• pulmonary fibrosis;
  • dimethylarginine dimethylaminohydrolase (DDAH);
  • nitric oxide pathway


  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References

Pulmonary fibrosis is a devastating and progressive parenchymal lung disease with an extremely poor prognosis. Patients suffering from idiopathic pulmonary fibrosis (IPF) display a compromised lung function alongside pathophysiological features such as highly increased production of extracellular matrix, alveolar epithelial cell dysfunction, and disordered fibroproliferation – features that are due to a dysregulated response to alveolar injury. Under pathophysiological conditions of IPF, abnormally high concentrations of nitric oxide (NO) are found, likely a result of increased activity of the inducible nitric oxide synthase (NOS2), giving rise to products that contribute to fibrosis development. It is known that pharmacological inhibition or knockdown of NOS2 reduces pulmonary fibrosis, suggesting a role for NOS inhibitors in the treatment of fibrosis. Recent reports identified a critical enzyme, dimethylarginine dimethylaminohydrolase (DDAH), which is exceedingly active in patients suffering from IPF and in mice treated with bleomycin. An up-regulation of DDAH was observed in primary alveolar epithelial type II (ATII) cells from mice and patients with pulmonary fibrosis, where it co-localizes with NOS2. DDAH is a key enzyme that breaks down an endogenous inhibitor of NOS, asymmetric dimethylarginine (ADMA), by metabolizing it to l-citrulline and dimethylamine. DDAH was shown to modulate key fibrotic signalling cascades, and inhibition of this enzyme attenuated many features of the disease in in vivo experiments, suggesting a possible new therapeutic strategy for the treatment of patients suffering from IPF.

Pulmonary fibrosis

  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References


Idiopathic pulmonary fibrosis (IPF) belongs to the family of idiopathic interstitial pneumonias (IIPs); it represents the most aggressive form of a diffuse parenchymal lung disease (DPLD) and affects probably up to 500 000 patients in the western world [1, 2]. With the exception of pirfenidone, there are currently no approved therapies for IPF, and IPF is still a disease with significant morbidity and mortality (median survival probably 2–3 years [3]). Patients suffering from IPF exhibit exertional dyspnoea alongside a non-productive cough, progressively diminished physical exercise capacity, and, in the later stages, the need for long-term oxygen treatment [4]. Further aggravation of the disease is fuelled by recurrent respiratory infections, which are in part due to a compromised immune defence of the lung. Recurrent injury of the alveolar epithelium, alveolar inflammation, and a profound proliferation of fibroblasts characterize IPF and result in a progressive loss of functional alveolar capillary units and lung architecture, as well as scarring of the lung [5, 6]. Today, the best treatment option for patients suffering from IPF is still lung transplantation, which, however, is only available for a small minority of patients and is itself limited by fibroproliferative changes based on the chronic rejection process.

Current understanding

Idiopathic pulmonary fibrosis is a devastating lung disease with an increasing incidence and prevalence with ageing and a median survival of 2–3 years. The pathogenesis of IPF is not fully understood: initially, IPF was thought to result from generalized pulmonary inflammation leading to fibrosis. But the current paradigm has shifted towards alveolar epithelial cell dysfunction and disordered fibroproliferation [5]. Additionally, an interplay between environmental (eg cigarette smoking) and genetic factors seems to be responsible for this severe disorder. A growing body of evidence suggests that alveolar epithelial type II cell micro-injury and apoptosis are key events in the initiation and progression of lung fibrosis [7, 8]. This results in the responsive proliferative activation of type II cells themselves, of yet to be defined epithelial progenitor cells, and in a paracrine fashion of the surrounding or invading mesenchymal cells, largely myofibroblasts. These peculiar fibroblast foci represent the hallmark lesions of IPF, as they secrete excessive amounts of extracellular matrix (ECM) components, thereby destroying the lung parenchyma [9]. The occurrence of fibroblast foci represents an important prognostic factor and correlates with survival in IPF [10]. Hence, IPF might be considered an imperfect or aberrant repair process that derails and becomes pathogenic. Several soluble mediators, such as transforming growth factor (TGF)-β or interleukin (IL)-1β, have been assigned a clear pathogenic role in IPF and experimental models thereof.

