Effect of NAD+ boosting on kidney ischemia-reperfusion injury

Abstract

Acute kidney injury (AKI) is associated with a very high mortality and an increased risk for progression to chronic kidney disease (CKD). Ischemia-reperfusion injury (IRI) is a model for AKI, which results in tubular damage, dysfunction of the mitochondria and autophagy, and in decreased cellular nicotinamide adenine dinucleotide (NAD+) with progressing fibrosis resulting in CKD. NAD+ is a co-enzyme for several proteins, including the NAD+ dependent sirtuins. NAD+ augmentation, e.g. by use of its precursor nicotinamide riboside (NR), improves mitochondrial homeostasis and organismal metabolism in many species. In the present investigation the effects of prophylactic administration of NR on IRI-induced AKI were studied in the rat. Bilateral IRI reduced kidney tissue NAD+, caused tubular damage, reduced α-Klotho (klotho), and altered autophagy flux. AKI initiated progression to CKD, as shown by induced profibrotic Periostin (postn) and Inhibin subunit beta-A, (activin A / Inhba), both 24 hours and 14 days after surgery. NR restored tissue NAD+ to that of the sham group, increased autophagy (reduced p62) and sirtuin1 (Sirt1) but did not ameliorate renal tubular damage and profibrotic genes in the 24 hours and 14 days IRI models. AKI induced NAD+ depletion and impaired autophagy, while augmentation of NAD+ by NR restored tissue NAD+ and increased autophagy, possibly serving as a protective response. However, prophylactic administration of NR did not ameliorate tubular damage of the IRI rats nor rescued the initiation of fibrosis in the long-term AKI to CKD model, which is a pivotal event in CKD pathogenesis.

Fig 1. Experimental design of the model on prophylactic NAD⁺ boosting and bilateral renal ischemia-reperfusion injury (IRI).

Animals were randomized to sham or IRI and given NR (500 mg/kg/d) or vehicle (water) from 14 days before the IRI or sham surgery to one day after surgery. Both renal arteries were clamped for 45 minutes. The rats were sacrificed after 24 hours or 14 days after IRI. NR: nicotinamide riboside.

Fig 2. Ngal, an acute kidney injury marker and plasma biochemistry in sham animals and 24 hours and 14 days after IRI.

Kidney expression of Ngal (A) and plasma biochemistry (B, C, D) are presented. Ngal expression was not affected by NR in the sham groups. The levels were similarly significantly increased after 24 hours after ischemia reperfusion injury (IRI) in the vehicle and sham groups and decreased to the same level 14 days after IRI. Plasma creatinine was similar in sham rats whether given NR or vehicle. IRI resulted in a significant increase in plasma creatinine after 24 hours with no difference between vehicle and NR groups.14 days after IRI, plasma creatinine became normal with no difference between the two groups. Plasma phosphate remained stable 24 hours after IRI at the level of sham rats in both vehicle and NR groups, and became significantly reduced 14 days after IRI. Basal plasma calcium was the same in vehicle and NR sham rats. Twenty-four hours after IRI significant hypocalcemia developed but increased again in the long term IRI groups. Ngal (neutrophil gelatinase-associated lipocalin). Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001 (n = 7 in each group).

Fig 3. Effect of NR administration on NAD⁺ boosting.

NAD⁺, NADH levels and Sirtuin1 expression in the kidney. NAD⁺ and NADH levels were measured in the kidney of sham+vehicle, sham+NR and IRI+vehicle and IRI+NR at 24 hours after IRI or sham. (A-B) NAD⁺ and NADH levels were significantly elevated in NR groups compared with the sham groups. IRI+vehicle rats had a pronounced significant reduction in the NAD⁺ and NADH levels compared with the sham+vehicle group. Also, NADH levels were significantly decreased in the IRI+vehicle as compared with IRI+NR group. NR rescued the fall in NAD⁺ and NADH levels after IRI. (C) The NAD/NADH ratio was unchanged in all the groups. (D) The expression of Sirt1 was significantly increased in sham+NR rats. IRI resulted in a significant fall in Sirt1 levels to a similar level. Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001 (n = 7 in each group).

Fig 4. Effect of NAD⁺ boosting by NR on autophagy in the kidney.

Western blot analysis and quantification of P62 protein, a marker for autophagy, showed that NAD⁺ boosting in sham group did not interfere with the levels of P62. A significant increase in P62 level was found in the IRI+vehicle compared to the IRI+NR group after 24 hours (A and B). The ratio between LC3BII/LC3BI was unchanged in all groups. Western blot analysis and quantification of P62 levels and LC3BII/I ratio 14 days after IRI are presented in (C and D). The level of P62 became normalized in the IRI groups. The ratio of LC3BII/ LC3BI remained unchanged. Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001.

