Folate Treatment to Reduce Death Risk in the Year After Infection-Related Acute Kidney Injury

1. Background Information Acute Kidney Injury (AKI) affects over 13 million people each year and has a hospital mortality of 20-40%. It is characterized by an abrupt loss of kidney function and is associated with multiple long-term sequelae including chronic kidney disease (CKD) and reliance on renal replacement therapy (RRT). Patients with AKI place a substantial financial burden on healthcare resources . Approximately 19%-37% of patients with AKI do not recover kidney function within one year, and 33% of patients progress to acute kidney disease (AKD), and from this subset, nearly 27% develop CKD within 90 days post-AKI diagnosis. This highlights the critical importance of early detection and effective management of AKI to prevent progression.

1.1 Epidemiology of AKI AKI affects all countries, and its prevalence ranges from \<1% to 66%. Apart from population variation, different classification systems used in epidemiological studies contributes to the varied estimation of AKI incidence. Little epidemiological data is available for AKI in Hong Kong (HK). Szeto and Pang reported AKI was present in 9.1% of all adult hospital admissions in HK. The crude mortality rate of AKI in patients attending an emergency department (ED) in HK is 25%.

1.2 Etiology of AKI AKI is a complex syndrome. The causes of AKI can be classified into prerenal, intrinsic renal and postrenal. Prerenal causes involve reduced renal perfusion due to medications, heart failure, infections, sepsis or volume depletion (e.g., diuretic overuse, severe dehydration and diarrhea). Intrinsic renal causes involve direct damage to the kidney structures including pre-existing renal impairment (e.g., glomerulonephritis, interstitial nephritis and vasculitis), and nephrotoxic drugs. Postrenal AKI is due to obstruction of urinary flow (e.g., kidney stone and tumors), and is especially common in elderly men. Sepsis, infections and use of nephrotoxic drugs are common causes of AKI overall, leading to reduced renal blood flow. In intensive care unit (ICU) settings, AKI occurs in more than half of patients, and half of all patients with AKI in ICU have sepsis. Drug induced AKI occurs in 20% of hospital cases. Associated drugs include non-steroidal anti-inflammatory drugs (NSAIDs), angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers \[ARBs\], metformin, aminoglycosides, diuretics and iodine containing X-ray contrast.

The diverse etiologies of AKI along with other comorbidities make it difficult to determine and understand the pathophysiology of AKI. Recently, oxidative stress has been accepted as the primary mediator of adverse outcomes in AKI, playing a critical role in both initiating and further development of AKI. In the context of deficient renal blood flow in AKI, ischemia-reperfusion represents the most common mechanism in which oxygen and nutrient delivery as well as waste removal are impaired; this mismatch of oxygen supply and demand, and accumulation of waste lead to cellular injury and even death. Sepsis-induced AKI also triggers oxidative stress. Oxidative stress as a key component in the pathophysiology of AKI provides a target for potential therapeutics.

1.3 Treatment for AKI The KDIGO serum creatinine-based criteria is mostly used to stage AKI. The management of AKI is primarily supportive. The key to management is assuring hemodynamic stability and preventing hypovolemia so as to ensure sufficient renal perfusion. Depending on the cause and severity of AKI, treatments in hospital usually include: blood pressure and circulating electrolyte control; body fluid balance (e.g., with fluid replacement and/or diuretics); review and cessation of nephrotoxic medication; removing the obstruction if there is obstruction; and use of antibiotics. Malnutrition also affects patients with AKI, which requires a kidney-friendly diet, adequate nutrition and restricting potassium. There are no approved pharmacological agents for treating AKI.

1.4 Folate Folate (vitamin B9) occurs naturally in foods. Folic acid is a synthetic, parent compound of folate family. Folic acid gets metabolized into 5-methyltetrahydrofolate (5-MTHF). 5-MTHF is the biologically active form of folate and the predominant form of dietary folate in plasma.

