De novo NAD+ biosynthetic impairment in acute kidney injury in humans

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) extends longevity in experimental organisms, raising interest in its impact on human health. De novo NAD⁺ biosynthesis from tryptophan is evolutionarily conserved yet considered supplanted among higher species by biosynthesis from nicotinamide (NAM). Here we show that a bottleneck enzyme in de novo biosynthesis, quinolinate phosphoribosyltransferase (QPRT), defends renal NAD⁺ and mediates resistance to acute kidney injury (AKI). Following murine AKI, renal NAD⁺ fell, quinolinate rose, and QPRT declined. QPRT^+/- mice exhibited higher quinolinate, lower NAD⁺, and higher AKI susceptibility. Metabolomics suggested an elevated urinary quinolinate/tryptophan ratio (uQ/T) as an indicator of reduced QPRT. Elevated uQ/T predicted AKI and other adverse outcomes in critically ill patients. A phase 1 placebo-controlled study of oral NAM demonstrated a dose-related increase in circulating NAD⁺ metabolites. NAM was well tolerated and was associated with less AKI. Therefore, impaired NAD⁺ biosynthesis may be a feature of high-risk hospitalizations for which NAD⁺ augmentation could be beneficial.

Figure 1. Renal and urinary quinolinate elevation in ischemic AKI

(a) NAD+ biosynthetic pathways: (1) “de novo” from tryptophan (Trp) through the intermediate quinolinate (Quin); (2) via a “salvage” pathway from nicotinamide (Nam); (3) from nicotinic acid (NA); and (4) from nicotinamide riboside (NR). QPRT = quinolinate phosphoribosyltransferase. NAMPT = Nam phosphoribosyltransferase. NaMN = nicotinate mononucleotide, NMN = nicotinamide mononucleotide. (b) Volcano plot comparing urinary metabolites (n = 204) in mice 24h after sham operation or transient bilateral renal ischemia (AKI) (n = 4 animals/group). Vertical dashed lines indicate threshold for two-fold abundance difference. Horizontal dashed line indicates p = 0.05 threshold. X axis = log₂[fold change for right condition/left condition]. Red dot = quinolinate, red dot with black border = quinolinate/tryptophan ratio. Y axis = -log₁₀[p-value]. P-value computed by two-sided unpaired t-test without adjustment for multiple comparisons. (c,d) Urinary quinolinate (uQuin, a.u. = arbitrary unit) and urinary quinolinate/tryptophan (uQ:T) ratio from (b). (e,f) Renal tissue quinolinate and quinolinate/tryptophan (Q:T) ratio as a function of serum creatinine (sCr) determined 24h after no operation, sham operation or varying durations of transient renal ischemia (n = 13 animals). Correlations by Pearson method. (g,h) Renal tissue quinolinate content and renal quinolinate/tryptophan ratio in controls or mice 24h after 25 minutes transient renal ischemia (n = 10/group). (i,j) Renal tissue NAD+ (n = 8,9 respectively) and NADP+ content (n = 5,7 respectively) in controls or mice 24h after transient renal ischemia. Data in c,d,g–j displayed as mean ± SEM; pairwise comparisons by Mann-Whitney with two-sided *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2. QPRT mediates resistance to experimental AKI

(a) Mouse renal QPRT mRNA 24h after transient ischemia (n = 6 animals/group). (b) QPRT mRNA in littermate controls (WT) vs. QPRT^+/− mice (n =5 animals/group). (c) Volcano plot comparing urinary metabolites (n = 204) in littermate controls (WT = 4 animals) vs. QPRT^+/− mice (n = 5 animals). Vertical dashed lines indicate threshold for two-fold abundance difference. Horizontal dashed line indicates p = 0.05 threshold. X axis = log₂[fold change for right condition/left condition]. Red dot = quinolinate, red dot with black border = quinolinate/tryptophan ratio. Y axis = -log₁₀[p-value]. P-value computed by two-sided unpaired t-test without adjustment for multiple comparisons. (d,e) Urinary quinolinate (uQuin, a.u. = arbitrary unit) and urinary quinolinate/tryptophan (uQ:T) ratio from (c). (f) Tissue NAD+ content in littermate controls (WT) vs. QPRT^+/− mice (n =5 animals/group). (g) Renal function 24h after transient renal ischemia in littermate controls (WT) vs. QPRT^+/− mice receiving vehicle or Nam (400 mg/kg ip) −24h, −1h, and +4–6h relative to surgery (n = 10, 10, 8, and 9 animals/group respectively). (h) Representative examples from 3 independent animals per group of intratubular cast (arrowhead) and tubular necrosis (arrows) 24h after transient renal ischemia in littermate controls (WT) vs. QPRT^+/− mice. Scale bar = 20 μm. Data in a,b,d–f,g displayed as mean ± SEM; pairwise comparisons by Mann-Whitney with two-sided *p < 0.05, **p < 0.01.

