Kidney International, Vol. 61 (2002), pp. 876–881

Arginase in glomerulonephritis

SIMON N. WADDINGTON

Gene Therapy Group, Section of Cell and Molecular Biology, Faculty of Medicine, Imperial College School of ScienceTechnology and Medicine, London, England, United Kingdom

Arginase in glomerulonephritis. l-Arginine is converted to ni- examined. There appeared to be an increase in ornithinetric oxide and citrulline by the enzyme nitric oxide synthase decarboxylase at six hours and a delayed increase in(NOS). Its in vivo inhibition has led to the revelation of a ornithine aminotransferase, between days 3 and 21 [3].multitude of diverse, often conflicting functions in the inflam-

After injection of lipopolysaccharide (LPS) in rats, NOSmatory melee. l-Arginine is also converted to ornithine andII and Arginase I mRNA were co-induced within 12urea by the enzyme arginase as a part of the hepatic urea cycle.

However, a more holistic interpretation of the two pathways hours [4]. In contrast, in a model of sepsis, increasedand the associated metabolism (summarized in Fig. 1) has led expression of NOS II was associated with a loss of Argi-to its reassessment as a pathologically significant enzyme. This nase II expression in lung tissue [5].is reflected by the continued increase over the past five yearsof the number of publications discussing both nitric oxide and

Arginine-metabolizing pathways reciprocally inhibitarginase. The strong association between inflammation andeach other in vitrohigh arginase and NOS activity is epitomized by immune com-

plex-induced glomerulonephritis and other glomerulonephriti- These pathways have been shown to inhibit each otherdes. Arginase is encoded by two recently discovered genes

at multiple levels, including substrate competition and(Arginase I and Arginase II). There is now substantial evidenceprotein and mRNA stability and expression.for an interaction between both arginase isoforms and all three

NOS isoforms in pathological situations. This review considers NOS inhibits arginase. Glomeruli were isolated fromthe relationship between Arginases I and II and the inflamma- normal rats and nephritic rats four days after the induc-tion-associated isoform of NOS called NOS II. In particular, tion of accelerated nephrotoxic nephritis. Addition of theit consolidates the current understanding of arginase and asso-

NOS inhibitor NG-monomethyl-l-arginine (L-NMMA)ciated metabolic pathways, and highlights some of the issuessubstantially increased metabolism of arginine by argi-that are often overlooked in such studies.nase over the 48-hour period of culture [6]. Limitationof l-arginine flux through the arginase pathway by NOS

WHAT IS KNOWN ABOUT ARGINASE AND ITSis likely the result of two independent mechanisms. The

ASSOCIATED METABOLISM?first is competition for substrate, discussed later. The

Enzymes associated with arginine metabolism are second is inhibition of arginase by N�-hydroxy-l-argi-up-regulated at inflammatory sites nine, an intermediate in the conversion of arginine to

For example, NOS II and Arginase I have been shown NO. This has been shown to be secreted into the cultureto be up-regulated in glomeruli after induction of heter- supernatant of LPS-stimulated rabbit alveolar macro-ologous nephrotoxic nephritis [1]. A similar increase in phages and has been shown to increase from 4 to 16arginase activity and induction of NOS II was observed �mol/L in the plasma of LPS-treated rats [7]. In 1994in in situ glomerulonephritis [2], and accelerated nephro- Boucher and colleagues demonstrated potent inhibitiontoxic nephritis [3]. These results were confirmed by Ket- of macrophage arginase by N�-hydroxy-l-arginine [8].teler et al, who studied arginine metabolism in isolated This compound also inhibited arginase activity in ne-glomeruli from rats with anti-thymocyte serum-induced phritic glomeruli and in stimulated mesangial cells [6].glomerulonephritis. At day 1, there was a transient maxi- Another possible mechanism by which NOS activitymum of nitrite production, whereas arginase activity was might affect arginase activity is through the accumulationsustained until day 3 of the disease. Ornithine decarbox- of nitrite, a breakdown product of NO. Hrabak, Bajor andylase and ornithine aminotransferase, enzymes leading Temesi demonstrated significant inhibition of inducibleto polyamine and proline production, respectively, were nitric oxide synthase (iNOS) in macrophage lysates, but

not whole cells, by nitrite, with a Km of 4.9 mmol/L [9].A third product of NOS metabolism, citrulline, hasKey words: arginase, nitric oxide, arginine, glomerulonephritis, kidney,

inflammation. been shown to inhibit arginase activity. Shearer and co-workers showed that inhibition of arginase activity dur- 2002 by the International Society of Nephrology