Current treatment options

As a result of the authorization of pirfenidone in Europe through the European Medicines Agency at the end of 2011, there is now one drug available for IPF patients in Europe [11]. Preclinical data strongly suggest that fibroblast proliferation, expression of fibrosis-associated proteins and cytokines (TGF-β and PDGF), biosynthesis and accumulation of extracellular matrix, as well as accumulation of inflammatory cells and TNF-α synthesis are all reduced by pirfenidone [12-16]. Initially, there was only limited enthusiasm for this drug, as two pivotal trials conducted in the USA and Europe showed opposite results with regard to their primary endpoint [17]. However, when additional trials conducted in Japan are also included [11], it is evident that pirfenidone is effective in IPF, resulting in less-pronounced worsening of lung function and physical exercise. As IPF has been repeatedly compared to malignant diseases, the viewpoint of accepting rather small improvements by novel drugs is increasingly accepted [18]. Eventually, IPF will most likely deserve a multi-drug treatment approach similar to pulmonary hypertension in order to completely stop disease progression. To this end, it is worth mentioning that despite so many other drugs having failed in phase III (among these, also the long-used steroid/azathioprine combination [19-22], endothelin receptor antagonists, interferon gamma, and TNF-blockers), yet another anti-fibrotic drug (BIBF 1120, a novel triple kinase inhibitor blocking FGF, PDGF, and VEGF) has promising effects in phase II studies [23] and is currently undergoing rigorous evaluation in phase III trials. Likewise, anti-oxidant treatment modalities such as high-dose N-acetyl-cysteine (NAC), already suggested to be effective in the context of an earlier trial also including steroids and azathioprine [24], are currently still under separate investigation within the PANTHER trial and it is to be hoped that this regimen turns out to be effective in IPF also.

Nevertheless, the condition of patients on anti-fibrotic therapies may still worsen, and lung transplantation, if applicable at all, then represents the only conceivable and effective treatment. However, lung transplantation is limited by organ availability and the 5-year survival rate after lung transplantation in IPF is only 44% [25].

It therefore appears essential to enlarge the spectrum of putative treatment modalities for IPF by developing novel drugs that interfere on the level of the more preceding triggering events in IPF rather than the (late-stage) scarring process.


  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References

One of the most important regulators of vascular structure and tone is the nitric oxide (NO) signalling pathway. A potent source of NO is epithelial cells that are mechanically stretched by ventilation, thereby regulating the blood flow to ventilated areas of the lung [26]. NO is derived from nitric oxide synthases (NOS), which synthesize NO by converting l-arginine to citrulline. The three NOS isoforms identified to date are neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). Each NOS is a distinct gene product and differs from the others in subcellular localization, tissue distribution, and regulation [27]. NO is produced by a variety of cells within the respiratory tract, including airway epithelial cells, airway nerves, inflammatory cells, and vascular epithelial cells. Accordingly, NO production by NOS has been shown to be involved in key regulatory processes such as bronchomotor control [28, 29], inflammation [30, 31], and host defence [32]. NO exhibits distinct physiological functions among tissues and cell types: (1) NOS1 produces NO as a neurotransmitter in the central nervous system as well as a neurotransmitter in the gastrointestinal tract. It may also effect penile erection and sphincter relaxation. (2) NOS2 plays an important role in the inflammatory response and in cellular signalling. (3) NOS3 is involved in regulation of the cardiovascular system, stimulating the relaxation of endothelial tissue of blood vessels via the production of NO. Furthermore, NO produced by NOS3 inhibits the activation of platelet aggregation. The physiological actions of NO are mediated by its haem protein sensor, the soluble guanylyl cyclase (sGC), which is activated upon binding NO, resulting in the synthesis of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP is a second messenger with a variety of effector mechanisms (eg vascular tone, cellular apoptosis). As NO has many diverse but central physiological functions in combination with a high reactivity and high permeability as a gaseous molecule, it is tightly regulated to protect cells from damage and malfunction [33].

NO production, on the one hand, depends on the NOS activity and protein expression. On the other hand, NO production absolutely depends on the availability of l-arginine to NOS, since NOS shares l-arginine as a common substrate with arginase. In addition, endogenous NOS inhibitors such as asymmetric and symmetric dimethylarginine (ADMA, L-NMMA, and SDMA) were shown to regulate NO production [34]. These endogenous guanidino-methylated arginines, such as asymmetric and symmetric dimethylarginine (ADMA, SDMA), are released as a result of the cleavage of post-translationally methylated tissue proteins containing methylarginine residues via protein-arginine methyl transferases (PRMTs) [35]. To date, three forms of methylarginines have been found in mammalian cells: symmetric dimethylarginine (SDMA); N-monomethyl-l-arginine (L-NMMA); and asymmetric dimethylarginine (ADMA). Only the asymmetrically methylated arginine residues (L-NMMA and ADMA) are competitive inhibitors of the three nitric oxide synthase (NOS) enzymes, while the symmetrically methylated arginine (SDMA) has no effect on NOS. Initially, analogues of l-arginine that were chemically modified by methylation, such as N-monomethyl-l-arginine (L-NMMA), were used for pharmacological inhibition of NOS, until it was also found to be a naturally occurring compound. Due to the competition of ADMA and L-NMMA with arginine for the active site of NOS, accumulation of asymmetrically methylated arginine residues leads to inhibition of NO synthesis. In 1988, Palmer et al demonstrated the inhibition of NO formation by L-NMMA in cultured vascular endothelial cells [36].

