TMAO is a small, low-molecular-weight metabolite with an outsized reputation. It begins its life in the gut, where bacteria convert dietary precursors such as choline, phosphatidylcholine, betaine, and L-carnitine, abundant in red meat, eggs, dairy, and saltwater fish, into trimethylamine, or TMA. The liver then oxidizes TMA into TMAO via the enzyme flavin monooxygenase 3. From there, TMAO circulates in the blood and is excreted almost entirely by the kidneys, without undergoing any meaningful biotransformation along the way. Elevated plasma TMAO has been associated in numerous studies with cardiovascular disease, atherosclerosis, chronic kidney disease, and type 2 diabetes itself, and in diabetic patients specifically, higher TMAO has been tied to more cardiovascular events, faster loss of kidney function, and greater all-cause mortality.

The new analysis was prompted by a puzzling observation from a secondary analysis of the EMMY trial, in which patients treated with empagliflozin after a myocardial infarction showed a more pronounced rise in serum TMAO than those on placebo. That result raised an obvious question: does the same thing happen in people with type 2 diabetes, and does it extend to other drugs in the class, such as dapagliflozin? To find out, investigators combined individual patient data and stored samples from two prospective, randomised, placebo-controlled, double-blind crossover trials conducted at the University of Erlangen-Nuremberg, registered as NCT02471963 and NCT02383238. In each trial, patients were randomised after a run-in phase to receive either 25 mg empagliflozin or 10 mg dapagliflozin, or matching placebo, once daily for six weeks, followed by a one-week washout and a switch to the alternate treatment for another six weeks.

Fasting blood samples and 24-hour urine collections were obtained at baseline and at the end of both treatment periods, then stored at minus 80 degrees Celsius until analysis. TMAO concentrations were quantified with a validated liquid chromatography tandem mass spectrometry assay, using a deuterated internal standard and protein precipitation with methanol. After excluding a small number of samples that could not be quantified, the final analytical population comprised 123 patients, 69 from the empagliflozin trial and 54 from the dapagliflozin trial. The cohort had a mean age of 61.2 years, was 59 percent male, had a mean HbA1c of 6.7 percent, and a mean estimated glomerular filtration rate of 93.8 mL/min/1.73 m², with more than 70 percent of participants above 90. Compliance, assessed by tablet counts, averaged 99 percent in both trials.

The results were consistent in direction across the pooled population. Median plasma TMAO was 3.71 μmol/L at baseline and 3.60 μmol/L after placebo, but rose to 4.19 μmol/L after active treatment, a crossover difference of 0.36 μmol/L with a p-value of 0.006. The median intraindividual percentage change was 11.5 percent, significantly different from zero. When the drugs were analysed separately, the effect was clearer for dapagliflozin, where median TMAO climbed from 3.30 to 4.44 μmol/L, a 23.4 percent median increase, than for empagliflozin, which produced a numerically similar but statistically non-significant rise of 8.4 percent. The authors suggest the pattern points toward a possible class effect of SGLT-2 inhibitors, echoing the EMMY findings and extending them to a second drug and a different patient population.

Just as intriguing was what happened in the kidneys. Because TMAO is cleared almost exclusively by renal excretion, the researchers calculated its renal clearance from paired plasma and 24-hour urine measurements. Across all 123 patients, median renal clearance of TMAO fell from 95.8 mL/min on placebo to 83.9 mL/min on active drug, a statistically significant decline. In the dapagliflozin group the drop was significant on its own, falling from 109.2 to 97.8 mL/min, while the empagliflozin subgroup showed a non-significant downward trend. Yet the total amount of TMAO excreted in 24-hour urine barely changed, rising only non-significantly. That combination, lower clearance with unchanged total excretion, is a biochemical clue that the kidneys may not be the whole story.

