PPDPF: Preventing kidney disease through NAD⁺ regulation

Abstract

Researchers have discovered PPDPF, a critical cellular factor that controls NAD⁺ biosynthesis, whose function is important to prevent kidney diseases.

The prevalence of chronic kidney disease (CKD) increases with age and based on a report from the US Centers for Disease Control and Prevention an approximate prevalence of CKD was 38% among adults aged ≥70 years during the period 2017 to 2020, whereas it was 13.9% among adults aged ≥18 years during the same time period. Nonetheless, the pathogenesis of CKD still remains poorly understood. Recently, the community has agreed that systemic nicotinamide adenine dinucleotide (NAD⁺) decline is a driving force for age-associated functional decline and diseases. An interesting connection between the impairment of NAD⁺ metabolism, particularly de novo NAD⁺ biosynthesis (Fig. 1A), and acute kidney injury (AKI) was previously reported, and was the finding that oral nicotinamide administration decreases AKI events, compared to placebo (1). These results implicate a possibility that impaired NAD⁺ biosynthesis may promote long-term kidney dysfunction as well, leading to CKD.

Fig. 1. Mammalian NAD⁺ biosynthetic pathways and the importance of PPDPF in protecting kidney function.

(A) In mammals, nicotinamide (NIC), nicotinic acid (NA), and tryptophan are major precursors for NAD⁺ biosynthesis. Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are also ingested as NAD⁺ intermediates. Nicotinamide/nicotinic acid mononucleotide adenyltransferases (NMNAT1-3) are common key enzymes for all NAD⁺ biosynthetic pathways. NAD⁺-consuming enzymes mediate many fundamental biological processes. In the kidney, maintaining NAD⁺ counteracts its dysfunction in AKI and CKD. (B) The effect of PPDPF that has thiol-disulfide oxidoreductase activity on NMNAT1-3 localized in the nucleus, cytoplasm, and mitochondria, respectively. PPDPF is critical to maintain the NAD⁺ biosynthetic activities of NMNAT1-3. In the kidney, this function of PPDPF is important to protect kidney function from injuries that cause AKI or CKD. Illustration credit: Austin Fisher/Science Advances.

The history of NAD⁺ biology started almost 120 years ago when Harden and Young found a factor that enhanced fermentation in yeast extracts. Since then, this ever-evolving field has witnessed many incredible discoveries. Over the past quarter century, in particular, interest in NAD⁺ biology has grown dramatically. This period opened with an unexpected discovery of NAD⁺-dependent protein deacetylase activity of yeast and mammalian sirtuins by Imai and Guarente in 2000 (2). Then, in 2001, human nicotinamide/nicotinic acid mononucleotide adenyltransferase (NMNAT), a key NAD⁺ biosynthetic enzyme that is common in all different NAD⁺ biosynthetic pathways (Fig. 1A), were fully characterized by Ziegler and his colleagues (3). Following these critical discoveries, nicotinamide phosphoribosyltransferase (NAMPT), another key NAD⁺ biosynthetic enzyme that regulates a major NAD⁺ biosynthetic pathway in mammals (Fig. 1A), was identified and characterized by Leo’s and Imai’s groups (4, 5). The importance of NAD⁺ biosynthetic intermediates was also rediscovered. It was reported by Bieganowski and Brenner in 2004 that nicotinamide riboside is critical for NAD⁺ biosynthesis via nicotinamide riboside kinases (NRKs) (6) (Fig. 1A). Imai and colleagues also reported remarkable efficacies of nicotinamide mononucleotide (NMN) on metabolism and aging (7).

In this issue of Science Advances, a team led by Guan and Geng reports the first critical clue for the connection between a defect in NAD⁺ biosynthesis and the pathogenesis of CKD (8). The team identified an interesting kidney disease risk gene, PPDPF (Pancreatic Progenitor Cell Differentiation and Proliferation Factor), by combining single-cell RNA-seq data from kidney samples of unilateral ureteral obstruction (UUO) model mice and patients with AKI and kidney function GWAS data. Indeed, the expression levels of the Ppdpf/PPDPF gene and its protein were up-regulated in mouse AKI models and human patients with AKI. The up-regulation of Ppdpf/PPDPF is higher in healthy proximal tubule (PT) cells, compared to injured cells, in both animal models and human patients, suggesting that PPDPF is a protective factor for PT cells. To further investigate the function of PPDPF in vivo, the team generated Ppdpf knockout (KO) mice. Intriguingly, Ppdpf KO mice and cells showed significant mitochondrial dysfunction, particularly with reduced expression of mitochondrial complex I. Furthermore, Ppdpf KO cells showed significant decreases in NAD⁺ levels. This particular finding inspired the team to look deeper into mammalian NAD⁺ biosynthetic pathways. An important clue came when they analyzed the subcellular localization of PPDPF. They found that PPDPF showed an interesting colocalization with NMNATs in the nucleus, cytoplasm, and mitochondria. There are three distinct NMNATs: NMNAT1 has the strongest enzymatic activity and is localized in the nucleus, NMNAT2 is localized in the cytoplasmic membranous fractions via palmitoylation, and NMNAT3 is localized in mitochondria (Fig. 1B). Remarkably, PPDPF physically interacts with all three NMNATs, suggesting that PPDPF might regulate the enzymatic activity of NMNATs and facilitate the conversion of NMN to NAD⁺.