Once methylarginines are released from methylated proteins by proteolysis, they are eliminated from the body by a combination of renal excretion and metabolism. However, the clearance routes differ between the symmetrically and asymmetrically methylated forms: SDMA is cleared almost entirely by the kidney without further metabolism, whereas ADMA and L-NMMA are metabolized extensively. It has been described that in some species over 90% of the generated ADMA is metabolized rather than excreted [37]. L-NMMA and ADMA are mainly inactivated by hydrolysis to citrulline and dimethylamines by the action of dimethylarginine dimethylaminohydrolases (DDAH) 1 and 2 or are exported from the cell to the plasma and taken up by other cells via system y + carriers of the cationic amino acid transporter (CAT) family [38]. CAT is involved in both cellular release and uptake of ADMA. While ADMA and L-NMMA are potent direct inhibitors of all three NOS isoforms [35], SDMA attenuates NOS function indirectly by inhibition of the cationic amino acid transporter (hCAT), which mediates the intracellular uptake of arginine [39].

Since ADMA is metabolized by DDAH and its plasma levels are regulated by DDAH, DDAH can determine the bioavailability of NO [38]. Consequently, maintaining the homeostatic integrity of the cardiovascular system is a pivotal role of DDAH. Under conditions of NO over-production, S-nitrosylation of DDAH diminishes its activity, leading to an accumulation of ADMA. Subsequently, a type of regulatory feedback mechanism may result from NOS inhibition. In 1999, Leiper et al reported that DDAH has two isoforms, DDAH1 and DDAH2, which are expressed in bacteria, sheep, mice, rats, and humans [40]. Tojo et al found that DDAH and NOS isoforms are co-expressed in cells, leading to the idea that NO activity may be regulated by DDAH-induced changes in cellular ADMA concentration in a cell-specific manner [41]. Their findings were supported by the report by MacAllister et al in 1996 showing that reduced expression of DDAH results in elevated levels of ADMA and that pharmacological inhibition of DDAH by S-2-amino-4(3-methylguanidino)butanoic acid (4121 W) leads to increased ADMA concentrations and a reduction in NO-mediated vasodilation [42]. The specific affinity of DDAH for ADMA has been explained by its molecular structure, as DDAH1 has a substrate-binding pocket which exclusively allows the non-methylated nitrogen side-chain of ADMA to enter. DDAH activity was found mainly in the kidney, pancreas, liver, and brain [43]. Leiper et al found that DDAH1 is highly expressed in the brain and kidney, locations with high expression levels of NOS1. In contrast, DDAH2 is highly expressed in the heart, placenta, and kidney. While NOS1 is not detectable in the heart and placenta, NOS3 is highly expressed in these tissues. This phenomenon (substrate specificity, distribution in NO-generating systems) suggests a possible isoform-specific regulation of NOS via a modulation of the methylarginine concentration [40].

DDAH in pulmonary fibrosis

  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References

Both beneficial and deleterious effects of NO have been demonstrated in the airways. In several lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) or pulmonary fibrosis, markedly increased production of NO has been shown to occur [44]. Some studies suggest that pulmonary fibrosis results from abnormal alveolar wound repair and remodelling in the lung; it can be assumed that NO may contribute to the pathogenesis of pulmonary fibrosis [45] as experimental and clinical wound-healing studies indicate that NO is a critical mediator of wound repair [46]. The animal models of pulmonary fibrosis (eg bleomycin-induced lung injury) and general wound models also exhibit strong similarities with respect to inflammatory responses, coagulation cascades, and other downstream responses [47]. This finding led to the assumption that the NO response occurring in pulmonary fibrosis is not inevitably caused by a unique mechanism, but may rather represent a more general response to wound repair [48]. In pulmonary fibrosis, the increased production of NO has been linked to increased expression of NOS2 in the alveolar epithelium. In 2004, Jang et al demonstrated an increased level of NO metabolites in bronchoalveolar lavage (BAL) fluids and overexpression of NOS2 in murine fibrotic lungs in response to bleomycin challenge [49]. From experimental studies, it is known that pharmacological inhibition or knockdown of NOS2 reduces pulmonary fibrosis, suggesting a role for NOS inhibitors in the treatment of fibrosis [50]. Several inhibitors of NOS2 have been described such as L-NIL, 1400 W or BYK191023 [51, 52], displaying a promising in vivo activity suitable for mechanistic studies on the role of selective NOS2 inhibition as well as clinical development for the treatment of diseases with increased NOS2 activity.

