Nicotinamide Adenine Dinucleotide Augmentation in Diabetic Kidney Disease: Randomized Trial Study Protocol

Abstract

Introduction

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease (CKD) and kidney failure worldwide. Reduced nicotinamide adenine dinucleotide (NAD) levels are mechanistically linked to DKD pathogenesis; and NAD augmentation by administration of its precursor, nicotinamide mononucleotide (NMN), has attenuated albuminuria and kidney injury in preclinical models.

Methods

The NAD Augmentation in Diabetic Kidney Disease (NAD in DKD) trial is a phase 2a, randomized, multicenter, double-blind, placebo-controlled, parallel-group study evaluating the efficacy and safety of oral pharmaceutical-grade microcrystalline β-NMN (MIB-626) in adults with DKD. One hundred fifty-six participants aged ≥ 30 years with diabetes, urinary albumin-to-creatinine ratio (UACR) ≥ 100 mg/g, and estimated glomerular filtration rate (eGFR) > 25 ml/min per 1.73 m² were randomized to receive MIB-626 (1000 mg twice daily) or placebo for 24 weeks, followed by 12 weeks of postintervention follow-up. The randomization was stratified for biological sex, age (30–44, 45–65, and ≥ 66 years), and enrolling site. The primary end point was change in UACR from baseline to 24 weeks. Secondary outcomes included serum creatinine, cystatin C, and eGFR, biomarkers of kidney injury and biological age, glycemic control, measures of muscle performance and physical function, and the circulating NAD metabolome.

Conclusion

This phase 2a trial was designed to evaluate the safety and efficacy of NAD augmentation in improving UACR in albuminuric DKD. The results will inform whether NAD augmentation warrants further evaluation in larger trials statistically powered for long-term kidney outcomes. The study is registered at ClinicalTrials.gov (NCT05759468).

Graphical abstract

Epidemiology and Clinical Burden of DKD

CKD affects approximately 25% of adults aged 65 to 74 years and > 50% of those aged ≥ 75 years, with a prevalence that continues to increase globally.1, 2, 3 Diabetes mellitus is a leading cause of CKD.³ Global diabetes prevalence among adults aged 20 to 79 years is projected to increase from 536.6 million in 2021 to 783.2 million by 2045, and DKD affects approximately 2 in 5 adults with diabetes.⁴ DKD is the leading cause of kidney failure requiring kidney replacement therapy and a major contributor to premature cardiovascular and all-cause mortality among individuals with diabetes.^3,5,6

Current Therapeutic Landscape

Despite the introduction of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), glucagon-like peptide-1 (GLP-1) receptor agonists, sodium-glucose cotransporter-2 (SGLT2) inhibitors, and mineralocorticoid receptor antagonists (MRAs), a substantial residual risk of kidney failure persists.7, 8, 9, 10, 11, 12 These agents can independently reduce albuminuria by primarily targeting glomerular hemodynamic, metabolic, and antiinflammatory mechanisms; however, DKD often continues to progress despite their use, leaving a substantial unmet need for therapies that address alternative pathophysiologic mechanisms. Critically, none of these agents target the tubulo-podocyte signaling axis. The NAD- sirtuin (SIRT) 1 mechanism is one such target: in animal models, it modulates tubulo-podocyte epigenetic signaling, raising the hypothesis that therapeutic NAD augmentation may provide renoprotection complementary to the current standard of care.13, 14, 15, 16

In this trial, participants receive background standard-of-care therapy for CKD, including ACE inhibitors, ARBs, and, where clinically indicated, SGLT2 inhibitors or GLP-1 receptor agonists.^12,17,18 NAD augmentation using oral administration of its precursor, NMN, is hypothesized to act as a mechanistically complementary intervention targeting the SIRT-NAD-mitochondrial axis, a pathway distinct from glomerular hemodynamics, tubular glucose handling, and incretin signaling. Whether this preclinical rationale translates into incremental kidney benefit in humans receiving contemporary background therapy remains unknown and is the central question addressed by this trial.13, 14, 15, 16^,19,20

Scientific Premise: The NAD-SIRT Pathway and Renal Pathophysiology

Disrupted NAD homeostasis has been implicated in DKD, with proposed effects on mitochondrial dysfunction, oxidative stress, inflammation, and SIRT signaling.21, 22, 23, 24, 25 Mitochondria-targeted interventions aimed at restoring NAD-dependent bioenergetics have emerged as a promising therapeutic strategy in this context.²⁶ NMN treatment has been shown to increase NAD levels in the blood and kidneys of mouse models of DKD and cisplatin-induced focal segmental glomerulosclerosis.²⁷ Pioneering work by Hasegawa et al.¹⁵ demonstrated that SIRT1 epigenetically regulates claudin-1 expression in podocytes via DNA (cytosine-5)-methyltransferase 1–mediated gene methylation, establishing a paracrine tubule-to-podocyte signaling axis mediated by the NAD-SIRT1 pathway.¹⁹ Podocyte-specific SIRT1 overexpression prevented renal abnormalities in murine models of DKD, while disruption of SIRT1 expression worsened them¹³ (Figure 1). Reduced NAD levels preceded the development of glomerulopathy and albuminuria in preclinical DKD models.¹⁵ Translating these preclinical observations to humans, transcriptomic analysis of kidney biopsies from patients with DKD demonstrated significant downregulation of kynurenine 3-monooxygenase, a rate-limiting enzyme in the de novo NAD biosynthetic pathway across both glomerular and tubulointerstitial compartments, with kynurenine 3-monooxygenase expression declining progressively as eGFR decreases. Increased urinary kynurenine excretion in patients with diabetes further corroborated impairment of this pathway in human disease.¹⁶ Consistent with functionally depleted NAD in the human diabetic kidney, immunohistochemical analysis of renal biopsies from patients with DKD demonstrated significantly reduced protein expression of SIRT3, a NAD⁺-dependent mitochondrial deacetylase, compared with controls without diabetes, accompanied by upregulation of the stimulator of interferon genes inflammatory pathway.²⁰

Figure 1.

