Biotechnological production of reduced and oxidized NAD+ precursors.

Dysregulation of nicotinamide adenine dinucleotide (NAD+) homeostasis by increased activity of NAD+ consumers or reduced NAD+ biosynthesis plays an important role in the onset of prevalent, often age-related, diseases, such as diabetes, neuropathies or nephropathies. To counteract such dysregulation, NAD+ replenishment strategies can be used. Among these, administration of vitamin B3 derivatives (NAD+ precursors) has garnered attention in recent years. However, the high market price of these compounds and their limited availability, pose important limitations to their use in nutritional or biomedical applications. To overcome these limitations, we have designed an enzymatic method for the synthesis and purification of (1) the oxidized NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), (2) their reduced forms NMNH and NRH, and (3) their deaminated forms nicotinic acid mononucleotide (NaMN) and nicotinic acid riboside (NaR). Starting from NAD+ or NADH as substrates, we use a combination of three highly overexpressed soluble recombinant enzymes; (a) a NAD+ pyrophosphatase, (b) an NMN deamidase, and (c) a 5'-nucleotidase, to produce these six precursors. Finally, we validate the activity of the enzymatically produced molecules as NAD+ enhancers in cell culture.

Reduced nicotinamide mononucleotide is a new and potent NAD+ precursor in mammalian cells and mice.

Nicotinamide adenine dinucleotide (NAD+ ) homeostasis is constantly compromised due to degradation by NAD+ -dependent enzymes. NAD+ replenishment by supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD+ pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD+ precursor for the first time. We show that NMNH increases NAD+ levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT-independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD+ surge in whole blood, which is accompanied by increased NAD+ levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors.

Metabolic Flexibility and Innate Immunity in Renal Ischemia Reperfusion Injury: The Fine Balance Between Adaptive Repair and Tissue Degeneration.

Renal ischemia reperfusion injury (IRI), a common event after renal transplantation, causes acute kidney injury (AKI), increases the risk of delayed graft function (DGF), primes the donor kidney for rejection, and contributes to the long-term risk of graft loss. In the last decade, epidemiological studies have linked even mild episodes of AKI to chronic kidney disease (CKD) progression, and innate immunity seems to play a crucial role. The ischemic insult triggers an acute inflammatory reaction that is elicited by Pattern Recognition Receptors (PRRs), expressed on both infiltrating immune cells as well as tubular epithelial cells (TECs). Among the PRRs, Toll-like receptors (TLRs), their synergistic receptors, Nod-like receptors (NLRs), and the inflammasomes, play a pivotal role in shaping inflammation and TEC repair, in response to renal IRI. These receptors represent promising targets to modulate the extent of inflammation, but also function as gatekeepers of tissue repair, protecting against AKI-to-CKD progression. Despite the important considerations on timely use of therapeutics, in the context of IRI, treatment options are limited by a lack of understanding of the intra- and intercellular mechanisms associated with the activation of innate immune receptors and their impact on adaptive tubular repair. Accumulating evidence suggests that TEC-associated innate immunity shapes the tubular response to stress through the regulation of immunometabolism. Engagement of innate immune receptors provides TECs with the metabolic flexibility necessary for their plasticity during injury and repair. This could significantly affect pathogenic processes within TECs, such as cell death, mitochondrial damage, senescence, and pro-fibrotic cytokine secretion, well-known to exacerbate inflammation and fibrosis. This article provides an overview of the past 5 years of research on the role of innate immunity in experimental and human IRI, with a focus on the cascade of events activated by hypoxic damage in TECs: from programmed cell death (PCD) and mitochondrial dysfunction-mediated metabolic rewiring of TECs to maladaptive repair and progression to fibrosis. Finally, we will discuss the important crosstalk between metabolism and innate immunity observed in TECs and their therapeutic potential in both experimental and clinical research.

ß-Cyclodextrin counteracts obesity in Western diet-fed mice but elicits a nephrotoxic effect.

Obesity has become a worldwide health crisis and is associated with a plethora of comorbidities. The multi-organ effects of obesity have been linked to ectopic lipid accumulation. Thus, there is an urgent need to tackle the obesity crisis by developing effective lipid-lowering therapies. 2-hydroxypropyl-ß-Cyclodextrin (2HP-ß-CD) has been previously shown to reduce lysosomal cholesterol accumulation in a murine model of Niemann Pick Type C (NPC) disease. Using a murine model of Western diet-induced obesity (DIO), we report the effects of 2HP-ß-CD in counteracting weight gain, expansion of adipose tissue mass and ectopic lipid accumulation. Interestingly, DIO caused intracellular storage of neutral lipids in hepatic tissues and of phospholipids in kidneys, both of which were prevented by 2HP-ß-CD. Importantly, this report brings attention to the nephrotoxic effects of 2HP-ß-CD: renal tubular damage, inflammation and fibrosis. These effects may be overlooked, as they are best appreciated upon assessment of renal histology.

TREM1/3 Deficiency Impairs Tissue Repair After Acute Kidney Injury and Mitochondrial Metabolic Flexibility in Tubular Epithelial Cells.