The most obvious candidate explanation is the well-known initial dip in estimated glomerular filtration rate that occurs when SGLT-2 inhibitor therapy begins, a reversible haemodynamic effect of tubuloglomerular feedback. The study did observe a small but significant eGFR decline overall, and TMAO levels correlated negatively with eGFR at all time points, as expected from prior literature. But the eGFR dip was marginal, particularly in the dapagliflozin group, which showed the larger TMAO changes, and crucially, there was no correlation between individual patients’ eGFR changes and their TMAO changes. A reduced filtration rate alone, the authors conclude, cannot adequately explain the rise in plasma TMAO.

That leaves transporters and production. TMAO is eliminated primarily by glomerular filtration with a substantial contribution from transporter-mediated secretion in the proximal tubule, where the organic cation transporter 2 shuttles TMAO from blood into tubular cells and the multidrug and toxin extrusion protein 1 exports it into the urine. Animal work has shown that SGLT-2 inhibition broadly downregulates renal transport proteins, and drug interactions with OCT2 or MATE1 could theoretically reduce TMAO secretion. But if transporter-mediated excretion were truly impaired, the total urinary excretion of TMAO should have fallen, and it did not. The authors therefore favour a different hypothesis: SGLT-2 inhibitors may increase the production of TMAO, most plausibly through changes in the gut microbiome, even though some prior studies had suggested these drugs shift the microbiome in ways that should lower TMAO-producing bacteria. A subgroup analysis of the 20 participants who took antibiotics during the trials found no significant difference in TMAO changes, and the crossover design, in which each patient served as their own control, makes dietary shifts an unlikely confounder, although diets were not formally documented.

The clinical implications remain deliberately open. TMAO is widely characterised as a risk marker, and possibly a risk factor, for cardiovascular and kidney disease, so a drug-induced rise of roughly 10 to 20 percent would seem, on its face, unwelcome. Yet SGLT-2 inhibitors have delivered unmistakable benefits on cardiovascular outcomes and mortality in enormous outcome trials, in diabetic and non-diabetic patients alike, benefits that clearly operate through mechanisms beyond glucose control. Whether the TMAO increase is a harmless bystander, a counterbalancing signal, or something that modulates the drugs’ net benefit is unknown, and the authors call for studies examining outcomes in relation to baseline and on-treatment TMAO levels, as well as cohorts with much higher starting concentrations.

The study’s limitations temper any strong conclusions. Six weeks per treatment period is short, so long-term trajectories of TMAO, whether the rise plateaus, persists, or reverses, cannot be determined. No second baseline was established after the washout, so carryover effects may have blunted the measured effect in some patients. All participants had preserved kidney function, with eGFR above 60, leaving the question unanswered for the many diabetic patients with renal impairment, who typically have the highest TMAO levels. Compliance was assessed by counting returned tablets, and diet went unrecorded. Still, the pooled crossover data are internally consistent, and the message is clear enough to unsettle a comfortable assumption: a drug class celebrated for protecting the heart and kidneys may simultaneously raise circulating levels of one of the most talked-about microbiome-derived risk markers in cardiovascular medicine, and the reason why remains an open and compelling scientific question.

Subject of Research: Effects of SGLT-2 inhibitors on plasma TMAO concentrations and renal clearance in patients with type 2 diabetes

Article Title: Effects of SGLT‐2 Inhibitors on Plasma Concentrations and Renal Clearance of the Risk Marker Trimethylamine‐N‐Oxide in Patients With Type‐2 Diabetes

Article References: Effects of SGLT‐2 Inhibitors on Plasma Concentrations and Renal Clearance of the Risk Marker Trimethylamine‐N‐Oxide in Patients With Type‐2 Diabetes. (n.d.). https://doi.org/10.1002/edm2.70318

Image Credits: AI Generated

DOI: 10.1002/edm2.70318

Keywords: SGLT-2 inhibitors, TMAO, type 2 diabetes, empagliflozin, dapagliflozin, gut microbiome, renal clearance, cardiovascular risk, kidney function, eGFR, clinical trial, biomarkers