Proposed NAD–SIRT1–DNMT1 epigenetic mechanism in DKD. In healthy podocytes, adequate NAD availability supports SIRT1 activity, promoting histone H3/H4 deacetylation and maintaining DNMT1 activity. This preserves methylation of the CLDN1 locus, suppresses claudin-1 and proinflammatory/prosclerotic gene expression, maintains podocyte structure, and prevents albuminuria. In DKD, reduced NAD availability may impair SIRT1 signaling, allowing histones to remain acetylated, decreasing DNMT1 activity, and leading to CLDN1 hypomethylation. These epigenetic changes may upregulate claudin-1 and pro-inflammatory/pro-sclerotic pathways, contributing to podocyte dysfunction and albuminuria. NAD augmentation is hypothesized to restore NAD-dependent SIRT1 activity and DNMT1-mediated epigenetic regulation, suppress maladaptive gene expression, preserve podocyte structure, and reduce albuminuria. DKD, diabetic kidney disease; DNMT1, DNA methyltransferase 1; NAD, nicotinamide adenine dinucleotide; SIRT1, sirtuin 1.

The SIRT-NAD pathway has emerged as a particularly important regulator of aging mechanisms.²⁸ NAD-dependent enzymes are involved in mitochondrial energy production, DNA repair, chromosomal integrity, regulation of gene expression, innate immune responses, and cellular regeneration, which are all recognized pillars of aging.29, 30, 31, 32, 33 These observations support the inclusion of physical function and aging-related biomarkers as exploratory secondary outcomes; however, this phase 2a trial is not designed to determine whether NAD augmentation improves other organ-system outcomes or reduces the risk of age-related conditions.29, 30, 31

Preclinical Evidence and the Legacy Effect

In a mouse model of DKD, administration of NMN for 2 weeks increased SIRT1 expression, attenuated the increase in UACR, provided histopathologic protection, and reduced mortality without affecting glycemic control.¹⁴ In this preclinical model, the benefits appeared to persist 14 weeks after treatment cessation, suggesting a possible posttreatment legacy effect attributed to epigenetic modulation by NAD-dependent SIRT deacetylase.¹⁴ Whether an analogous durable effect occurs in humans with DKD remains unknown. This response has been postulated to be mechanistically grounded in SIRT1-mediated DNA (cytosine-5)-methyltransferase 1 activity: NAD-dependent SIRT deacetylation remodels CpG methylation at the claudin-1 promoter in podocytes, a chromatin-level change that persists well beyond the period of NAD repletion and is not observed with drugs that affect renal hemodynamics or glycemic control.^13,15 A phase 1 trial provided preliminary evidence that nicotinamide supplementation attenuated acute kidney injury in patients undergoing cardiac bypass surgery.³⁴

Trial Rationale

Building upon these preclinical and phase 1 data, we designed the NAD in DKD trial—the first randomized controlled trial of pharmaceutical-grade microcrystalline formulation of β-NMN (MIB-626) for the treatment of DKD. In pharmacokinetic studies, we have shown that 1.0 g twice daily regimen of β-NMN safely increased blood NAD levels by >200% above baseline.^35,36 At the doses used in this trial, MIB-626 has not been associated with flushing, hepatotoxicity, or insulin resistance reported previously with high-dose niacin.^35,37,38 The primary objective of this trial is to determine whether oral β-NMN treatment significantly reduces UACR compared with placebo in adults with DKD. We hypothesize that β-NMN treatment will improve UACR without significantly affecting glycemic control. In addition, we will explore if such improvements in UACR during MIB-626 treatment persist 3 months after treatment cessation (i.e., a legacy effect).¹⁴ Secondary objectives include evaluating the effects of β-NMN on kidney function measures, including serum creatinine, cystatin C, and eGFR; kidney injury and inflammatory biomarkers, including kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, soluble tumor necrosis factor receptor 1, and soluble tumor necrosis factor receptor 2; and measures of muscle performance and physical function. An additional objective is to evaluate the effects of β-NMN treatment on consensus biomarkers of aging (e.g., interleukin-6, tumor necrosis factor-α, insulin-like growth factor–1, and urinary F2-isoprostane). In addition, we will assess the effects of MIB-626 treatment on blood NAD levels, plasma concentrations of NAD metabolites, glycemic control, lipids, and blood pressure. Here, we describe the design and methods of this trial.

Methods

Overall Study Design

The NAD in DKD trial, also known as the Kidney Insufficiency and NAD Treatment in Diabetes (KIND) Study, is an investigator-initiated, phase 2a, randomized, double-blind, placebo-controlled, parallel-group trial funded by the National Institute on Aging. The Mass General Brigham Institutional Review Board served as the central institutional review board, with additional approval obtained from the institutional review boards at each participating site. The trial is registered on ClinicalTrials.gov (NCT05759468). The protocol adheres to SPIRIT guidelines, and reporting will follow the CONSORT statement.