Long-term sequelae of acute kidney injury (AKI) are associated with incomplete recovery of renal function and the development of chronic kidney disease (CKD), which can be mediated by aberrant innate immune activation, mitochondrial pathology, and accumulation of senescent tubular epithelial cells (TECs). Herein, we show that the innate immune receptor Triggering receptor expressed on myeloid cells-1 (TREM-1) links mitochondrial metabolism to tubular epithelial senescence. TREM-1 is expressed by inflammatory and epithelial cells, both players in renal repair after ischemia/reperfusion (IR)-induced AKI. Hence, we subjected WT and TREM1/3 KO mice to different models of renal IR. TREM1/3 KO mice displayed no major differences during the acute phase of injury, but increased mortality was observed in the recovery phase. This detrimental effect was associated with maladaptive repair, characterized by persistent tubular damage, inflammation, fibrosis, and TEC senescence. In vitro, we observed an altered mitochondrial homeostasis and cellular metabolism in TREM1/3 KO primary TECs. This was associated with G2/M arrest and increased ROS accumulation. Further exposure of cells to ROS-generating triggers drove the cells into a stress-induced senescent state, resulting in decreased wound healing capacity. Treatment with a mitochondria anti-oxidant partly prevented the senescent phenotype, suggesting a role for mitochondria herein. In summary, we have unraveled a novel (metabolic) mechanism by which TREM1/3 deficiency drives senescence in TECs. This involves redox imbalance, mitochondrial dysfunction and a decline in cellular metabolic activities. These finding suggest a novel role for TREM-1 in maintaining tubular homeostasis through regulation of mitochondrial metabolic flexibility.

NLRX1 does not play a role in diabetes nor the development of diabetic nephropathy induced by multiple low doses of streptozotocin.

Diabetic nephropathy (DN) is a microvascular complication of diabetes mellitus that results in both tubular and glomerular injury. Low-grade inflammation and oxidative stress are two mechanisms known to drive the progression of DN. Nucleotide-binding leucine-rich repeat containing family member X1 (NLRX1) is an innate immune receptor, uniquely located in mitochondria, that has been found to regulate inflammatory responses and to dampen renal oxidative stress by regulating oxidative phosphorylation. For this reason, we investigated the role of NLRX1 in the development of DN in a Type 1 Diabetes mouse model. We analyzed the effect of NLRX1 deficiency on diabetes development and the accompanied renal damage, inflammation, and fibrosis. We found that multiple low doses of streptozotocin induced body weight loss, polydipsia, hyperglycemia, glycosuria, and a mild DN phenotype in wildtype and NLRX1-deficient mice, without significant differences between these mouse strains. Despite increased NLRX1 expression in diabetic wildtype mice, NLRX1 deficiency did not affect the diabetic phenotype induced by streptozotocin treatment, as reflected by similar levels of polyuria, microalbuminuria, and increased renal markers of oxidative stress and inflammation in wildtype and NLRX1-deficient mice. The present findings show that NLRX1 does not mediate the development of streptozotocin-induced diabetes and diabetic-induced nephropathy in mice after multiple low doses of streptozotocin. This data implies that, while NLRX1 can be triggered by cellular stress, its regulatory and functional effects may be dependent on the specific physiological conditions. In the case of DN, NLRX1 may be neither helpful nor harmful, but rather a marker of metabolic stress.

Author Correction: Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the 'resistant' C57Bl/6J mouse strain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the 'resistant' C57Bl/6J mouse strain.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease. Animal models are essential tools for designing new strategies to prevent DN. C57Bl/6 (B6) mice are widely used for transgenic mouse models, but are relatively resistant to DN. This study aims to identify the most effective method to induce DN in a type 1 (T1D) and a type 2 diabetes (T2D) model in B6 mice. For T1D-induced DN, mice were fed a control diet, and randomised to streptozotocin (STZ) alone, STZ+unilateral nephrectomy (UNx), or vehicle/sham. For T2D-induced DN, mice were fed a western (high fat) diet, and randomised to either STZ alone, STZ+UNx, UNx alone, or vehicle/sham. Mice subjected to a control diet with STZ +UNx developed albuminuria, glomerular lesions, thickening of the glomerular basement membrane, and tubular injury. Mice on control diet and STZ developed only mild renal lesions. Furthermore, kidneys from mice on a western diet were hardly affected by diabetes, UNx or the combination. We conclude that STZ combined with UNx is the most effective model to induce T1D-induced DN in B6 mice. In our hands, combining western diet and STZ treatment with or without UNx did not result in a T2D-induced DN model in B6 mice.

TLR9 Mediates Remote Liver Injury following Severe Renal Ischemia Reperfusion.

Ischemia reperfusion injury is a common cause of acute kidney injury and is characterized by tubular damage. Mitochondrial DNA is released upon severe tissue injury and can act as a damage-associated molecular pattern via the innate immune receptor TLR9. Here, we investigated the role of TLR9 in the context of moderate or severe renal ischemia reperfusion injury using wild-type C57BL/6 mice or TLR9KO mice. Moderate renal ischemia induced renal dysfunction but did not decrease animal well-being and was not regulated by TLR9. In contrast, severe renal ischemia decreased animal well-being and survival in wild-type mice after respectively one or five days of reperfusion. TLR9 deficiency improved animal well-being and survival. TLR9 deficiency did not reduce renal inflammation or tubular necrosis. Rather, severe renal ischemia induced hepatic injury as seen by increased plasma ALAT and ASAT levels and focal hepatic necrosis which was prevented by TLR9 deficiency and correlated with reduced circulating mitochondrial DNA levels and plasma LDH. We conclude that TLR9 does not mediate renal dysfunction following either moderate or severe renal ischemia. In contrast, our data indicates that TLR9 is an important mediator of hepatic injury secondary to ischemic acute kidney injury.