We obtained banked citrated plasma samples collected from patients with SCD (n = 63) during steady state, as well as race, age and sex‐matched HbAA controls (n = 27) enrolled in a prior clinical trial (NCT02946905). All participants were of African descent. Among the patients with SCD, 38 had severe disease (HbSS and HbS/β⁰ thalassaemia) and 25 ‘mild’ disease (HbSC and HbS/β⁺ thalassaemia). See Appendix S1 for details of the clinical parameters of the participants. The patients' clinical laboratory data were obtained at the time of their clinic visit. The frozen plasma samples were then batch tested for PNP by ELISA according to the manufacturer's protocol.

Humanised transgenic Townes AA mice (Hba^tm1(HBA)Tow Hbb^{tm3(HBG1,HBB)Tow}) and SS mice (Hba^tm1(HBA)Tow Hbb^{tm2(HBG1,HBB*)Tow}) have been previously described [40, 41, 42]. Six to seven‐week‐old male AA and SS mice were obtained from Jackson Laboratory, and the University of Pittsburgh Animal Care and Use Committee (IACUC) approved the animal use protocol. At 10 weeks of age, the AA and SS mice were randomised to receive 8‐AG (80 mg/kg/day) in drinking water (AA+8‐AG group, n = 9 and SS + 8‐AG group, n = 11) or had ad libitum access to tap water (AA group, n = 9 and SS group, n = 11) for 20 weeks. An additional subset of 10‐week‐old male SS mice was treated with 8‐AG (60 mg/kg/day) in drinking water (SS + 8‐AG group n = 11) or consumed tap water (SS group, n = 11) for 8 weeks.

To determine 8‐AG metabolism and to investigate the effects of PNP inhibition on purine metabolome, urine samples were analysed for urinary adenosine, inosine, guanosine, guanine, hypoxanthine, xanthine, 8‐AG, and 8‐aminoguanine using ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS), as previously described [43].

Plasma haemoglobin and AST, urinary haemoglobin, creatinine and albumin, and liver iron content were determined using colorimetric assays or ELISA kits according to manufacturers' protocols. The kits and reagents used are listed in Table S1.

Closed chest catheterization for in vivo right ventricle (RV) pressure measurements was performed at the University of Pittsburgh Vascular Medicine Institute (VMI) Small Animal Hemodynamic Core Lab, based on a previously documented protocol [44, 45] and as described in Appendix S1. Non‐invasive transthoracic echocardiography of the left ventricle (LV) was performed at the University of Pittsburgh VMI Echocardiography Core using the Vevo 3100 ultrasound machine (FUJIFILM Visual Sonics, Toronto, Canada) as previously described [41, 46]. Focus was on the left heart, as the non‐invasive echo of the right ventricle has a limited visual window and inconsistent reproducibility (details are in Appendix S1).

At the end of each treatment period, the mice were euthanized, and the organs were immediately removed and processed as follows The organs were weighed and bisected or divided into two groups—one was snap‐frozen in liquid nitrogen for biochemical tests and the other was fixed in 10% formalin and embedded in paraffin for histology. Paraffin blocks were prepared and four‐micrometre serial tissue sections from the liver, spleen and right lung were deparaffinised and stained with haematoxylin and eosin (H&E). Lung sections were also stained with pentachrome, and liver and spleen sections for trichrome and Prussian blue. The entire slide sections were scanned, digitised and analysed using the Aperio ScanScope XT system (Aperio Inc., Vista, CA) and Aperio Image Scope software.

Data were analysed using GraphPad Prism for Windows v.10.4.0 (Boston, MA) and NCSS 2022 statistical software (East Kaysville, UT). The results are presented as mean ± SEM and compared using student's T‐tests for unpaired data and two‐factor ANOVA for four groups with post hoc analyses. Spearman Rho test for non‐parametric data was used for correlations. p < 0.05 was considered statistically significant.

Plasma PNP levels were markedly elevated in the patients compared to controls (SCD: 2772 ± 362 vs. CON: 1310 ± 188.1 pg/mL, p = 0.001, Figure 1A) and those with severe disease had higher PNP levels (Severe: 3558 ± 570.9 vs. Mild: 1681 ± 240.9 pg/mL, p = 0.003, Figure 1B) and LDH levels (Severe: 373 ± 21.0 U/L vs. Mild: 249.6 ± 14.0 U/L, p < 0.0001, Figure 1C) than those with mild disease. The PNP levels in the patients with SCD correlated positively with LDH (r = 0.6032, p < 0.0001, Figure 1D) and platelet counts (r = 0.2543, p = 0.0461, Figure 1F), and negatively with haemoglobin (r = −0.4523, p = 0.0002, Figure 1E). Plasma PNP levels also positively correlated with % reticulocytes (Figure 1G), ferritin (Figure 1H), total bilirubin (Figure 1I) and AST (Figure 1J). As expected, the correlations were generally stronger in the patients with severe disease (Table S3).