The trial is being conducted across the following 3 academic centers: (i) the Research Program in Men's Health: Aging and Metabolism at Brigham and Women's Hospital, Harvard Medical School, Boston, MA; (ii) the Division of Nephrology at Boston Medical Center, Boston University School of Medicine, Boston, MA; and The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA. All participants provided written informed consent prior to enrollment.

Study Setting and Participant Recruitment

Potential participants were identified through the following: (i) proactive outreach via the electronic medical record databases to identify adults with International Classification of Diseases 10th or 11th Revision codes for diabetes mellitus and elevated UACR; (ii) referrals from nephrology, endocrinology, and primary care clinics; and (iii) direct advertising through institutional websites and social media. Institutional review board–approved recruitment materials were used for all recruitment methods.

Potential participants were prescreened by telephone. Those who qualified in the telephone prescreening, were screened in person, at which time eligibility was formally determined based on medical history, physical examination, review of records, and laboratory testing, including 2 fasting morning UACR measurements on separate days, serum creatinine, eGFR, glycated hemoglobin (HbA1c), and routine blood counts and chemistries.

Eligibility Criteria

The inclusion and exclusion criteria are presented in detail in Table 1. Briefly, eligible participants were men and women, aged ≥ 30 years, with type 1 or type 2 diabetes mellitus, defined by self-report of diabetes and current use of prescribed diabetes medication, International Classification of Diseases 10th or 11th Revisions coding for diabetes plus current medication, HbA1c > 6.4%, or 2 fasting glucose values > 125 mg/dl, along with an average UACR ≥ 100 mg/g creatinine (each value ≥ 60 mg/g) and an eGFR > 25 ml/min per 1.73 m². Participants with UACR > 300 mg/g creatinine were required to be on an ACE inhibitor, ARB, or SGLT2 inhibitor. Participants were required to be able to speak English, Spanish, or Haitian Creole.

Table 1.

Eligibility criteria

Inclusion criteria

1.Community-living adults, aged ≥ 30 yrs

2.Diagnosis of type 1 or type 2 diabetes mellitus, defined by any of the following: (a) self-report of diabetes and current use of prescribed diabetes medication; (b) ICD-10 or ICD-11 diagnosis of diabetes with current use of diabetes medication documented in the electronic medical record; or (c) HbA1c > 6.4% or 2 fasting glucose values > 125 mg/dl.

3.Albuminuria defined as ≥ 2 morning UACR measurements ≥ 100 mg/g creatinine (each ≥ 60 mg/g), with ≥ 1 value obtained during screening; a value within 3 mos before screening is acceptable.

4.For participants with type 2 diabetes and UACR > 300 mg/g creatinine: current treatment with an ACE inhibitor, ARB, or SGLT2 inhibitor.

5.eGFR > 25 ml/min per 1.73 m2

6.HbA1c < 10%

7.Able to speak English or Spanish or Haitian Creole

8.Willing and able to provide written informed consent.

9.For female participants: not pregnant and not planning pregnancy during the study.

Exclusion criteria

1.Morning UACR > 5000 mg/ g creatinine

2.Laboratory abnormalities: (a) AST or ALT >3× upper limit of normal; (b) eGFR < 25 ml/min per 1.73 m2; (c) hematocrit < 0.34 or > 0.50 L/L.

3.Major adverse cardiovascular event in the preceding 3 mos

4.Participation in an investigational drug or biologic study within the past 3 mos or within 5 half-lives of the investigational agent, whichever is shorter.

5.Current alcohol or substance use disorder or dependence (DSM 5 criteria).

6.Major depressive disorder, bipolar disorder, schizophrenia, or current psychotic symptoms or behavioral problems that could interfere with study procedures. If stable over the past 2 yrs, they are allowed.

7.Acute illness requiring hospitalization within the past 3 mos, or any acute illness (including COVID-19) within the past month

8.History of anaphylaxis from vitamin B3 derivatives

9.BMI > 42.5 kg/m2

10.Diagnosis of type 3 diabetes or gestational diabetes.

ACE, angiotensin-converting enzyme; ALT, alanine aminotransferase; ARB, angiotensin receptor blocker; AST, aspartate aminotransferase; BMI, body mass index; DSM-5, Diagnostic and Statistical Manual of Mental Disorders, 5th Edition; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; ICD, International Classification of Diseases; NSAIDs, nonsteroidal antiinflammatory drugs; NMN, nicotinamide mononucleotide; UACR, urinary albumin-to-creatinine ratio.

Participants were excluded if they had a UACR > 5000 mg/g creatinine, aspartate aminotransferase or alanine aminotransaminase > 3× the upper limit of normal, a hematocrit outside the range of 0.34 to 0.50 L/L, or HbA1c > 10%. In addition, a major adverse cardiovascular event within the 3 months preceding enrollment or a body mass index > 42.5 kg/m² precluded participation.

Additional exclusions included participation in an investigational drug trial within 3 months or 5 half-lives (whichever was shorter), a known history of anaphylaxis to vitamin B3 derivatives, and active major untreated psychiatric illness. Pregnancy was exclusionary. Use of niacin, NMN, or nicotinamide riboside supplements was prohibited throughout the study period. Participants receiving GLP-1 receptor agonists or SGLT2 inhibitors were permitted to enroll, provided they had been on a stable dose for ≥4 weeks before screening. Baseline use of ACE inhibitors or ARBs, SGLT2 inhibitors, GLP-1 receptor agonists, and MRAs, including nonsteroidal MRAs and changes in these medications during the trial, will be captured through concomitant medication review and considered in sensitivity analyses.