ADMA, being an endogenous inhibitor of NOS2, blocks NO synthesis from NOS2 [53] and is expected to show beneficial effects in fibrosis per se. In contrast, ADMA was reported to promote pulmonary fibrosis by up-regulation of arginase-1 and collagen synthesis [44]. It was shown that the lung is a major source of ADMA and that the clearance of this methylation product is mainly mediated by DDAH1 [54]. Moreover, in several studies, ADMA has also been identified as a risk factor for endothelial dysfunction-associated diseases such as hypercholesterolaemia, diabetes mellitus, and pulmonary hypertension in humans [55-57]. In 2009, Wells et al demonstrated that direct infusion of ADMA results in increased collagen deposition in murine lungs and an enhanced arginase activity [44], a feature of experimental pulmonary fibrosis. These findings clearly show a role for ADMA in the pathogenesis of pulmonary fibrosis. However, the causal relationship between lung injury, ADMA metabolism, and remodelling remains to be studied in future experiments [53] as the mechanisms are still unknown.

In 2011, Pullamsetti et al reported that DDAH is expressed in alveolar type II (ATII) cells of healthy lungs, and also reported increased expression and activity levels of DDAH in ATII cells in a murine model of pulmonary fibrosis (eg bleomycin-induced lung fibrosis) and in the lungs of patients with IPF [48]. It was therefore hypothesized that DDAH may be an important regulator of ATII cell biology. It was demonstrated that DDAH2 and NOS2 co-localize in the same cell compartment of the lung and have a close spatial relationship to thickened septa and scar tissue. This is to be expected if DDAH regulates NOS2 in the fibrotic lung [48]. These findings suggest a crucial role for DDAH in the regulation of NOS2 under conditions of pulmonary fibrosis. Further studies with DDAH1-overexpressing mice [58, 59], which displayed markedly more severe bleomycin-induced pulmonary fibrosis than wild-type controls, corroborated that DDAH contributes to the fibrotic process. Experimental studies confirmed the role of DDAH signalling in pulmonary fibrosis in vivo: bleomycin-challenged mice treated with the selective DDAH inhibitor L-291 displayed reduced pulmonary fibrosis, reduced collagen deposition and overall cellularity, and their lung function was restored to near-normal levels without affecting inflammatory cell infiltration, with the exception that L-291 reduced neutrophils in NOS2-deficient but not in wild-type mice [48]. These functional changes are characteristic of reduced levels of endogenous NO, and the same results were observed when ATII cells were treated with ADMA. Additionally, treatment of bleomycin-challenged mice with ADMA and L-NIL, a highly selective NOS2 inhibitor, as well as treatment of NOS2-deficient mice challenged with bleomycin with the selective DDAH inhibitor L-291 demonstrated that in bleomycin-induced fibrosis, NOS2 inhibition decreased fibrosis and an even stronger reduction was observed after simultaneous inhibition of DDAH [48]. In conclusion, DDAH inhibition reduces fibroblast-induced collagen production in an ADMA-independent manner and reduces abnormal epithelial proliferation in an ADMA-dependent manner (Figure 1). Thus, Pullamsetti et al stated that DDAH inhibition may offer a therapeutic approach for attenuation of pulmonary fibrosis [48].


Figure 1. A model for the role of DDAH and ADMA in pulmonary fibrosis depicting the mechanisms whereby DDAH produces proliferative and fibrotic effects in both ADMA-dependent and ADMA-independent manners. Elevated ADMA levels inhibit NOS activity leading to (1) reduced production of NO and (2) increased l-arginine bioavailability for metabolism by arginase. This results in (1) decreased production of NO and (2) increased arginase activity, both contributing to elevated production of collagen and aggravation of pulmonary fibrosis.

Download figure to PowerPoint

Several mechanisms may explain the enhanced expression of DDAH in pulmonary fibrosis and its role in the regulation of NO homeostasis. In 2003, Ueda et al reported that cytokines increase DDAH expression and activity [60]. In corroboration, Pullamsetti [48] and co-workers found that TGF-β1 up-regulates DDAH2 expression in a time-dependent manner in vitro. TGF-β1 is a pro-fibrotic cytokine which is up-regulated at the sites of fibrogenesis and is involved in a variety of cellular responses that contribute to fibrosis [61]. Additionally, another well-known fibrogenic cytokine, IL-6, also mediates the transcriptional modulation of DDAH2 in ATII cells [62]. Epithelial cell damage, aberrant tissue regeneration, epithelial–mesenchymal transition, and fibroproliferation, all of which are pathomechanisms found in pulmonary fibrosis, may be regulated by DDAH as pro-fibrotic mediators induce DDAH2 in vitro.