876

Waddington: Arginase in glomerulonephritis 877

Fig. 1. Arginine and associated metabolicpathways. (1) argininosuccinate lyase; (2) argi-nine transporters; (3) arginine decarboxylase;(4) l-arginine:glycine amidinotransferase; (5)arginine-tRNA synthetase; (6) arginase; (7)nitric oxide synthase; (8) ornithine decarbox-ylase; (9) ornithine aminotransferase; (10) or-nithine transcarbamylase.

ing increased iNOS activity was unlikely to be due to lower NO production [20]. Macrophages transfectedwith Arginase promoted growth of breast tumor cells insubstrate competition. However, they showed that LPS

increased intracellular citrulline concentrations above co-culture systems. This effect was down-regulated bythe arginase inhibitor l-norvaline [21]. In contrast, Argi-100 �mol/L, a concentration found to significantly inhibit

arginase activity in macrophage lysates [10]. nase I transfection of RAW 264.7 macrophages increasedthe efferent pathways but did not affect NO productionArginase inhibits NOS. Evidence that arginase could

inhibit the cytotoxic effects of macrophages via NO was [22]. There is also some in vivo evidence for inhibitionof NO production by arginase. For example, diabeticfirst provided by Hibbs, Vavrin and Taintor [11]. A simi-

lar effect was seen in glomerular cultures. NO is gener- tissue in the corpus cavernosum had reduced ability toconvert l-arginine to l-citrulline. This was reversed byated by nephritic glomeruli isolated 24 hours after induc-

tion of accelerated nephrotoxic nephritis; the addition addition of the arginase inhibitor 2(S)-amino-6-boron-hexanoic acid (ABH) [23]. In a model of experimentalof arginase to these cultures completely abrogated its

production [12]. trypanosomiasis, macrophages from infected mice haveincreased arginase activity. Addition of arginase inhibi-The Km of arginase for l-arginine is reported to be

around 7 to 18 mmol/L [13], whereas that of iNOS is tors restored trypanosome killing in vitro. Injection ofarginine into the peritoneal cavity of infected mice in-3 to 9 �mol/L and 130 �mol/L for purified and intact

macrophage enzyme activity, respectively [14–16]. The creased NO production and parasite killing, an effectreversed by addition of L-NMMA [24].concentration of arginine in rat plasma is around 250

�mol/L, and therefore, ornithine production by arginase An additional mechanism by which arginase activityinhibits NO production is through urea generation. NOwill be affected by variations around this level, whereas

the arginine concentration should, in theory, drop con- production in RAW 264.7 macrophages was found to beinhibited by 10 mmol/L urea, a concentration similar tosiderably before NO production is affected [17]. Modo-

lell and colleagues provided evidence supporting this that found in uremic humans [25].Cross-inhibition of other pathways. Several other mech-statement [18]. Mouse bone marrow-derived macro-

phages were stimulated with interleukin-4 (IL-4) for 24 anisms of cross-inhibition have been described. NO hasbeen shown to inhibit ornithine decarboxylase in thehours, after which LPS was added and the culture contin-

ued for a further 24 hours. Cells cultured in medium MCT kidney tubule cell line [26] and in vascular smoothmuscle cells [27]. Inactivation of ornithine decarboxylasecontaining 400 �mol/L arginine had large amounts of

arginase but did not generate NO in response to LPS, in has been shown to occur by specific S-nitrosylation ofthe enzyme [28].contrast to high NO output in 2000 �mol/L. The authors

concluded that arginase was depriving NOS of its sub- Agmatine has been shown to attenuate polyamine syn-thesis by inhibition of ornithine decarboxylase and alsostrate [18]. Corroborative observations were made by