Randomization

Eligible participants were assigned in a 1:1 ratio to MIB-626 or a matched placebo using a computer-generated block randomization scheme with randomly varying block sizes. Stratification was performed by sex (male, female), age group (30–44, 45–65, and ≥66 years), and enrolling site.

Allocation concealment was maintained throughout enrollment. The allocation sequence was created by an unblinded study biostatistician using R (www.r-project.org)³⁹ and deployed through a secure, centralized web-based system. Participants were then issued a sequential randomization number, which served as the sole identifier linking each individual to their assigned intervention for the duration of the trial.

Masking

The trial is double-blind: participants and study staff were unaware of the intervention assignment. The randomization schedule was masked from all personnel except the unblinded biostatistician, the Investigational Drug Pharmacy Services staff, and the Data and Safety Monitoring Board (DSMB). MIB-626 and placebo tablets are matched in appearance and packaging. The Investigational Drug Pharmacy Services pharmacist dispenses study medication according to the randomization code.

Study Intervention

MIB-626 is a Good Manufacturing Practice–grade microcrystalline β-NMN formulation manufactured by Metro International Biotech, LLC, and compressed into 500-mg tablets.³⁵ Participants assigned to the active arm receive two 500-mg tablets orally twice daily at 12-hour intervals (2000 mg total/d). The control group receives matching placebo tablets on the same schedule as the active treatment arm. On day 1, the first dose was administered under direct staff supervision, and participants were observed for 30 minutes following administration.

The 1000 mg twice daily dose is based on our phase 1a and phase 1b trials in which this regimen was safe, raised intracellular NAD by >250% above baseline, achieved steady state levels by day 8, and was associated with no serious adverse events or grade 2 toxicities.³⁶ Participants were instructed to maintain their regular diet and physical activity levels throughout the study period.

Study Outcomes

Study outcomes are summarized in Table 2.

Table 2.

Study end points

End point category	Outcome measure	Description	Time frame
Primary end point	Change in UACR	Change from baseline in UACR over the 6-mo intervention period (baseline to 6 mos) to assess treatment effect on albuminuria, a validated surrogate marker of DKD progression that responds to therapeutic interventions within mos.	Baseline, weeks 6, 12, 18, 24
Secondary end point	≥ 30% reduction in UACR	Proportion of participants achieving ≥ 30% reduction in UACR from baseline at 6 mos, a clinically meaningful threshold associated with improved renal outcomes.	Baseline, weeks 6, 12, 18, 24
	Kidney injury biomarkers (KIM-1, NGAL, sTNFR1, sTNFR2)	Change from baseline in biomarkers of kidney injury and inflammation over the 6-mo intervention period (baseline to 6 mos) associated with DKD progression.	Baseline, weeks 12, 24
	Serum creatinine	Change from baseline in serum creatinine over the 6-mo intervention period (baseline to 6 mos) to evaluate kidney function using a standard clinical marker.	Baseline, weeks 6, 12, 18, 24
	Cystatin C	Change from baseline in cystatin C over the 6-mo intervention period (baseline to 6 mos) to provide a muscle mass–independent estimate of kidney function.	Baseline, weeks 6, 12, 18, 24
	eGFR	Change from baseline in eGFR over the 6-mo intervention period (baseline to 6 mos) to evaluate kidney function using a standard estimated measure derived from serum creatinine.	Baseline, weeks 6, 12, 18, 24
	Muscle performance (strength, power, endurance)	Change from baseline in maximal voluntary strength, peak power, and endurance (repetitions to failure) using Keiser resistance equipment over the 6-mo intervention period (baseline to 6 mos) to assess skeletal muscle function.	Baseline, weeks 12, 24
	6-min walk distance	Change from baseline in 6-min walk distance over the 6-mo intervention period (baseline to 6 mos) to assess performance-based physical function.	Baseline, weeks 12, 24
	Late-life function and disability instrument (LLFDI)	Change from baseline in LLFDI scores over the 6-mo intervention period (baseline to 6 mos) to assess patient-reported physical function and disability.	Baseline, weeks 12, 24
	Inflammatory and aging biomarkers (IL-6, TNF-α, IGF-1, T3, urinary F2-isoprostanes)	Change from baseline in circulating biomarkers of inflammation, oxidative stress, and aging pathways over the 6-mo intervention period (baseline, interim visits, and 6 mos) relevant to DKD and geroscience.	Baseline, weeks 12, 24
	NAD metabolome (NMN, NAD, related metabolites)	Change from baseline in NAD-related metabolites in blood measured by LC–MS/MS over the 6-mo intervention period and during follow-up (baseline to 6 mos, with assessment at 9 mos) to confirm target engagement and pharmacodynamic effects.	Baseline, weeks 12, 24
	Glycemic control (HbA1c, fasting glucose)	Change from baseline in HbA1c and fasting glucose over the 6-mo intervention period (baseline to 6 mos) to assess whether renal and metabolic effects occur independently of glucose lowering.	Baseline, weeks 6, 12, 18, 24
	Blood pressure	Change from baseline in blood pressure measured under standardized conditions over the 6-mo intervention period (baseline to 6 mos) to assess cardiovascular and renal risk modification.	Baseline, weeks 6, 12, 18, 24
	Treatment adherence	Proportion of prescribed doses taken, assessed by pill counts and/or electronic monitoring over the 6-mo intervention period to evaluate treatment fidelity.	6 mos
Exploratory end point	eGFR trajectory	Change from baseline in eGFR over the 6-mo intervention period (baseline to 6 mos) to explore trends in kidney function and inform design and sample size estimation for future phase 3 trials.	Baseline, weeks 6, 12, 18, 24, 36
	Posttreatment NAD levels (legacy effect)	Change in NAD and related metabolites from the end of intervention (6 mos) to 3 mos posttreatment (9 mos total) to evaluate sustained (“legacy”) effects of NMN.	Weeks 24, 36

DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; IGF-1, insulin-like growth factor 1; IL-6, interleukin-6; KIM-1, kidney injury molecule-1; LC–MS/MS, liquid chromatography–tandem mass spectrometry; NAD, nicotinamide adenine dinucleotide; NGAL, neutrophil gelatinase-associated lipocalin; NMN, nicotinamide mononucleotide; sTNFR1, soluble tumor necrosis factor receptor 1; sTNFR2, soluble tumor necrosis factor receptor 2; T3, triiodothyronine; TNF-α, tumor necrosis factor–α; UACR, urinary albumin-to-creatinine ratio.

Primary Outcome

The primary end point is the change from baseline in UACR over the 24-week intervention period. UACR is a highly meaningful and commonly used outcome in phase 2 DKD trials because albuminuria is a cardinal feature of DKD that predicts doubling of serum creatinine, rate of decline of eGFR, start of kidney replacement therapy, as well as cardiovascular and all-cause mortality. Furthermore, improvements in UACR by drugs, such as ACE inhibitors, ARBs, and SGLT2 inhibitors, are associated with slower rates of decline in eGFR and progression to kidney failure in patients with type 2 diabetes mellitus, and lower risk of cardiovascular events. The magnitude of early reduction in UACR during the first months of treatment is strongly associated with reduced risk of progression of DKD and cardiovascular events. In an individual participant data analysis of 48 randomized controlled trials (studies) involving 85,681 participants, treatment effects on 6-month UACR was predictive of kidney failure or doubling of serum creatinine concentration, supporting the use of albuminuria change as a surrogate end point in CKD clinical trials.⁴⁰ UACR will be measured at baseline and during weeks 6, 12, 18, 24, and 36. This phase 2a trial is not statistically powered or sufficiently long to determine effects on sustained eGFR decline, or the progression to end-stage renal disease or kidney replacement therapy. These outcomes would require larger and longer trials.

Secondary Outcomes

Secondary outcomes encompass a range of functional, biochemical, and molecular end points. The proportion of participants achieving a ≥30% reduction in UACR was prespecified as a supportive analysis. A 30% reduction in UACR is highly meaningful because this magnitude of reduction in UACR is associated with a significant reduction in DKD progression and cardiovascular risk,^41,42 is achievable with several drugs approved for this indication,^7,41,42 is unlikely to be due to test-retest variability observed in patients with DKD,⁴³ and has been proposed to the US Food and Drug Administration as a surrogate end point in a workshop sponsored jointly by the National Kidney Foundation, US Food and Drug Administration, and the European Medicines Agency.⁴⁴

Kidney function measures, including serum creatinine, cystatin C, and eGFR, will be assessed as changes from baseline. Kidney injury and inflammatory biomarkers, including kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, soluble tumor necrosis factor receptor 1, and soluble tumor necrosis factor receptor 2, will be assessed over the intervention period at baseline and at weeks 12 and 24.

Maximal voluntary muscle strength, muscle power, and endurance (repetitions to failure at 80% 1-repetition maximum) in the leg press exercise, and both performance-based (6-minute walk distance) and self-reported (Late Life Function and Disability Index) measures of physical function will be assessed at baseline and at weeks 12, 24, and 36. Maximal voluntary strength will be assessed using the 1-repetition maximum method on pneumatic-resistance leg press and chest press machines (Keiser Sport, Fresno, CA)⁴⁵ using standardized seat positions, range of motion, and testing procedures. Following a warm-up set at approximately 50% of the estimated 1-repetition maximum, resistance will be progressively increased until only 1 full-range-of-motion repetition can be completed with proper technique. For muscle fatigability, the participants will perform as many full-range-of-motion repetitions as possible at 80% of their 1-repetition maximum using standardized cadence and technique. Total repetitions completed will be recorded. The 6-Minute Walk Test will be conducted in a long, flat indoor corridor using standardized instructions and encouragement. Participants will be instructed to walk as far as possible over 6 minutes, with rests permitted as needed. Total distance walked will be recorded in meters.⁴⁶

Circulating aging biomarkers include HbA1c, insulin-like growth factor–1, triiodothyronine, interleukin-6, tumor necrosis factor–alpha, and urinary F2-isoprostane. Changes in blood NAD levels and circulating concentrations of NMN; nicotinamide; nicotinamide riboside; N1-methyl-2-pyridone, 4-carboxamide;, and 1-methylnicotinamide will be measured using validated liquid chromatography with tandem mass spectrometry assays. Fasting glucose and blood pressure under standardized conditions will be measured as secondary end points.

Exploratory Outcomes

The exploratory outcomes include the longitudinal trajectory of eGFR during the intervention and posttreatment follow-up periods, and the incidence of progression to end-stage renal disease and kidney replacement therapy to inform end point selection and sample size estimation for a future phase 3 trial. In addition, the persistence of changes in UACR and blood NAD levels from week 24 to week 36 as a potential legacy effect of the intervention will be evaluated.