A true surprise in this study is the discovery that PPDPF has thiol-disulfide oxidoreductase activity and enhances the activity of all three NMNATs. Cellular factors that could regulate the enzymatic activities of NAD⁺ biosynthetic enzymes have not been previously identified, and therefore, this discovery is a critical breakthrough in identifying such a regulatory mechanism for NMNATs. The team used a series of in silico and in vitro biochemical analyses and convincingly demonstrated the thiol-disulfide oxidoreductase activity of PPDPF. Thiol-disulfide oxidoreductase activity is important to catalyze the proper folding of proteins through the formation of disulfide bonds. Indeed, this particular enzymatic activity that PPDPF possesses was required to maintain the enzymatic activities of all three NMNATs (Fig. 1B). Consistently, knockdown of PPDPF resulted in significant decreases in the activity of NMNATs and NAD⁺ levels in human cells. These defects were rescued by the wild-type PPDPF but not enzymatically defective point mutants. All these findings clearly demonstrate that PPDPF is an important regulatory factor for NMNAT enzymatic activity through its physical interaction with these specific NAD⁺ biosynthetic enzymes.

The team also addressed whether Ppdpf deficiency exacerbated kidney dysfunction and caused CKD in mice. Ppdpf KO mice developed signs of kidney dysfunction when they reached 12 months of age. Furthermore, Ppdpf KO mice displayed severer pathologies and defects in gene expression, compared to control mice, in cisplatin- and UUO-induced CKD models. Particularly, in the folic acid–induced kidney disease model, NAD⁺ administration, but not NMN administration, ameliorated the signs of renal injuries in Ppdpf KO mice. Consistently, in the same disease model, kidney-specific Ppdpf overexpression significantly rescued NMNAT enzymatic activity and NAD⁺ levels and mitigated the pathologies of renal injury. These results demonstrate that PPDPF is a critical factor to maintain kidney function and counteract its dysfunction caused by acute and chronic renal injuries.

This study raises an interesting question regarding the compartmentalization of NAD⁺ metabolism. It has been suggested that mitochondrial NMNAT3 does not usually catalyze NAD⁺ biosynthesis so that mitochondria depend on the NAD⁺ transporter SLC25A51 to maintain their NAD⁺ levels (9). This means that the effect of PPDPF on NAD⁺ biosynthesis could be more significant through nuclear NMNAT1 and cytosolic NMNAT2. If this is the case, defects in PPDPF amounts and/or function could primarily cause significant reduction in nuclear and cytoplasmic NAD⁺ levels, which might secondarily reduce mitochondrial NAD⁺ levels. Therefore, in addition to mitochondrial dysfunction, PPDPF defects are expected to cause nuclear and cytoplasmic dysfunctions independently. As a result, extra caution is necessary to dissect which compartment’s NAD⁺ decrease triggers pathogenic changes in kidney function for AKI and CKD.

Another interesting question is whether NMN administration could be effective to ease kidney dysfunction in AKI and CKD due to the up-regulation of PPDPF. NMN administration could potentially boost kidney NAD⁺ levels more efficiently than a normal, healthy condition because PPDPF up-regulation enhances the enzymatic activities of all three NMNATs (Fig. 1B). Although this appears to be the case in mice, it will be critical to conduct a human clinical trial for the efficacy of NMN on patients with AKI and CKD.

The study by Guan and Geng adds a different player to the systemic regulatory mechanisms for NAD⁺ biosynthesis. Recently, a newly formulated concept, NAD World 3.0, was proposed (10). This concept suggests that multilayered feedback regulations for systemic NAD⁺ biosynthesis play a critical role in the control of mammalian aging and longevity and maintaining each feedback loop is critical to counteract age-associated organ dysfunctions and diseases. It is conceivable that cellular factors, such as PPDPF, could also make an important contribution to the maintenance of these systemic feedback loops and keep adequate NAD⁺ levels for organs to resist the wearing effects of time. Further exploration of the NAD World will surely provide more surprises to this ever-evolving field of NAD⁺ biology.