Chang, Liao and Kuo, who examined these enzyme inter- by suppression of the putrescine transporter. Agmatinealdehyde, a metabolite of agmatine, has been shown toactions in J774A.1 mouse macrophages [19]. Cells were

cultured for 12 hours in different concentrations of l-argi- be an inhibitor of iNOS in vitro and in vivo. It is likelythat these effects are responsible for the inhibition ofnine. The arginase inhibitor l-norvaline increased NO

production by 55% and 28% at 0.05 mmol/L and 0.1 cell proliferation and improvement in renal function inthymocyte-1 (Thy-1) glomerulonephritis [29].mmol/L l-arginine, respectively. Outside this range of

arginine concentrations, l-norvaline had no effect. Conversely, polyamines have been shown to inhibitNO production. Szabo and colleagues studied the affectRecently, elegant experiments have been performed

using gene delivery technology. Endothelial cells trans- of polyamines and polyamine derivatives on NO produc-tion in LPS-stimulated J774.2 macrophages finding that,fected with Arginase I or Arginase II had significantly

Waddington: Arginase in glomerulonephritis878

in order of decreasing potency, spermine, spermidine, Arginine-associated enzymes undergotemporal regulationputrescine and cadaverine inhibited nitrite production

in culture. The inhibitory action of polyamines on iNOS Albina and colleagues first demonstrated temporalappeared to depend on the presence of spermine oxidase variation in the activity of NOS II and arginase in a rat[30]. Hrabak and colleagues examined the effect of vari- wound model of implanted polyvinyl sponges. Over theous inhibitors on iNOS activity in rat macrophage lysates first 12 hours, NOS activity prevailed. From 12 hours to[15]. Putrescine inhibited iNOS activity with a Ki of 0.7 three days, the NOS activity rapidly decreased and themmol/L; l-ornithine was also a weak inhibitor. Blachier, arginine concentration rose. From three to ten days, the

arginine concentration fell slowly as wound fluid arginaseMignon and Soubrane analyzed iNOS activity in homog-increased. Disappearance of NOS activity by three daysenates of livers from rats given LPS [31]. NO productioncould not be explained by a dearth of arginine, as culturewas significantly inhibited by spermine at 150 �mol/Lof sponges in high-arginine medium did not elicit nitriteand 500 �mol/L, but by putrescine and spermidine onlyor citrulline synthesis [39, 40].at the higher concentration. Sooranna and Das showed

Cook and co-workers reported a similar pattern ofthat putrescine significantly inhibited nitrite productionenzyme activity [41]. Glomeruli were isolated from theby cultures of the choriocarcinoma cell line BeWo [32].kidneys of rats with acute in situ glomerulonephritis.NOS II activity was induced and arginase activity wasArginase activity can modulate efferent pathwaysincreased in nephritic glomeruli at days 1, 4 and 7, com-Many studies have provided circumstantial evidencepared to normal glomeruli. Whereas the greatest NOSthat arginase activity may be rate-limiting for the produc-activity was at 24 hours, arginase activity peaked at fourtion of proline and polyamines. In a physiological con-days; the activity of both fell rapidly from four to seventext, up-regulation of Arginase II was found to coincidedays. These results were confirmed by Ketteler et al,

with development of connective tissue (rich in proline)who studied arginine metabolism in isolated glomeruli

in Xenopus laevis [33]. In the lactating mammary gland, from rats with anti-thymocyte serum-induced glomerulo-a concerted increase in arginase, ornithine aminotrans- nephritis [3]. At day 1, there was a transient maximumferase and ornithine decarboxylase was consistent with of nitrite production, whereas arginase activity was sus-the increased production of proline and polyamines tained until day 3 of the disease. Ornithine decarboxylase[34, 35]. A pathological up-regulation of these three and ornithine aminotransferase, enzymes leading toenzymes also was observed in anti-thymocyte serum- polyamine and proline production, respectively, wereinduced glomerulonephritis [3]. examined. There appeared to be an increase in ornithine