In pulmonary fibrosis, it is still unclear whether hyperplastic epithelial cells play a beneficial (providing a source for alveolar epithelial cell renewal) or a detrimental role (acting as producers of pro-fibrotic signals). Such uncertainty also stems from the profound differences between clinical IPF and the widely used bleomycin model, especially with regard to the kinetics of epithelial injury (chronically modest in IPF; acutely massive in the bleomycin model). It was demonstrated by Kim et al in 2006 that alveolar type II epithelial cells undergo an epithelial–mesenchymal transition. This can contribute significantly to the pool of expanded fibroblasts after lung injury [63]. In hyperplastic epithelial cells, increased DDAH2 expression was reported by Pullamsetti et al., who also demonstrated that proliferation of ATII cells is markedly inhibited and apoptosis is increased by DDAH [48]. Treatment with the specific DDAH inhibitor L-291 led to decreased proliferation and increased apoptosis of ATII cells, which were isolated from murine bleomycin-treated lungs in vitro, and inhibited cell proliferation in vivo. This led to the assumption that L-291 blocks ATII cell hyperplasia and activation, attenuating bleomycin-induced fibrosis [48]. Pullamsetti et al also investigated the effects of a DDAH2-selective siRNA on alveolar epithelial cell proliferation and apoptosis in vitro and found that not only the pharmacological inhibition of DDAH by L-291, but also the DDAH2-selective siRNA attenuated alveolar epithelial cell proliferation and apoptosis, showing the role of DDAH in these processes [48].

A key finding of the experimental studies of Pullamsetti et al is that collagen deposition, as well as overall cellularity, is markedly decreased by DDAH inhibition and that treatment of bleomycin-challenged mice with the DDAH inhibitor prevented pulmonary fibrosis, with near-normalization of lung function. They assumed that DDAH inhibition may provide a new therapeutic approach for patients with IPF as DDAH inhibition has no effect on inflammatory cell influx but modulates key fibrotic signalling cascades [48]. The effect of DDAH on collagen deposition may not be mediated by its inhibition of ADMA as exogenous applied ADMA decreases proliferation and induces apoptosis, but only partially affects lung compliance and has no influence on collagen deposition [48]. Studies conducted in activated human lung fibroblasts confirmed that the DDAH inhibitor and exogenous ADMA exert differential effects on extracellular matrix (ECM) production and only the DDAH inhibitor was able to decrease collagen production [48].

Role of DDAH in other forms of fibrosis

  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References

Liver cirrhosis

Liver cirrhosis represents the terminal stage of chronic liver diseases and is characterized by the replacement of liver tissue by fibrosis, scar tissue, and regenerative nodules, leading to a loss of liver function. These changes in tissue structure lead to impaired perfusion of the liver, possibly causing portal hypertension. As endothelial dysfunction and NO deficiency in the intrahepatic circulation represent a major feature of the disease, dimethylarginines may also play an important role in the pathophysiology of liver cirrhosis [64]. Indeed, the literature supports the view that increased vascular resistance is due partly to reduced intrahepatic NOS3 activity [65]. Furthermore, an alteration of hepatic DDAH expression and/or activity in liver diseases leads to high intrahepatic ADMA levels, endothelial dysfunction, and increased intrahepatic resistance as suggested by Mookerjee et al [66]. Lluch et al reported in 2004 that cirrhotic patients exhibit elevated peripheral ADMA and NO levels and suggested that ADMA might oppose the peripheral vasodilation caused by excessive systemic NO production during liver cirrhosis [67].

Wound healing

Both inflammation and resolution of the inflammatory response are processes that are involved in wound healing. The complex wound-healing process is divided into three phases: (1) infiltration of inflammatory cells; (2) degranulation of platelets; and (3) proliferation of fibroblasts and epithelial cells. NO is one important factor regulating wound healing processes. NOS2 is up-regulated following tissue injury [46] and evidently may be beneficial to normal healing. It was demonstrated that the wound fluid of diabetic patients with foot ulcers contains decreased levels of NO and increased levels of ADMA, indicating impaired wound healing [68]. Furthermore, patients with Peyronie's disease were protected against fibrosis and abnormal wound healing when NOS2 was induced in the fibrotic plaques [69]. As DDAH metabolizes ADMA and therefore regulates its plasma levels, DDAH may play an important role in wound healing.

Renal (interstitial) fibrosis and chronic kidney disease (CKD)

A major feature of CKDs is the deficiency of endogenous NO favouring hypertension, platelet aggregation, and vascular disease development. Vallance et al reported elevated plasma levels of ADMA in patients with CKD [70]. As ADMA is one of the most potent endogenous inhibitors of NOS, it contributes to the excess of cardiovascular morbidity and mortality in CKD patients [71-73]. ADMA represents an independent risk marker for the progression to end-stage renal disease, as demonstrated by cohort studies in patients with CKD [74]. As the kidney plays a central role in ADMA metabolism by DDAH, reduced levels of DDAH were found to contribute to ADMA accumulation and subsequent elevation of blood pressure in an experimental model of CKD [75]. It seems plausible that DDAH may exhibit a protective role against renal damage in CKD by suppressing the inhibitory effect of ADMA on NO generation [76]. Therefore, substitution of DDAH protein or enhancement of its activity may become a new therapeutic approach for the treatment of CKD.


