L-citrulline attenuates arrested alveolar growth and pulmonary hypertension in oxygen-induced lung injury in newborn rats

Abstract

Bronchopulmonary dysplasia (BPD) is characterized by arrested alveolar development and complicated by pulmonary hypertension (PH). NO promotes alveolar growth. Inhaled NO (iNO) ameliorates the BPD phenotype in experimental models and in some premature infants. Arginosuccinate synthetase (ASS) and arginosuccinate lyase (ASL) convert L-citrulline to L-arginine; L-citrulline is regenerated during NO synthesis from L-arginine. Plasma levels of these NO precursors are low in PH. We hypothesized that L-citrulline prevents experimental O2-induced BPD in newborn rats. Rat pups were assigned from birth through postnatal day (P) 14 to room air (RA), RA + L-citrulline, 95% hyperoxia (BPD model), and 95%O2 + L-citrulline. Rat pups exposed to hyperoxia had fewer and enlarged air spaces and decreased capillary density, mimicking human BPD. This was associated with decreased plasma L-arginine and L-citrulline concentrations on P7. L-citrulline treatment significantly increased plasma L-arginine and L-citrulline concentrations and increased ASL protein expression in hyperoxia. L-citrulline preserved alveolar and vascular growth in O2-exposed pups and decreased pulmonary arterial medial wall thickness (MWT) and right ventricular hypertrophy (RVH). Increased lung arginase (ARG) activity in O2-exposed pups was reversed by L-citrulline treatment. L-citrulline supplementation prevents hyperoxia-induced lung injury and PH in newborn rats. L-citrulline may represent a novel therapeutic alternative to iNO for prevention of BPD.

Figure 1. Schematic of the citrulline-arginine-NO pathway in endothelial cells

This cartoon depicts the intracellular enzymes that convert citrulline to arginine (ASS and ASL) as well as the enzymes eNOS that convert arginine to NO and citrulline, and ARG II, that converts arginine to ornithine and urea.

Figure 2. Effect of L-citrulline administration from postnatal days (P) 4 to 14 on amino acid plasma levels and NOx levels in normoxic control rats and O₂-induced BPD rats

Plasma levels of L-citrulline (A), L-arginine (B), ornithine (C) and NOx (D) were measured on P7, 10 and 14 (N=5/group, *P<0.05 and **P<0.0005, normoxia vs hyperoxia, §P<0.001, normoxia (■) and hyperoxia (◆) vs normoxia+L-Citrulline (⨯) and hyperoxia+L-Citrulline (▲). Inset shows plasma levels of L-Citrulline in normoxia (■) and hyperoxia (◆) only

Figure 3. L-Citrulline prevents alveolar simplification in O₂-induced BPD in newborn rats

Representative H&E–stained lung sections show larger and fewer alveoli in hyperoxia-exposed lungs (A), resulting in a significantly higher mean linear intercept (Lm) (B) and lower septal counts than in control (normoxia) animals (C). Treatment with L-citrulline preserved alveolar structure and significantly improved Lm and septal counts as compared with the experimental BPD model. N=5/group, **P<0.001 hyperoxia vs normoxia, *P<0.01 hyperoxia vs normoxia+L-Citrulline and hyperoxia+L-Citrulline for Lm. For septal counts, §P<0.0001 hyperoxia vs normoxia, **P<0.001 hyperoxia vs normoxia+L-Citrulline and hyperoxia vs L-Citrulline, *P<0.01 normoxia vs hyperoxia+L-Citrulline.

Figure 4. L-Citrulline improves lung angiogenesis in O₂-induced BPD in newborn rats

Capillary density, as quantified by the number of barium-filled pulmonary arteries counted per high-power field, was significantly decreased in hyperoxia-exposed lungs compared with room air controls. Treatment with L-citrulline preserved lung capillary density in experimental BPD. N=5/group, §P<0.0001 hyperoxia vs normoxia and normoxia+L-Citrulline, **P<0.001 hyperoxia vs hyperoxia+L-Citrulline, *P<0.02 normoxia+L-Citrulline vs hyperoxia+L-Citrulline.

Figure 5. L-Citrulline prevents pulmonary hypertension in O₂-induced BPD in newborn rats

A. Hyperoxia-exposed rats had significant RVH as indicated by the increase in RV/LV+S ratio compared with normoxic controls. L-Citrulline reduced RVH. N=5/group, *P<0.05 hyperoxia vs other groups. (B) Representative H&E stained lung sections showing pulmonary arteries displaying a thickened medial arterial wall in hyperoxic rat lungs compared with normoxic controls (scale bar is 65μm, original magnification is 4×). (C) L-Citrulline significantly reduced the %MWT compared with pulmonary arteries from untreated hyperoxic rat lungs. N=5/group, §P<0.0001 hyperoxia vs normoxia and normoxia+L-Citrulline, **P<0.002 hyperoxia vs hyperoxia+L-Citrulline, *P<0.05 normoxia vs hyperoxia+L-Citrulline.

Figure 6. Effects of hyperoxia and postnatal age on protein abundance in rat lung homogenates

(A) Representative Western blots demonstrating the abundance of eNOS, ASL, ASS and β-Actin in protein lysates from normoxic (lanes 1–3) and hyperoxic (lanes 4–6) rat lungs on P7, P10 and P14. Blots are representative of two separate studies performed on a total of 6–8 normoxic and hyperoxic neonatal rat lungs at each postnatal time point.(B). Summary densitometry data showing the effects of hyperoxia (□) on eNOS, ASS and ASL protein expression relative to β-actin at each of three postnatal ages. *P<0.05 different from age-matched normoxic control (■) group.

Figure 7. Effects of L-citrulline treatment on the abundance of proteins in the citrulline, arginine, NO regenerating pathway on P7 and P10

(A and C) Representative western blots demonstrating the abundance of eNOS, ASS, ASL, Arg II and β-actin in protein lysates from L-citrulline-treated and untreated normoxic (left panel, Lane 1–3: normoxia; Lane 4–6: normoxia+L-Cit) and hyperoxic (right panel, Lane 1–3: hyperoxia; Lane 4–7: hyperoxia+L-Cit) rat lungs on P7 (7A) and P10 (7C). Blots are representative of two separate studies performed on a total of 6–8 untreated and citrulline-treated normoxic and hyperoxic neonatal rat lungs. (B and D) Summary densitometry showing the effect of L-citrulline (□) on lung protein abundance of eNOS, ASS, ASL and Arg II on P7 (7B) and P10 (7D). *P<0.05 different from untreated normoxic or hyperoxic control (■) group.

Figure 8. Effects of hyperoxia and L-citrulline treatment on protein abundance of arginase II and arginase activity in P7 rat lung homogenates

(A) Representative western blots of three separate studies performed on a total of 6 untreated (lane 1–2) and citrulline-treated (lane 3–4) normoxic and untreated (lane 5–6) and citrulline-treated (lane 7–8) hyperoxic neonatal rat lungs. (B) Summary densitometry showing that both hyperoxia and L-citrulline independently and synergistically increased arginase II expression in neonatal rat lungs. *P<0.05 different than normoxia; **P<0.05 different from all other groups. (C) L-Citrulline decreases pulmonary arginase activity in O₂-induced BPD in newborn rats. Hyperoxic exposed rats had significantly increased lung arginase activity on postnatal day 7 (P7). L-Citrulline normalized lung arginase activity in hyperoxic exposed rats. Arginase activity was unchanged in L-citrulline treated control rats. N=5/group, *P<0.05.