A systematic review was conducted in accordance with the PRISMA guidelines and registered in PROSPERO (CRD42024589377). The investigators searched six databases (PubMed, MEDLINE, CINAHL, CENTRAL, Embase, and Web of Science) from their inception until March 2024, identifying 36 randomized controlled trials (RCTs) that evaluated folate use in patients with CKD or AKI. Of these, 22 RCTs compared folate use with no use in patients with CKD, with treatment durations ranging from 4 weeks to 4.5 median years, and majority use folic acid. Notably, no published data focused on AKI populations. Among included RCTs, 20 reported that folate use significantly reduced homocysteine (Hcy) levels but not directly reduced cardiovascular events in CKD patients. Only 5 trials assessed markers of kidney disease progression including 3 measured serum creatinine and 2 measured estimated glomerular filtration rate (eGFR) with inconsistent findings. The largest study, China Stroke Primary Prevention Trial with 1671 CKD patients, demonstrated that folate use slowed the annual decline in eGFR compared to controls (0.96 ± 5.81% vs. 1.72 ± 6.08%, respectively). Similarly, Chang et al. observed a modest reduction in serum creatinine in end-stage renal disease (ESRD) patients receiving folate (10.94 ± 2.01 vs. 11.30 ± 2.31 in controls). Safety analyses across all 22 RCTs revealed no severe adverse events attributable to folate, and no significant differences in adverse outcomes were noted between the treatment and control groups.

Low doses: In a rat model of AKI with ischemia-reperfusion, 5-MTHF at a low dose of 3 μg/kg body weight improved kidney function, reduced plasma creatinine levels and alleviate oxidative stress within 24 hours. The expression of neutrophil gelatinase-associated lipocalin (NGAL), a marker of proximal tubular injury, was significantly reduced after folate treatment. 5-MTHF activated the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant defense system. Nrf2 activation regulates gene expression of detoxification enzymes and antioxidant proteins, which is vital to restore antioxidant defense against oxidative stress injury. Oxidative stress is the mainstay responsible to the adverse outcomes in AKI. The expression of antioxidant enzymes, superoxide dismutase-1 (SOD-1), glutathione synthesizing enzymes and heme oxygenase-1 (HO-1), were all upregulated after folate treatment.

High doses: Folic acid, a precursor of folate, at high doses, e.g., 250 mg/ /kg body weight, is used to induce AKI in animals. High doses of folic acid remain unmetabolized and cause AKI due to the rapid onset of folic acid crystals within renal tubules with a consequent acute tubular necrosis, epithelial regeneration and renal cortical scarring. This is only experimental in animals, and high levels of folic acid have not been observed in the clinic kidney diseases.

Preliminary data The investigators conducted a retrospective study to characterize the epidemiology and outcomes of adult patients with presumed infections and diagnosed with AKI at Queen Mary Hospital (QMH). The study utilized data from the Clinical Data Analysis and Reporting System (CDARS) of the Hospital Authority (HA) for the period between January 1, 2023, and December 31, 2023. Inclusion criteria required patients to be aged 18 years or older, have an infection, and be diagnosed with AKI based on the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. A total of 2585 patients met the eligibility criteria, with a median age of 72 \[IQR: 61, 84\] years and 56.0% being male. The median length of hospital stay was 13 \[IQR: 6, 26\] days. Notably, 61.2% (1581 patients) recovered quickly within one day, and by 7 days 23.4% (605 patients) did not recover and majority of them (543 patients) remained unrecovered by 28 days. 28.1% (727 patients) had sepsis, and 13.8% (357 patients) had pre-existing CKD. Among patients without pre-existing CKD, 1.5% (33 patients) developed CKD at 3 months post AKI diagnosis. The one-year mortality was 23.2% (600 patients).