Harder evidence has been provided by in vitro studies. decarboxylase at six hours and a delayed increase inTransfecting RAW cells with Arginase I increased polya- ornithine aminotransferase between days 3 and 21.mine synthesis [22] (with no effect on NO). Transfecting

There is an underlying pattern to expression of theseendothelial cells with Arginase I and Arginase II in-enzyme systemscreased proline and polyamine synthesis and decreased

The prevailing view is that NOS II and arginase char-NO production [20]. In a similar study, transfecting vas-acterize, and are inherent to, distinct phases of the in-cular smooth muscle cells with Arginase I or treatingflammatory process. Early flux of arginine through NOSthem with IL-4 resulted in increased urea and polyamineII is associated with cytotoxicity, promotion of apoptosisproduction and cellular proliferation [27]. The trans-[42] and nitrosylation of structural components includingforming growth factor-� (TGF-�)–induced increase inbasement membrane. In the aforementioned studies onputrescine and l-proline generation by vascular smoothimplanted sponge models, the authors proposed thatmuscle cells were profoundly inhibited by the arginaseNOS activity, probably from neutrophils, would contrib-inhibitor HOArg [36]. Buga and colleagues demon-ute to microbiostasis and vasodilation in the early wound

strated that inhibition of arginase by HOArg retarded[39, 40].

Caco-2 tumor cell proliferation. Since this effect was In contrast, the delayed flux of arginine through argi-reversed by the addition of ornithine, putrescine, spermi- nase is linked to cell proliferation via polyamine synthe-dine or spermine, arginase appeared to be required by sis [27, 43], collagen synthesis [36, 44] and moderationthese cells to synthesize polyamines for optimal cell of apoptosis [45]. For example, TGF-� increases fibrosisgrowth [37]. Durante et al demonstrated that the polar and wound repair as well as orchestrates the up-regula-phospholipid lysophosphatidylcholine increases arginase tion of a whole armature of arginine metabolism includ-activity, arginine uptake and putrescine production of ing arginase, ornithine aminotransferase and collagensmooth muscle. The arginase inhibitor HOArg was [36]. In contrast, TGF-� down-regulates iNOS inductionshown to strongly inhibit putrescine production in cells in rat renal mesangial cells [46] as well as many other

cell types.[38].

Waddington: Arginase in glomerulonephritis 879

Recently, it has been suggested that NOS II and argi- CAVEATSnase activity are intrinsic to the T-helper 1 and 2 cell There are several issues that often have been omitted(Th1 and Th2) immune responses, respectively. As the from discussion in studies on arginine metabolism, espe-Th1/Th2 dichotomy of murine CD4� T cells became cially in in vivo models.more widely accepted, interest in regulation of arginine

Arginase activity may be increased systemicallymetabolism by cytokines secreted by these subsets in-creased. Th1 cells produce interferon-� (IFN-�), whereas There are three separate points to consider. The firstTh2 cells generate IL-4 and IL-10. Following evidence is that local inflammation may lead to increased arginasethat IL-4 and IL-10 strongly suppressed the induction of activity elsewhere. For example, examination of theiNOS [47, 48], Modolell and Corraliza demonstrated spleens of mice 12 to 48 hours following surgical traumatheir effect on arginase activity in cell lysates of bone revealed increased Arginase I mRNA and protein ex-

pression and arginase activity with no effect on Arginasemarrow-derived murine macrophages [18, 49]. They de-II or NOS II mRNA [52]. The same group also observedmonstrated an increase in cellular arginase content whenan increase in renal arginase activity [53].macrophages were cultured with IL-4 and IL-10. These