Figure 3. Urinary quinolinate/tryptophan (uQ:T) elevation in human AKI

(a) De novo biosynthesis of NAD+ from tryptophan. IDO=indole dioxygenase; TDO-tryptophan dioxygenase; AFMID = arylformamidase; KMO = kynurenine monoxygenase; KYNU = kynureninase; HAAO = hydroxyanthranilate dioxygenase; Quinolinate is then ribosylated by QPRT = quinolinate phosphoribosyltransferase. (b–f) Urinary metabolites in cardiac surgery patients. Measurements were compared by time relative to cardiopulmonary bypass and whether subjects developed AKI (n=6/group). CPB = during cardiopulmonary bypass. ICU = immediate post-operative period during which patients were still intubated for mechanical ventilation. +6h = 6 hours since surgery completion. Otherwise, times are day relative to surgery. For b–f, significance was assessed by two-way ANOVA with two-sided p-value indicating treatment effect and data displayed as mean ± SEM. (g–i) Urinary metabolites in a prospective cohort study of ICU patients (n = 215 subjects, 51 of whom subsequently developed AKI and 164 without AKI). (g,h) Urinary quinolinate and uQ:T in those who did or did not develop AKI. Data displayed as median ± interquartile range and compared by Wilcoxon Rank Sum test. (i) Odds ratios for incident AKI (n = 215 subjects) according to quartiles of uQ:T determined by multivariate logistic regression. Model 1 is unadjusted. Model 2 is adjusted for the following demographics and comorbidities: age, gender, race, baseline estimated glomerular filtration rate, hypertension, and diabetes mellitus. Model 3 is further adjusted for the following severity of illness covariates: ICU type, need for mechanical ventilation, and APACHE II score. Quartile 1 (Q1) was the reference in all models. Error bars for 95% confidence interval. (j) Forest plot for incident AKI and other outcomes as listed per standard deviation (SD) of log-transformed uQ:T. Error bars for 95% confidence interval. Two-sided **p < 0.01, ***p < 0.001.

Figure 4. Participants and outcomes of oral Nam Phase 1 pilot study in cardiac surgery patients

(a) Nam can be converted to NAD+ through the intermediate nicotinamide mononucleotide (NMN) or methylated and oxidized for excretion through the intermediate N1-methyl nicotinamide (MNA). (b) Flow diagram for participants in oral Nam Phase 1 pilot study (clinicaltrials.gov entry NCT02701127). (c–f) Serum Nam, urine Nam, serum MNA, and serum NMN temporal profiles by treatment arm with data displayed as mean ± SEM. N per group as indicated in (b). P-values for c–f are pairwise Mann-Whitney comparisons of areas under the curve (AUCs, μM * days) for each treatment arm relative to placebo. For f, only the 3 gm/d arm was significantly different vs. placebo. (g,h) Temporal profiles of the cardiac injury marker Troponin T by treatment arm displayed as mean ± SEM and AUCs (ng/ml * days) with treatment groups combined (n = 14 in placebo, n = 27 in Nam treatment groups combined). (i,j) Serum creatinine (sCr) by treatment arm displayed as mean ± SEM and AUCs (mg/dl * days) with treatment groups combined (n = 14 in placebo, n = 27 in Nam treatment groups combined). AUCs for troponin T and sCr were compared to placebo by Mann-Whitney.