Measurement of Blood NAD and NMN, and Plasma Concentrations of NAD-Related Metabolites

For blood NAD, blood samples in K2EDTA vacutainers are immediately precipitated with 4% TCA, centrifuged, and supernatants stored at −80 °C. Blood and plasma NAD metabolites (NAM, nicotinamide riboside, N1-methyl-2-pyridone, 4-carboxamide, 1-methylnicotinamide) will be quantified in plasma using validated liquid chromatography with tandem mass spectrometry assays that have been published.^35,36

Schedule of Study Procedures

The assessment schedule is summarized in Table 3. Potential participants underwent screening within 45 days before the baseline visit. Baseline procedures included vital sign assessment, phlebotomy for biomarker analysis, and evaluations of physical function. Following these assessments, subjects were randomized, and the initial dose of the study medication was administered under clinical supervision on day 1. In-person assessments, including a physical examination, electrocardiogram, and full biomarker panels, with muscle performance and physical function tests, are performed at baseline and at weeks 12, 24, and 36.

Table 3.

Schedule of study assessments

Procedure	Prescreen	Screen	Baseline studies	Day 1	Week 1	Week 6	Week 12	Week 18	Week 24	Week 36
Telephone prescreening	X
Informed consent form		X
Eligibility checklist			X
Randomization				X
Intervention period
Study medication dispensing				X		X	X	X
Reminders about study medication and dietary adherence				X	X	X	X	X	X
UACR, creatinine, eGFR		X	X			X	X	X	X	X
Glycemic control: A1c, fasting glucose		X	X			X	X	X	X	X
Safety labs: CBC, chemistry panel, urinalysis, lipids		X	X		X	X	X	X	X	X
Standardized blood pressure measurement		X		X		X	X	X	X	X
Body weight		X	X		X	X	X	X	X	X
ECG		X					X		X	X
Blood for NMN, NAD, and NAD metabolites		X		X			X		X	X
General physical examination		X					X		X	X
Biomarkers of aging			X				X		X	X
Biomarkers of kidney function			X				X		X	X
Physical function (LLFDI, 6MWD, muscle strength, power, and endurance)			X				X		X	X
AE/ SAE recording		X			X	X	X	X	X	X
Concomitant medications		X			X	X	X	X	X	X
Study medication accountability/ adherence				X	X	X	X	X	X

6MWD, 6-minute walk distance; AE, adverse event; CBC, complete blood count; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; IL-6, interleukin-6; LLFDI, Late-Life Function and Disability Instrument; NAD, nicotinamide adenine dinucleotide; NMN, nicotinamide mononucleotide; PBMC, peripheral blood mononuclear cell; SAE, serious adverse event; TNF-α, tumor necrosis factor alpha; UACR, urinary albumin-to-creatinine ratio.

Safety Assessment and Monitoring

An independent DSMB composed of experts in nephrology, clinical trials, and biostatistics reviews safety data and study progress every 6 months. All adverse events and serious adverse events are captured, coded by Medical Dictionary for Regulatory Activities, and summarized by System Organ Class. Safety laboratory assessments, including hematology and blood chemistries, are performed at weeks 1, 6, 12, 18, 24, and 36. Prespecified stopping rules are in place (alanine aminotransaminase or aspartate aminotransferase >3× upper limit of normal).

Diabetes and hypertension management, including medication and dietary adjustments, remained under the care of participants’ primary care clinicians and/or diabetes or hypertension specialists. HbA1c, fasting glucose, and blood pressure were reviewed by the study clinician at each trial site in a blinded manner as safety measures. Prespecified glycemic rescue thresholds included HbA1c > 10%, measured or self-reported glucose > 250 mg/dl, or hypoglycemia with glucose < 70 mg/dl on ≥2 occasions in the same week. If these thresholds were met, the participants were asked to contact their primary care team for medication adjustment and offered referral to diabetes specialty care. For blood pressure, if measured or reported systolic blood pressure was > 180 mm Hg or diastolic blood pressure was > 100 mm Hg on >2 occasions over 2 consecutive weeks, the participants were asked to contact the primary care team for medication adjustment and offered referral to a hypertension specialist at the participant’s usual-care institution. Because blood pressure control can affect albuminuria, standardized blood pressure measurements and antihypertensive medication use are being recorded and will be summarized; sensitivity analyses will consider blood pressure and changes in antihypertensive therapy as covariates.

Statistical Considerations

Sample Size Determination

We conservatively assumed that NMN treatment for 24 weeks will produce a mean 35% reduction in UACR versus approximately 5% with placebo, yielding a net 30% greater reduction, with interindividual SD ≤ 50%. This magnitude of change is clinically meaningful: in trials of ACE inhibitors, ARBs, GLP-1 receptor agonists, and SGLT2 inhibitors, UACR reductions of this magnitude were associated with lower rates of eGFR decline, kidney failure, and cardiovascular mortality.^41,47, 48, 49 In the preclinical model of DKD, UACR improvements with NMN treatment were substantially greater (40%–75%).¹⁴

In a simple between-arm comparison using a 2-sided unpaired t test, 118 evaluable participants provide 90% power to detect a net 30% UACR reduction attributable to NMN (80% power to detect a net 24% reduction). The longitudinal mixed-model design is expected to provide greater power than this conservative estimate, because serial intraindividual correlations will be substantial (anticipated rho ≥ 0.50 for biomarkers, 0.8–0.9 for physical function). Assuming 15% missingness, the sample size was initially set at 140 participants.

In November 2025, because the study was very close to meeting its enrollment target of 140, 13 participants had discontinued the study before study completion or were lost to follow-up (missingness rate 13%). Due to high rates of enrollment, many subjects were still early in their intervention period. To protect statistical power should the missingness rate unexpectedly exceeded 15%, the sample size was increased to 156 with DSMB's approval.