Fig 5. Effect of NAD⁺ boosting by NR on the mitochondrial oxidative phosphorylation (OXPHOS) complexes.

Western blot (A) and quantifications (B) of the subunits of the mitochondrial OXPHOS complex I-V demonstrated that subunits II, III, and V were significantly increased by IRI but not further affected by NAD⁺ boosting. The levels of OXPHOS complex I-V were normalized to the housekeeping protein, PARK7. Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001.

Fig 6. Effect of NAD⁺ boosting by NR on Klotho and Napi2c in the kidney.

Western blot of klotho protein in the kidney of sham+vehicle, sham+NR and IRI+vehicle and IRI+NR at 24 hours (A) and its quantification (B) normalized to actin. NAD⁺ boosting did not affect klotho levels in the sham groups. Klotho became significantly decreased by IRI in both the vehicle and NR groups after 24 hours. (C) Western blot of klotho in the kidney of IRI+vehicle and IRI+NR after 14 days and its quantification (D) showed that klotho in the IRI+NR 14 days group was significantly reduced as compared with the sham group. (E) The mRNA levels of Kl were similarly reduced at the protein level. (F) mRNA of Napi2c was significantly decreased 24 hours after IRI in both groups and administration with NR resulted in a further decrease. Napi2c: sodium phosphate cotransporter 2. Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001.

Fig 7. Effect of NAD⁺ boosting by NR on genes related to the de novo NAD⁺ pathway in the kidney.

The expression levels of genes related to the de novo biosynthesis pathway of NAD⁺ were examined. QPRT is a key enzyme in the NAD⁺ de novo pathway for converting Quinolinic acid to NAD⁺. It was significantly downregulated by IRI to the same extent in both the IRI+Vehicle and IRI+NR 24 hour groups. The levels became normalized or upregulated 14 days after IRI (A). The expression level of pgc-1α was also significantly downregulated after 24 hours in the IRI+vehicle and IRI+NR groups, as compared to the sham groups (B). (C) Cox4 was significantly downregulated in both IRI groups after 24 hours compared to the sham groups and became normal after 14 days (C). The two antioxidants Sod2 and Cat, downstream for pgc-1α, were significantly downregulated in both IRI groups after 24 hours compared with the sham groups. Finally, were also levels of Sod2 downregulated in the IRI+NR-14 days group compared to sham+NR (D and E). Qprt: Quinolinate Phosphoribosyltransferase; Pgc-1α: Peroxisome proliferator-activated receptor gamma coactivator 1α; Cox4: Cytochrome c oxidase subunit 4; Sod2: Superoxide dismutase 2; Cat: Catalase. Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001.

Fig 8. NAD⁺ boosting by NR and expression of fibrogenic markers in the kidney.

The four figures depict the expression level of Tgfb1, Inhba, Bmp7, and Postn. The relative mRNA level in those genes was not changed by NR administration in sham groups. Significant upregulation to the similar level of the expression of Tgfb1, Inhba and Postn was seen after 24 hours in both IRI groups compared with sham groups. Tgfb1, Inhba and Postn were still upregulated after 14 days post-IRI in both groups compared with sham groups (A—C). The level of Bmp7 was significantly downregulated at 24 hours post-IRI in both groups compared with sham groups. The Bmp7 level was normalized after 14 days post-IRI compared with sham (D). Tgfb1: Transforming growth factor-beta1; Inhba: Inhibin subunit beta A; Postn: Periostin; Bmp7: Bone morphogenetic protein 7. Mean ± SD; * p < 0.05, ** p < .001, *** p < 0.0001.

Fig 9. Effect of boosting NAD⁺ by NR on the kidney histological appearance in IRI.

Representative kidney cortex sections from sham rats, and IRI rats after 24 hours and 14 days treated with NR or vehicle, H&E (400) (A-F). Significant damage of the kidney cortex was seen in both IRI groups after 24 hours with inflammation, cast-formation, and severe appearance of necrosis in the tubular cells, mild endothelial swelling and tubular/interstitial inflammation, and hemorrhage in the tissue. The glomeruli were not significantly affected. Fourteen days after IRI the tubular changes were less severe, but development of interstitial fibrosis appeared. The histological changes were scored with focus on tubular, glomerular, endothelial and tubulo-interstitial damages 24 hours and 14 days after IRI in NR and sham rats, demonstrating that even though severe changes were induced, there was no significant improvement of boosting NAD⁺ by NR (G-J). EGTI damage score: Endothelial, Glomerular, Tubular, and Interstitial damage score. Mean ± SD; *p <0.05, **p <0.001, ***p <0.0001.