  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References

Over the last 10 years, biological functions and mechanisms of the NO–ADMA–DDAH pathway have been revealed, and much is now known about the regulation of DDAH in experimental models. However, the clinical significance of this pathway in pulmonary fibrosis remains to be investigated further. Zakrzewicz and Eickelberg stated that elevated serum/alveolar/pulmonary ADMA levels lead to vascular and/or interstitial remodelling in the lung, but the causal relationship between lung injury, ADMA metabolism, and remodelling remains to be studied in future experiments as the mechanisms are still unknown [53]. In 2011, Pullamsetti et al identified DDAH as one of the critical enzymes in IPF which contributes to the disease development and progression [48]. DDAH is excessively active in patients with IPF as well as in mice subjected to IPF-like lung injury by bleomycin instillation. Once the activity of DDAH is inhibited, many features of the disease are attenuated. Still, further investigations are needed to evaluate the clinical relevance of the NO–ADMA–DDAH pathway and to test new compounds in clinical studies to stimulate the development of a new therapeutic approach targeting DDAH for the treatment of human pulmonary fibrosis in order to lessen this devastating disease.

Author contribution statement

  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References

WJ contributed to literature searches and writing; SSP contributed the section on DDAH in pulmonary fibrosis and to proofreading; JC and NW contributed to proofreading and editing; AG contributed the clinical background and to proofreading; and RTS supervised editing of all the text.