Statistical and Analytical Methods

A formal statistical analysis plan will be prepared and finalized before database lock and unblinding. All analyses will use R version 4.1.0 or later (R Foundation for Statistical Computing, Vienna, Austria).³⁹ All hypothesis tests are 2-sided with a type I error rate of 0.05. No adjustments for multiple comparisons are planned. Accordingly, the P-values for secondary and exploratory analyses will be considered nominal and supportive and interpreted with caution. No interim efficacy analyses will be conducted. The DSMB monitors safety, accrual, adherence, and data quality on an ongoing basis.

Analysis Populations

The intention-to-treat population will include all randomized participants. It will be used for primary efficacy analyses, with each participant analyzed in the arm to which they were randomized, regardless of adherence. The safety population will include all randomized participants who receive ≥1 dose of the study medication. The per-protocol population includes intention-to-treat participants who complete the study without major protocol deviations and adhered to ≥80% of study medication.

Primary Efficacy Analysis

The primary analysis will use a mixed models repeated measures framework implemented as a linear mixed-effects regression model on all available data. Time will be treated as a discrete variable; neither equality of effects at different time points nor linearity of changes is assumed. The model will control for stratification factors (sex, age group, trial site). The primary end point is the between-arm difference in UACR over the 24-week treatment period, estimated by a treatment contrast from the mixed models repeated measures. Model-based point estimates and 95% confidence intervals will be reported for all time points.

Secondary and Exploratory Analyses

In supportive analysis of the primary end point, we will compare the proportion achieving ≥30% UACR reduction using a modified Poisson regression model with robust variance estimated using generalized estimating equations, providing relative risk estimates. We anticipate that approximately one-third of NMN-treated participants will achieve this threshold, compared with <5% of placebo participants. Secondary outcomes, including kidney biomarkers, eGFR, physical function, NAD metabolome, and glycemic outcomes, will be analyzed using parallel linear mixed effects models with the same covariate structure. Sensitivity analyses controlling for factors that were imbalanced at randomization may be employed based on clinical judgment. The association between steady-state NAD and NAD metabolite concentrations and changes in UACR will be assessed using linear regression. Legacy effect analyses will be used to compare NAD levels and kidney biomarkers at week 36 relative to week 24 within each arm. Missing data will be handled using mixed models repeated measures, with multiple imputation by chained equations considered as a sensitivity analysis.

Prespecified exploratory subgroup analyses of the primary end point will examine whether the treatment effect on UACR differs by baseline use of SGLT2 inhibitors, GLP-1 receptor agonists, and MRAs, including nonsteroidal MRAs. Subgroup-specific treatment estimates and interaction P-values will be reported; however, because the trial is not powered for interaction testing, these analyses will be interpreted as exploratory and hypothesis-generating. Sensitivity analyses will additionally adjust for on-treatment blood pressure.

Exploratory posttreatment analyses will compare NAD levels, kidney biomarkers, and UACR at week 36 relative to week 24 within each arm to evaluate the persistence of pharmacodynamic and biomarker changes after treatment cessation. Missing data will be handled using mixed models repeated measures, with multiple imputation by chained equations considered as a sensitivity analysis.

The association between steady-state NAD and NAD metabolite concentrations and changes in UACR will be assessed using linear regression.

Safety outcomes will be summarized by system organ class and preferred term using the Medical Dictionary for Regulatory Activities. Mean changes in laboratory measures from baseline to final visit will be compared between groups using analysis of covariance. Binary safety end points will be compared using Chi-square or Fisher exact test.

Current Enrollment Status

Screening for the trial began in July 2023, and enrollment was completed in February 2026. A total of 156 participants were randomized across 3 sites: 123 at Brigham and Women’s Hospital, 21 at The Lundquist Institute, and 12 at Boston Medical Center. As of May 15, 2026, 82 participants had completed the study, and 47 participants are currently in the intervention or the postintervention follow-up period. The DSMB has regularly conducted scheduled safety reviews approximately every 6 months and has not recommended stopping or modifying the trial.

The baseline characteristics of the study participants are described in Table 4.

Table 4.

Baseline characteristics of the study population (N = 156)

Characteristics	n
Age group (yrs)
30–44	6 (3.8%)
45–65	63 (40.4%)
≥ 66	87 (55.8%)
BMI (kg/m2)	29.5 (5.0)
Sex, n (%)
Male	120 (76.9)
Female	36 (23.1)
Ethnicity
Hispanic or Latino, n (%)	32 (20.5%)
Race, n (%)
American Indian or Alaska Native	2 (1.3%)
Asian	11 (7.1%)
Black or African American	25 (16.0%)
Native Hawaiian or Other Pacific Islander	1 (0.6%)
White	105 (67.3%)
> 1 Race	2 (1.3%)
Other	10 (6.4%)
Systolic blood pressure (mm Hg)	130 (20)
Diastolic blood pressure (mm Hg)	71 (14)
UACR (mg/ g of creatinine)	258 (161–686)
HbA1c (%)	7.2 (1.0)
Fasting glucose (mg/dl)	133 (42)
Hemoglobin (g/dl)	13.7 (1.7)
eGFR (ml/min per 1.73 m2)	63 (24)
Serum creatinine (mg/dl)	1.3 (0.5)

BMI, body mass index; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; UACR, urinary albumin-to-creatinine ratio.

Data are presented as n (%) for categorical variables, mean (SD) for normally distributed continuous variables, and median (interquartile range) for nonnormally distributed variables. Percentages may not total 100% because of rounding.