A retrospective analysis comparing folic acid users (206 patients) and non-users (2379 patients) was conducted. Folic acid was prescribed at the same time when AKI occurred. Majority (178 patients) received an oral dose of 5 mg folic acid daily. The median treatment period was 35.5 days \[IQR 13.25, 83\]. Results revealed that folic acid users had slightly higher rates of AKI recovery at 24 hours (64.1% vs. 60.9%) and 7 days (79.1% vs. 76.4%), and had a significantly lower one-year mortality \[11.6% vs. 24.2% (24 vs. 576 patients), P \< 0.001\], compared with non-users. The progression to CKD at 3 months and cardiovascular events over one year post AKI showed no significant differences. The investigators conducted a multivariate logistic regression analysis to analyze the confounding factors including age, gender, CKD, diabetes mellitus, hypertension, heart failure, ischemic heart disease, stroke and sepsis, and confirmed the use of folic acid contributed to 57% \[OR 0.428 (95% CI: 0.275, 0.666), P = 0.0002\] of reducing mortality risk. Age, stroke and sepsis increased mortality risk; gender, diabetes mellitus, hypertension, heart failure, and ischemic heart disease did not significantly affect patient mortality; and CKD unexpectedly decreased mortality risk by 28% (P= 0.0325). Patients with CKD are often under more intense medical surveillance and management, and receive more comprehensive care, which may contribute to better overall health and increased survival.

The discrepancy between one-year mortality reduction and non-significant AKI recovery may reflect the multifactorial nature of mortality in AKI patients. While early renal recovery is important, long-term survival is influenced by systemic inflammation, cardiovascular events, and nutritional status. Folate's known roles in endothelial function, homocysteine metabolism, and oxidative stress modulation may confer survival benefits independent of short-term renal recovery.

The investigators hypothesize that the observed mortality reduction is mediated by mechanisms beyond immediate renal function improvement. Sepsis, the leading cause of AKI in critically ill patients, triggers a systematic inflammatory response and a surge in reactive oxygen species that cause widespread cellular damage. Folate has been shown to alleviate oxidative stress in a rat model of AKI. Folate's benefit may lie in attenuating systemic oxidative stress. Moreover, AKI recovery defined by serum creatinine has inherent limitations. Creatinine is a delayed and imperfect marker of kidney function, especially in sepsis. Septic AKI involves complex pathophysiology including tubular injury, microvascular dysfunction, and systemic inflammation, which is not adequately represented by creatinine levels alone. A prospective randomized controlled trial is important to confirm or refute the observation, and to investigate the causal link between folate and improved survival.

To further explore the influence of CKD status, subgroup analyses were conducted. Among 357 patients with CKD, folic acid users (n = 55) had relatively higher AKI recovery rates at 24 hours (74.5% vs. 73.2%), 7 days (87.3% vs. 84.4%), and 28 days (89.1% vs. 85.1%), along with significantly lower one-year mortality (5.5% vs. 20.5%, P = 0.0069). In the cohort without CKD (n = 2228), folic acid users (n = 151) had comparable AKI recovery rates to non-users at 24 hours (62.9% vs. 61.8%), 7 days (78.8% vs. 79.0%), and 28 days (78.8% vs. 79.8%), and still showed significantly lower one-year mortality (13.9% vs. 24.7%, P = 0.

• AKI Recovery Rate at 7 Days [Срок оценки: 7 days after randomization]
• Progression to Chronic Kidney Disease (CKD) at 3 Months [Срок оценки: 3 months (90 days) after randomization]
• Incidence of Major Adverse Cardiovascular Events (MACE) at 1 Year [Срок оценки: Up to 365 days from randomization]
• Incidence of Adverse Events [Срок оценки: Up to 365 days from randomization]
• Serum Folate Levels Comparison [Срок оценки: Baseline and 3 months after randomization]
• Change in Serum Creatinine [Срок оценки: Baseline, up to 30 days from randomization, 3 months after randomization, and 1 year after randomization]
• Change in Estimated Glomerular Filtration Rate (eGFR) [Срок оценки: Baseline, up to 30 days from randomization, 3 months after randomization, and 1 year after randomization]
• Change in Urine Output [Срок оценки: Up to 30 days from randomization]
• Change in Blood Urea Nitrogen (BUN) [Срок оценки: Baseline, up to 30 days from randomization, 3 months after randomization, and 1 year after randomization]
• Incidence of Proteinuria [Срок оценки: Up to 30 days from randomization and at 3 months after randomization]
• Time to AKI Recovery [Срок оценки: Up to 30 days from randomization]
• Incidence of Renal Replacement Therapy (RRT) Initiation [Срок оценки: Up to 90 days from randomization]