The second point pertains to the effect of arginase onresults lent credibility to the hypothesis that Th2 cyto-immune function at sites distal to the site of inflamma-kines were not simply reducing macrophage activationtion. There is conflicting evidence suggesting that argi-but inducing an alternative phenotype. In subsequentnase can both inhibit [54] and promote [55] lymphocytestudies, macrophages were cultured with Th1 or Th2 cellproliferation. Hacker-Shahin and Droge injected spleenclones, or with in vitro polarized Th1 or Th2 cells. Bothcells from one strain of mouse into the peritoneal cavitysystems showed the same pattern; Th1 and Th2 cellsof another [56]. Spleen cells were isolated from theseexclusively induced iNOS and arginase, respectively. Ar-responder mice and tested for their immunological re-ginase was induced to a far greater extent than with Th2sponse to spleen cells of the donor strain (mixed leuko-cytokines alone, and supernatant from Th2 cells alsocyte reaction). Both putrescine and ornithine augmentedinduced arginase. Antibodies against IL-4 and IL-10 sub-the immunogenicity of the cells to the same extent.stantially abrogated the stimulatory effect of the super-Therefore, independently of the local inflammation, thenatant on arginase activity [50]. Recently, it has beensystemic immune response may be subject to regulationshown that this phenotypic dichotomy also extends toby arginine concentrations. This point should be consid-murine macrophages independently of the effect ofered when systemically administering compounds thatT- or B-lymphocytes. C57Bl/6 mice and BALB/c micemodulate activity of arginine-associated enzymes.are prototypical Th1 and Th2 strains, respectively; mac-

This leads to the third point, that the sum of systemicrophages from C57Bl/6 NUDE or SCID mice and

activity of arginase and other arginine-associated en-BALB/c NUDE or SCID mice demonstrated their re-

zymes will influence plasma amino acid levels. For exam-spective Th1 and Th2 phenotypes even in the absence ple, one day after induction of accelerated nephrotoxicof T or B lymphocytes [51]. nephritis, plasma arginine levels were significantly re-

We have observed similar characteristics in isolated duced compared to normal rats [12]. These concentra-glomeruli and mesangial cells. Four days after induction tions are likely to affect the concentrations at the in-of accelerated nephrotoxic nephritis, addition of IL-4 flammatory site as well as in distal organs.caused a large increase in arginase activity in glomerularcultures independent of the profound decrease in NOS The use of specific NOS inhibitors may cross-inhibitactivity over the 48-hour culture period [6]. The same other enzymes or enzyme systemsobservations were made on glomeruli isolated seven days These enzymes, by virtue of their metabolic proximity,after the induction of disease (unpublished data). When act on molecules possessing similar structural character-isolated mesangial cells were stimulated with a combina- istics. For example, the NOS inhibitors L-NIO andtion of cyclic adenosine 3�,5�-monophosphate (cAMP) L-NMMA inhibit uptake of arginine in endothelial cellsand IL-1�, IL-4 exerted the same effect of independently [57] since they are transported by the same cationic y�

increasing arginase activity and inhibiting NOS activity. transporter system as l-arginine [58]. A similar effectThis was accompanied by an increase in cell lysate argi- was demonstrated in rat renal brush border membranenase activity [6]. iNOS and Arginase I mRNA were unde- vesicles [59]. L-NMMA also was shown to inhibit arginasetectable in unstimulated glomeruli whereas Arginase II activity in rat macrophages, most likely by inhibition ofmRNA was detectable. iNOS and Arginase I mRNA arginine uptake [60]. �-Difluoromethylornithine (DFMO)were induced by the cAMP agonist cholera toxin. Addi- is considered to be a potent ornithine decarboxylasetion of IL-4 increased expression of Arginase I mRNA inhibitor, however, it also has been shown to inhibit

arginase activity [61]. Many novel inhibitors of arginaseand abolished expression of iNOS mRNA [1].

Waddington: Arginase in glomerulonephritis880

Reprint requests to Dr. Simon N. Waddington, Gene Therapy Groupand associated enzymes have not been rigorously testedSection, Cell and Molecular Biology, Faculty of Medicine, Imperial

on their metabolic neighbors, upon which they may be College School of Science Technology and Medicine, Sir AlexanderFleming Building, Imperial College Road, London, England SW7 2AZ,having inhibitory effects or even acting as substrates.United Kingdom.E-mail: s.waddington@ic.ac.ukModulation of the NOS pathway may channel

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