  1. Top of page
  2. Abstract
  3. Pulmonary fibrosis
  4. The NO–ADMA–DDAH axis
  5. DDAH in pulmonary fibrosis
  6. Role of DDAH in other forms of fibrosis
  7. Outlook
  8. Author contribution statement
  9. References
  • 1
    Coultas DB, Zumwalt RE, Black WC, et al. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med 1994; 150: 967972.
  • 2
    Hodgson U, Laitinen T, Tukiainen P. Nationwide prevalence of sporadic and familial idiopathic pulmonary fibrosis: evidence of founder effect among multiplex families in Finland. Thorax 2002; 57: 338342.
  • 3
    American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med 2002; 165: 277304.
  • 4
    King TE Jr, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet 2011; 378: 19491961.
  • 5
    Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001; 134: 136151.
  • 6
    Thannickal VJ, Toews GB, White ES, et al. Mechanisms of pulmonary fibrosis. Annu Rev Med 2004; 55: 395417.
  • 7
    Korfei M, Ruppert C, Mahavadi P, et al. Epithelial endoplasmic reticulum stress and apoptosis in sporadic idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2008; 178: 838846.
  • 8
    Mahavadi P, Korfei M, Henneke I, et al. Epithelial stress and apoptosis underlie Hermansky–Pudlak syndrome-associated interstitial pneumonia. Am J Respir Crit Care Med 2010; 182: 207219.
  • 9
    Katzenstein AL, Myers JL. Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am J Respir Crit Care Med 1998; 157: 13011315.
  • 10
    King TE Jr, Schwarz MI, Brown K, et al. Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am J Respir Crit Care Med 2001; 164: 10251032.
  • 11
    Taniguchi H, Ebina M, Kondoh Y, et al. Pirfenidone in idiopathic pulmonary fibrosis. Eur Respir J 2010; 35: 821829.
  • 12
    Gurujeyalakshmi G, Hollinger MA, Giri SN. Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level. Am J Physiol 1999; 276: L311L318.
  • 13
    Iyer SN, Gurujeyalakshmi G, Giri SN. Effects of pirfenidone on transforming growth factor-beta gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis. J Pharmacol Exp Ther 1999; 291: 367373.
  • 14
    Iyer SN, Gurujeyalakshmi G, Giri SN. Effects of pirfenidone on procollagen gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis. J Pharmacol Exp Ther 1999; 289: 211218.
  • 15
    Oku H, Shimizu T, Kawabata T, et al. Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis. Eur J Pharmacol 2008; 590: 400408.
  • 16
    Schaefer CJ, Ruhrmund DW, Pan L, et al. Antifibrotic activities of pirfenidone in animal models. Eur Respir Rev 2011; 20: 8597.
  • 17
    Noble PW, Albera C, Bradford WZ, et al. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet 2011; 377: 17601769.
  • 18
    Vancheri C, du Bois RM. Progression-free end point for idiopathic pulmonary fibrosis trials: lessons from cancer. Eur Respir J 2012; DOI: 10.1183/09031936.00115112
  • 19
    Behr J. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med 2012; 367: 869; 870–871.
  • 20
    Izumi S, Iikura M, Hirano S. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med 2012; 367: 870; 870–871.
  • 21
    Raghu G, Anstrom KJ, King TE Jr, et al. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med 2012; 366: 19681977.
  • 22
    Uruga H, Hanada S, Kishi K. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med 2012; 367: 870; 870–871.
  • 23
    Richeldi L, Costabel U, Selman M, et al. Efficacy of a tyrosine kinase inhibitor in idiopathic pulmonary fibrosis. N Engl J Med 2011; 365: 10791087.
  • 24
    Demedts M, Behr J, Buhl R, et al. High-dose acetylcysteine in idiopathic pulmonary fibrosis. N Engl J Med 2005; 353: 22292242.
  • 25
    Thabut G, Christie JD, Ravaud P, et al. Survival after bilateral versus single-lung transplantation for idiopathic pulmonary fibrosis. Ann Intern Med 2009; 151: 767774.
  • 26
    Ghofrani H, Osterloh I, Grimminger F. Sildenafil: from angina to erectile dysfunction to pulmonary hypertension and beyond. Nature Rev Drug Discov 2006; 5: 689702.
  • 27
    Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001; 357: 593615.
  • 28
    Hogman M, Frostell C, Arnberg H, et al. Inhalation of nitric oxide modulates methacholine-induced bronchoconstriction in the rabbit. Eur Respir J 1993; 6: 177180.
  • 29
    Nijkamp FP, van der Linde HJ, Folkerts G. Nitric oxide synthesis inhibitors induce airway hyperresponsiveness in the guinea pig in vivo and in vitro. Role of the epithelium. Am Rev Respir Dis 1993; 148: 727734.
  • 30
    Chang RH, Feng MH, Liu WH, et al. Nitric oxide increased interleukin-4 expression in T lymphocytes. Immunology 1997; 90: 364369.
  • 31
    Huang FP, Niedbala W, Wei XQ, et al. Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages. Eur J Immunol 1998; 28: 40624070.
  • 32
    Green SJ, Meltzer MS, Hibbs JB Jr, et al. Activated macrophages destroy intracellular Leishmania major amastigotes by an l-arginine-dependent killing mechanism. J Immunol 1990; 144: 278283.
  • 33
    Knipp M. How to control NO production in cells: Nω,Nω-dimethyl-l-arginine dimethylaminohydrolase as a novel drug target. Chembiochem 2006; 7: 879889.
  • 34
    Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J 2012; 33: 829-837, 837a837d.
  • 35
    Tran CT, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atheroscler Suppl 2003; 4: 3340.
  • 36
    Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from l-arginine. Nature 1988; 333: 664666.
  • 37
    Leiper JM. The DDAH–ADMA–NOS pathway. Ther Drug Monit 2005; 27: 744746.
  • 38
    Palm F, Onozato ML, Luo Z, et al. Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. Am J Physiol Heart Circ Physiol 2007; 293: H3227H3245.
  • 39
    Closs EI, Basha FZ, Habermeier A, et al. Interference of l-arginine analogues with l-arginine transport mediated by the y + carrier hCAT-2B. Nitric Oxide 1997; 1: 6573.
  • 40
    Leiper JM, Santa Maria J, Chubb A, et al. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem J 1999; 343 (Pt 1): 209214.
  • 41
    Tojo A, Welch WJ, Bremer V, et al. Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. Kidney Int 1997; 52: 15931601.