First, we examined whether Townes mice efficiently metabolise 8‐AG (a pro‐drug) to 8‐aminoguanine, an endogenous purine metabolite and a potent PNP inhibitor [47]. After 8 and 20 weeks of 8‐AG treatment (Figure 2, top and bottom panel, respectively), the SS mice had very negligible urinary concentrations of 8‐AG, albeit higher in the treated SS mice than the untreated group, p < 0.001 (Figure 2A,C). The SS mice treated with 8‐AG for 8 or 20 weeks had 40–50‐fold higher urinary concentration of the active PNP inhibitor 8‐aminoguanine, while the urinary levels of 8‐aminoguanine remained low in the untreated SS mice, p < 0.001 (Figure 2B,D). This indicates efficient conversion of the pro‐drug and weak PNP inhibitor 8‐AG to the potent PNP inhibitor 8‐aminoguanine in 30‐week‐old Townes SS mice even at an advanced stage of disease (Figure 2C,D). Notably, in naïve mice (animals not receiving oral 8‐AG), no endogenous 8‐AG was measurable, whereas endogenous 8‐aminoguanine was detectable in the urine, confirming our previous findings that 8‐aminoguanine is an endogenous metabolite [48].

Analysis of the urinary purine metabolome revealed dysregulated purine metabolism in SS mice compared to age‐matched AA controls (Figure 3). The 30‐week‐old SS mice had reduced urinary adenosine (genotype effect p = 0.002) and lower adenosine/inosine ratio (p = 0.001; Figure 3A,E, respectively) and increased inosine (p = 0.021), hypoxanthine (p = 0.012) and xanthine levels (p = 0.017; Figure 3B–D, respectively), collectively suggesting accelerated purine metabolism and increased activity of ADA, PNP and XO [13, 14, 15]. The higher urinary hypoxanthine levels along with increased guanine levels (p = 0.001, Figure 3I) are also indicative of increased PNP activity as previously reported in SCD patients [13] and in accordance with the elevated plasma PNP levels in patients from the current study (Figure 1). The 20‐week treatment with 8‐AG reduced hypoxanthine (p = 0.036, Figure 3C) and guanine (p = 0.047, Figure 3I) and increased the inosine/hypoxanthine ratio (p = 0.048, Figure 3F) and guanosine/guanine ratio (p = 0.001, Figure 3J), suggesting an efficient inhibition of PNP by 8‐AG in SCD mice (see Figure S1C,G,I,J). Furthermore, 8‐AG treatment did not alter xanthine levels in the treated SS mice (Figure S1D), suggesting no effects of PNP inhibition on XO activity, but reduced the hypoxanthine/xanthine ratio (p = 0.004, Figure 3G), most likely as an indirect effect of 8‐AG due to reduced hypoxanthine and subsequently reduced downstream production of xanthine.

Complete deficiency of PNP (a very rare, inherited disorder) leads to severe combined immunodeficiency, lymphopenia, autoimmune hemolytic anaemia and thrombocytopenia in humans [49]. Thus, we monitored mice blood counts at 0, 10, and 20 weeks of treatment (Figure 4 and Figure S2). As expected, compared to untreated AA mice, the untreated SS mice had higher total white blood cell (WBC, Figure 4A), lymphocyte (Figure 4B), neutrophil (Figure 4C), monocyte (Figure 4D), and eosinophil (Figure 4E) counts. Notably, 8‐AG treatment did not significantly reduce the circulating levels of immune cells in both AA and SS mice.

To further investigate the impact of 8‐AG treatment, we analysed serial RBC parameters in both Townes AA and SS mice during the 20‐week treatment with 8‐AG versus control animals consuming tap water (n = 9–11 per group). As expected, because of chronic hemolysis, the SS mice were anaemic throughout the study period compared to the AA mice (Figure 4F–H and Figure S2). However, whereas the untreated SS mice had worsening anaemia, the progression of anaemia halted at around the 10th week of treatment in the 8‐AG‐treated SS mice suggesting reduced hemolysis. To confirm reduction of hemolysis with 8‐AG treatment, we analysed mouse plasma samples for free haemoglobin and AST and urine for free haemoglobin levels (Figure 4J–L). Plasma free haemoglobin was reduced following 20 weeks of 8‐AG treatment in the SS mice (Figure 4J). Levels of both plasma AST and urinary haemoglobin in the SS mice treated for 8 and 20 weeks were lower than in untreated SS mice (Figure 4K,L), however, the results did not reach statistical significance.

Compared to AA controls, SS naïve mice had reduced platelet counts. 8‐AG had no effect on platelet counts in AA mice, but importantly led to a transient increase in platelet counts in SS mice at 10 weeks (treatment p = 0.005; time ×treatment interaction p = 0.032; Figure 4I).