All data were collected at baseline prior to randomization, with treatment allocation remaining blinded at the time of analysis.

Perspective

The NAD in DKD trial is the first randomized, placebo-controlled trial to evaluate the safety and efficacy of pharmaceutical-grade β-NMN (MIB-626) in adults with DKD. The scientific rationale draws on converging evidence from preclinical and early-phase human studies. NAD levels decline in DKD, a change associated with reduced SIRT1 activity, impaired tubulo-podocyte crosstalk, mitochondrial dysfunction, and progressive albuminuria and glomerulosclerosis.13, 14, 15^,19,20 In a mouse model of DKD (Leprˆdb/dbˆ), NMN attenuated UACR and histopathologic injury without affecting glycemic control, supporting a mechanism distinct from that of existing therapies.¹⁴ Critically, the beneficial effects of a 2-week NMN course persisted for 14 weeks after cessation—a legacy effect mediated by epigenetic remodeling¹⁴—which, if replicated in humans, would have major implications for the therapeutic deployment of NAD augmentation. Whether such a legacy effect occurs in humans remains unknown and is addressed here as an exploratory objective in this trial.

The proposed randomized controlled trial has several important attributes of a rigorous trial design: concealed block randomization, a double-blind, placebo-controlled, parallel-group design, multisite enrollment, stratification by key prognostic factors, a validated and clinically meaningful primary end point, and independent DSMB oversight. A major strength is the use of pharmaceutical-grade, Good Manufacturing Practice–manufactured MIB-626, overcoming a central limitation of earlier NMN studies that used variable-quality dietary supplement formulations. The 1000 mg twice-daily dose is supported by our own phase 1 data demonstrating safety and a >200% increase in blood NAD levels.³⁵ The rigorous sample collection procedure ensures preanalytical stability of NAD and the application of validated liquid chromatography with tandem mass spectrometry assays provides high precision and accuracy in the measurements of NAD and its metabolites.

The inclusion of secondary outcomes spanning physical function, muscle endurance, and aging biomarkers will assess the multiorgan potential of NAD augmentation. Patients with DKD frequently have sarcopenia and reduced physical capacity. NMN's demonstrated improvement of endothelial function and exercise performance in aged mice⁵⁰ provides a biologically plausible basis for these secondary hypotheses. The measurement of steady-state NAD 3 months posttreatment will evaluate for legacy effect.

Several design features warrant comment because of their limitations. First, the primary end point is change in UACR, a surrogate end point appropriate for a phase 2a proof-of-concept trial; the study is neither large nor long enough to assess patient-important kidney outcomes, such as sustained eGFR decline, progression to end-stage renal disease or need for kidney replacement therapy. The 24-week intervention duration was selected to assess changes in UACR and kidney injury biomarkers over a timeframe consistent with prior DKD trials, while requiring stable background albuminuria-lowering therapies, including ACE inhibitors or ARBs, before randomization to minimize confounding from concomitant therapy changes.51, 52, 53 Prespecified exploratory sensitivity analyses will evaluate the influence of background use of ACE inhibitors or ARBs, SGLT2 inhibitors, GLP-1 receptor agonists, and MRAs because these therapies can independently reduce albuminuria; however, the trial is not powered to definitively assess treatment-effect modification. Because UACR is blood pressure sensitive, changes in antihypertensive therapy during follow-up may influence albuminuria; we will frequently record blood pressure under standardized conditions, and sensitivity analyses will consider on-treatment blood pressure and blood pressure medications. The trial's findings do not apply to individuals with very advanced CKD, UACR > 5000 mg/g, poorly controlled diabetes (HbA1c > 10%), severe obesity (body mass index > 42.5 kg/m²), or recent major adverse cardiovascular events, who were excluded from the study. Whether NAD augmentation could benefit nonalbuminuric DKD, in which tubulointerstitial injury and vascular disease may be prominent, remains unknown. Finally, because numerous secondary and exploratory end points are included without adjustment for multiple comparisons, these analyses should be interpreted with caution as hypothesis-generating. If this trial demonstrates acceptable safety and efficacy in reducing UACR, these data will provide the rationale for larger trials with longer duration to assess the efficacy of NAD augmentation in improving patient-important kidney outcomes, such as progression to end-stage renal disease and kidney replacement therapy.

In conclusion, the NAD in DKD trial will evaluate the safety and efficacy of pharmaceutical-grade β-NMN (MIB-626) in reducing UACR in adults with albuminuric DKD. The trial will provide data on UACR, kidney injury biomarkers, physical function, aging-related biomarkers, and the NAD metabolome to inform the design of larger trials powered for long-term kidney outcomes.

Disclosure

SB reports receiving research grants from the National Institute on Aging (NIA), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the National Institute of Child Health and Human Development (NICHD), FPT, and Metro International Biotech; and receiving consulting fees from Eli Lilly and Company, Besins Healthcare, Arvinas, and Pfizer. These grants are managed by Brigham and Women's Hospital and overseen by the Office of Industry Interaction of Mass General Brigham. MEM reports receiving research funding from Abbott Industries. JF receives research support from the National Institutes of Health (NIH). KFR reports receiving research grants and contracts, paid directly to their institution, from the National Institutes of Health (NIH), the NFL Players Association, and Best Buy Health, Inc. SSW reports research support from NIH; consulting fees from Vertex, Pepgen, and Delix; and serving as an expert witness on litigation related to voclosporin. All the other authors declared no competing interests.