  • 42
    MacAllister RJ, Parry H, Kimoto M, et al. Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase. Br J Pharmacol 1996; 119: 15331540.
  • 43
    Murray-Rust J, Leiper J, McAlister M, et al. Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase. Nature Struct Biol 2001; 8: 679683.
  • 44
    Wells SM, Buford MC, Migliaccio CT, et al. Elevated asymmetric dimethylarginine alters lung function and induces collagen deposition in mice. Am J Respir Cell Mol Biol 2009; 40: 179188.
  • 45
    Geiser T. Idiopathic pulmonary fibrosis – a disorder of alveolar wound repair? Swiss Med Wkly 2003; 133: 405411.
  • 46
    Schwentker A, Vodovotz Y, Weller R, et al. Nitric oxide and wound repair: role of cytokines? Nitric Oxide 2002; 7: 110.
  • 47
    Chambers RC. Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention? Br J Pharmacol 2008; 153 (Suppl 1): S367S378.
  • 48
    Pullamsetti SS, Savai R, Dumitrascu R, et al. The role of dimethylarginine dimethylaminohydrolase in idiopathic pulmonary fibrosis. Sci Transl Med 2011; 3: 87ra53.
  • 49
    Jang AS, Lee JU, Choi IS, et al. Expression of nitric oxide synthase, aquaporin 1 and aquaporin 5 in rat after bleomycin inhalation. Intensive Care Med 2004; 30: 489495.
  • 50
    Genovese T, Cuzzocrea S, Di Paola R, et al. Inhibition or knock out of inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury. Respir Res 2005; 6: 58.
  • 51
    Lehner MD, Marx D, Boer R, et al. In vivo characterization of the novel imidazopyridine BYK191023 [2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3 H-imidazo[4,5-b]pyridine], a potent and highly selective inhibitor of inducible nitric-oxide synthase. J Pharmacol Exp Ther 2006; 317: 181187.
  • 52
    Pullamsetti SS, Maring D, Ghofrani HA, et al. Effect of nitric oxide synthase (NOS) inhibition on macro- and microcirculation in a model of rat endotoxic shock. Thromb Haemost 2006; 95: 720727.
  • 53
    Zakrzewicz D, Eickelberg O. From arginine methylation to ADMA: a novel mechanism with therapeutic potential in chronic lung diseases. BMC Pulm Med 2009; 9: 5.
  • 54
    Li J, Wilson A, Gao X, et al. Coordinated regulation of dimethylarginine dimethylaminohydrolase-1 and cationic amino acid transporter-1 by farnesoid X receptor in mouse liver and kidney and its implication in the control of blood levels of asymmetric dimethylarginine. J Pharmacol Exp Ther 2009; 331: 234243.
  • 55
    Boger RH, Bode-Boger SM, Szuba A, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998; 98: 18421847.
  • 56
    Lin KY, Ito A, Asagami T, et al. Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 2002; 106: 987992.
  • 57
    Pullamsetti S,Kiss L, Ghofrani HA, et al. Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension. FASEB J 2005; 19: 11751177.
  • 58
    Dayoub H, Achan V, Adimoolam S, et al. Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence. Circulation 2003; 108: 30423047.
  • 59
    Hasegawa K, Wakino S, Tatematsu S, et al. Role of asymmetric dimethylarginine in vascular injury in transgenic mice overexpressing dimethylarginine dimethylaminohydrolase 2. Circ Res 2007; 101: e2e10.
  • 60
    Ueda S, Kato S, Matsuoka H, et al. Regulation of cytokine-induced nitric oxide synthesis by asymmetric dimethylarginine: role of dimethylarginine dimethylaminohydrolase. Circ Res 2003; 92: 226233.
  • 61
    Gauldie J, Sime PJ, Xing Z, et al. Transforming growth factor-beta gene transfer to the lung induces myofibroblast presence and pulmonary fibrosis. Curr Top Pathol 1999; 93: 3545.
  • 62
    Shahar I, Fireman E, Topilsky M, et al. Effect of IL-6 on alveolar fibroblast proliferation in interstitial lung diseases. Clin Immunol Immunopathol 1996; 79: 244251.
  • 63
    Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A 2006; 103: 1318013185.
  • 64
    Wiest R, Groszmann RJ. The paradox of nitric oxide in cirrhosis and portal hypertension: too much, not enough. Hepatology 2002; 35: 478491.
  • 65
    Shah V, Toruner M, Haddad F, et al. Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology 1999; 117: 12221228.
  • 66
    Mookerjee RP, Vairappan B, Jalan R. The puzzle of endothelial nitric oxide synthase dysfunction in portal hypertension: the missing piece? Hepatology 2007; 46: 943946.
  • 67
    Lluch P, Torondel B, Medina P, et al. Plasma concentrations of nitric oxide and asymmetric dimethylarginine in human alcoholic cirrhosis. J Hepatol 2004; 41: 5559.
  • 68
    Petrova N, Edmonds M. Emerging drugs for diabetic foot ulcers. Expert Opin Emerg Drugs 2006; 11: 709724.
  • 69
    Ferrini MG, Vernet D, Magee TR, et al. Antifibrotic role of inducible nitric oxide synthase. Nitric Oxide 2002; 6: 283294.
  • 70
    Vallance P, Leone A, Calver A, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992; 339: 572575.
  • 71
    Abedini S, Meinitzer A, Holme I, et al. Asymmetrical dimethylarginine is associated with renal and cardiovascular outcomes and all-cause mortality in renal transplant recipients. Kidney Int 2010; 77: 4450.
  • 72
    Young JM, Terrin N, Wang X, et al. Asymmetric dimethylarginine and mortality in stages 3 to 4 chronic kidney disease. Clin J Am Soc Nephrol 2009; 4: 11151120.
  • 73
    Zoccali C, Bode-Boger S, Mallamaci F, et al. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 2001; 358: 21132117.
  • 74
    Mihout F, Shweke N, Bige N, et al. Asymmetric dimethylarginine (ADMA) induces chronic kidney disease through a mechanism involving collagen and TGF-beta1 synthesis. J Pathol 2011; 223: 3745.
  • 75
    Matsuguma K, Ueda S, Yamagishi S, et al. Molecular mechanism for elevation of asymmetric dimethylarginine and its role for hypertension in chronic kidney disease. J Am Soc Nephrol 2006; 17: 21762183.
  • 76
    Matsumoto Y, Ueda S, Yamagishi S, et al. Dimethylarginine dimethylaminohydrolase prevents progression of renal dysfunction by inhibiting loss of peritubular capillaries and tubulointerstitial fibrosis in a rat model of chronic kidney disease. J Am Soc Nephrol 2007; 18: 15251533.