-1.mm tubule length-1): PCT, 122 +/- 15; cortical PST, 71 +/- 6; outer medullary PST, 41 +/- 4; all other segments, less than 6. Arginine synthesis changed almost proportionally with precursor concentration of less than or equal to 0.4 mM. We had shown previously that PST but not PCT was able to hydrolyze arginine into urea and ornithine. In this study arginine was further hydrolyzed in cortical (40%) and medullary (64%) PST but not in PCT. These observations suggest that the arginine formed in PCT contributes to the maintenance of the whole body arginine pool, whereas most of the arginine formed in PST might contribute, by its conversion to urea, to the process of urine concentration.

.-In the kidney, L-ornithine is reabsorbed along the proximal convoluted tubule (PCT), transported by basolateral carriers, and produced by arginase II (AII). Here, the renal metabolic fate of L-ornithine was analyzed in male and female rats. Kidneys and renal zones were dissected and used for Western blot analysis, immunofluorescence, and electron microscopic studies. Ornithine aminotransferase (OAT) and AII were localized using specific antibodies. Ornithine oxidation was determined by incubating microdissected tubules with L-[1-14 C] or L-[U-14 C]ornithine in the presence or absence of energy-providing substrates. Ornithine decarboxylase (ODC) mRNAs were localized by in situ hybridization. The 48-kDa OAT protein was detected in male and female kidneys, but its level was fourfold higher in the latter. OAT relative distribution increased from the superficial cortex toward the outer medulla to reach its highest level. Almost all OAT protein was localized in cortical and medullary proximal straight tubules (CPST and OSPST, respectively). In proximal straight tubule (PST), AII protein distribution overlapped that of OAT. No gender difference in AII protein level was found. OAT and AII were colocalized within PST mitochondria. 14 C]ornithine demonstrated the complete oxidation of ornithine. In conclusion, the OAT gene was expressed more in female rat proximal tubules than in male. Because OAT and AII proteins overlapped in PST mitochondria, L-argininederived ornithine may be preferentially converted to L-glutamate, as proven by ornithine oxidation. However, the coexpression of ODC, glutamate decarboxylase, and glutamine synthetase in PST suggests that L-ornithine can also be metabolized to putrescine, GABA, and L-glutamine. The fate of L-ornithine may depend on the cellular context. L-ornithine; L-arginine; proximal tubules; isolated nephron segments; Western blot analysis; immunofluorescence; electron microscopy; mitochondria; ornithine decarboxylase THE AMINO ACID L-ORNITHINE is absent from food and is not a constituent of proteins; therefore, vertebrate organisms are dependent on its formation. In kidneys, four sources of Lornithine can be considered: 1) L-ornithine is present in the blood and then transported across the basolateral membranes of a variety of renal cells (36,40); 2) L-ornithine is filtered in glomeruli and reabsorbed by specific carriers located in the apical membrane of the proximal convoluted tubule (PCT) (43); 3) in PCT, the enzyme L-arginine:glycine amidinotransferase (GAT; EC 2.1.4.1) metabolizes L-arginine in the presence of L-glycine and produces guanidinoacetic acid and Lornithine (42); and 4) in the whole proximal straight tubule (PST) of several mammals including rats, the extrahepatic arginase II isoenzyme (AII; EC 3.5.3.1) hydrolyzes L-arginine into urea and L-ornithine (23).L-Ornithine is known to play a pivotal role in several metabolic pathways as precursor for L-proline, L-glutamate, L-glutamine, L-citrulline, and L-arginine biosynthesis. Interestingly, renal tubular cells contain...