Humans with SCD have high rates of hemolysis and are at risk of pulmonary hypertension (PH) [50]. SCD mice also develop PH and RV dysfunction [51]. Thus, we investigated the effect of 8‐AG treatment on RV hemodynamics, measured by closed‐chest RV catheterization (Figure 5A–L), and on echocardiographic parameters of LV dysfunction (Figure S4) in subsets of Townes AA and SS mice treated with 8‐AG and control animals (n = 7–10 per group). Compared to AA controls, non‐treated SS mice did exhibit RV dysfunction in terms of contractile function (Figure 5E,F) and RV hypertrophy (Figure 5J,K). Following 20‐week treatment, 8‐AG tended to improve RV contractility in both AA and SS mice as evidenced by increased RV pressure rate of rise (dP/dt; p = 0.019; Figure 5D), a tendency towards increased contractility index (p = 0.061; Figure 5E), and a reduction in the systolic ejection period (p = 0.020; Figure 5G). In both AA and SS mice, 8‐AG improved RV relaxation as evidenced by increased −dP/dt (p = 0.049, Figure 5H) and a tendency to reduce RV Tau in 8‐AG treated animals (p = 0.059; Figure 5I). Most importantly, both short‐ and long‐term treatments with 8‐AG reduced RV hypertrophy (Figure 5L). Histopathological analysis of lung tissue from control and treated AA and SS mice revealed mild to moderate pathology with scattered mononuclear infiltrates, emphysema, perivascular edema and dilated and congested/clogged small‐size pulmonary arteries, and no signs of haemorrhages and infarcts in untreated SS mice (Figure S3). 8‐AG treatment did not modify these mild/moderate histopathological alterations.

Non‐invasive echocardiography revealed LV hypertrophy and dilated cardiomyopathy with preserved ejection fraction in SS mice compared to AA controls, and 20‐week treatment with 8‐AG did not affect LV function (Figure S4).

SCD‐related kidney disease occurs in 50% of patients, is an independent risk factor for premature death [52, 53], and is characterised by both glomerular and tubular damage [54]. In SS mice, 8‐AG significantly attenuated urinary N‐acetyl‐beta‐D‐glucosaminidase (NAG) activity (Figure 6D) and glutathione urinary excretion (Figure 6F) and, to a lesser degree, urinary kidney injury molecute‐1 (KIM‐1, Figure 6C), suggesting reduced tubular damage, and decreased albuminuria (Figure 6A,B), which is indicative of reduced glomerular damage. In SS mice, the progression of disease was associated with increased 8‐hydroxydeoxyguanosine (8‐OHdG) levels (Time: p = 0.001, Figure 6E), a marker of oxidative stress, and a time‐related reduction in erythrocytic glutathione, which should be expected to contribute to increased oxidative stress (Time: p = 0.001, Figure 6G). However, treatment with 8‐AG did not affect levels of 8‐OHdG or erythrocytic glutathione.

In patients with SCD, the prevalence of liver disease ranges from 10% to 30% at autopsy and is associated with increased mortality [55, 56]. As expected, compared to the 30‐week‐old Townes AA controls, the untreated SS mice had remarkably larger livers (Liver: AA = 2230 ± 73 vs. SS = 2886 ± 81 mg, p = 0.001; Liver Index: AA = 57 ± 1 vs. SS = 83 ± 2 mg/g b.w. p < 0.001). Compared to control AA mice (Figure 7B,F), SS mice had ischemic and necrotic areas with infiltrating inflammatory cells (Figure 7C). The central veins and sinusoids were congested with sickled RBCs (Figure 7E,G,I) and surrounded by inflammatory cells and pericellular (Figures 7E,I) and perivascular (Figure 7J) fibrotic changes. Furthermore, SS mice exhibited remarkable iron deposition in central and mid‐hepatic areas (Figure 7M), mainly around central veins and nearby sinusoids (Figure 7N). Both 8‐ and 20‐week treatment with 8‐AG significantly reduced hepatomegaly (treatment effect, p = 0.001 Figure 7A), tended to reduce ischemic/necrotic damage (Figure 7D), central veins and sinusoids congestion and liver iron content (Figure 7H,L, respectively) and perivascular fibrosis (Figure 7K). Townes SS mice had massive splenomegaly (Spleen AA = 165 ± 13 vs. SS = 1813 ± 144 mg; p < 001; Spleen index: AA = 4 ± 0.3 vs. SS = 51 ± 32 mg/g b.w., p = 0.0001). Histopathological changes included vascular congestion and loss of the splenic architecture in SS mice, and iron deposition in both AA and SS mice (Figure S5). Notably, both 8‐ and 20‐week 8‐AG treatment reduced splenomegaly in SS mice (p = 0.021, Figure 7O).