快樂小藥師 Im pharmacist nichts glücklich

INTRODUCTION

Patients with chronic kidney disease (CKD) are at increased risk for development of cardiovascular disease (CVD) beyond traditional risk factors. Yet the mechanism(s) through which CKD is linked to enhanced atherosclerotic heart disease is not fully elucidated. While a role for uremic toxins in the pathogenesis of cardiovascular and renal disease progression in CKD has been suggested for some time, the precise participants involved, and their mechanisms of action remain unclear. Recent studies have suggested involvement of gut microbiota in the generation of metabolites that display uremic toxicity¹. Further, perturbations of the composition of the gut microbial community in both human and experimental CKD are associated with significant elevations of gut-derived uremic toxins². Such alterations have also been associated with increased systemic inflammatory burden, and thus are suspected to potentially play a role in the pathogenesis of cardiovascular and renal disease progression in subjects with CKD. Bacterial structural components such as lipopolysaccharide³, and metabolites such as indoxyl sulfate, p-cresyl sulfate, amines, and ammonia, have been identified as potential microbial byproducts capable of initiating pro-inflammatory cytokine/chemokine cascades seen in the setting of CKD and end stage renal disease⁴. Thus, it has been hypothesized that gut-microbiota derived uremic toxins may serve as both therapeutic targets and/or assessment tools for renal diseases in this vulnerable population^1, 5–7. However, demonstration of a role for these uremic toxins in CVD pathogenesis has been indirect.

Our group recently identified a novel mechanistic link between gut microbiota metabolism of dietary trimethylamine-containing nutrients, and CVD pathogenesis^8–13 Specifically, gut microbiota-mediated metabolism of phosphatidylcholine, choline or L-carnitine each have been shown to ultimately produced trimethylamine-N-oxide (TMAO), and in multiple clinical studies, TMAO levels have been shown to be associated with cardiovascular risks^8–10, 13. Further, animal model studies have revealed TMAO is mechanistically linked to atherosclerosis development through multiple distinct pathways^8, 14. Animal model and human clinical studies demonstrate an obligatory role for gut microbiota in TMAO formation from TMA-containing nutrients including choline and phosphatidylcholine^9, 10, L-carnitine⁸, and more recently, γ-butyrobetaine¹⁵, and to a lesser degree, the choline oxidation metabolite betaine¹¹.

TMAO is cleared by the kidney, and prior studies have reported TMAO is elevated in subjects with impaired renal function^16–18. It is thus an attractive hypothesis that TMAO may represent an excellent mechanism-based marker of CVD risk in subjects with impaired renal function. In one large study of subjects with predominantly preserved renal function, we reported the prognostic value of TMAO for predicting three year risk for major adverse cardiovascular events (MACE = myocardial infarction, stroke or death) remained significant following adjustments for renal function ⁹. However, no studies to date have directly looked at the long-term mortality risk of TMAO among CKD subjects. Interestingly, plasma TMAO levels in apparently healthy donors was identified in a prospective multicenter study aimed at identifying donor factors associated with delayed graft function in renal transplant recipients¹⁹. A direct role for TMAO in impacting renal functional impairment has not been reported. Herein, we sought to further examine both the prognostic value of TMAO among CKD subjects and the potential contribution of dietary-induced, gut microbiota-associated TMAO formation in the development and progression of CKD.

METHODS

RESULTS

In the CKD cohort, higher TMAO levels (quartiles 4 versus 1) were associated with a 2.8-fold increase in risk for all-cause mortality at 5 years (unadjusted HR 2.76, 95%CI 1.74–4.37, p<0.001). After adjusting for traditional CVD risk factors, log-transformed hsCRP, and log-transformed eGFR, higher TMAO levels still were associated with a 1.9-fold poorer 5-year survival (adjusted HR 1.93, 95%CI 1.13–3.29, p<0.05; Table 2, Kaplan-Meier curve shown in Figure 1B). When stratified according to median levels (7.9 μM), higher TMAO conferred a 1.7-fold increase in risk for all-cause mortality (HR 1.70, 95%CI 1.25–2.30, p<0.001), and remained significant after adjusting for traditional risk factors and log-transformed hsCRP (adjusted HR 1.72, 95%CI 1.16–2.34, p<0.001), as well as with addition of cystatin C to the model (adjusted HR 1.45, 95%CI 1.05–2.02, p<0.05). Using median cohort cut-offs with low cystatin C (<1.4 mg/dL) and low TMAO (<7.9 μM) as reference, those with concomitant high cystatin C and high TMAO had a 3-fold increase in mortality risk (HR 3.01, 95%CI 1.97–4.59, p<0.001). These findings are consistent with the notion that elevated TMAO is associated with poor prognosis in patients with established CKD.

Increased TMAO levels in non-CKD patients with elevated Cystatin C

Within the non-CKD cohort (n=3,166), the prognostic value of elevated TMAO (quartile 4 vs 1) remained predictive of 5-year mortality risk (HR 2.21, 95%CI 1.57–3.12, p<0.001), as well as after adjusting for traditional risk factors, log-transformed hsCRP, and log-transformed eGFR (adjusted HR 1.47, 95%CI 1.02–2.12, p<0.05, Table 2B). These findings were similar when restricted to subjects with preserved eGFR (≥60 ml/min/1.73m²) plus normal cystatin C (<1.4mg/dL, n=3,151). Elevated TMAO levels are associated with higher 5-year mortality risk among subjects with either normal or elevated cystatin C levels (Figure 2). Using median cohort cut-offs with low cystatin C (<0.9 mg/dL) and low TMAO (<3.4μM) as reference, those with concomitant high cystatin C and high TMAO had a 3.7-fold increase in mortality risk (HR 3.67, 95%CI 2.57–5.23, p<0.001).

Dietary choline and dietary TMAO both promote renal fibrosis and dysfunction in animal models

To directly test the hypotheses that either dietary TMAO itself, or dietary nutrients that contribute to gut microbiota dependent production of TMAO, can impact development and progression of CKD, we performed animal model studies. Conventionally housed 8-week old male mice (C57BL/6J background) were fed ad libitum a chemically defined diet comparable to normal chow (0.08gm% choline), or the same diet supplemented with either choline (1.0% final) or TMAO (0.12%), as described under Methods. After 6 weeks we observed significant (p<0.01) increases in TMAO levels in both the TMAO-supplemented and choline-supplemented groups of mice (Figure 3A), with TMAO levels observed within the range of values detected among CKD subjects studied (97.5 percentile 77.6μM, 99 percentile 96.3μM). Importantly, elevated TMAO levels were associated with corresponding increases in tubulointerstitital fibrosis and collagen deposition (Figure 3B–C) and phosphorylation of Smad3, an important regulator of the pro-fibrotic TGF-β/Smad3 signaling pathway during fibrotic kidney disease²³ (Figure 3E). Furthermore, TMAO-fed and choline-fed mice experienced increased kidney injury marker-1 (KIM-1, Figure 4A). Extending the TMAO/choline feeding to 16 weeks was associated with increased serum cystatin C levels compared to chow-fed mice (Figure 4C). Upon further examination, striking dose-dependent relationships were noted between plasma TMAO levels and monitored indices of renal histopathological (Figure 3D, 3F) and functional impairment (Figure 4B).

DISCUSSION

There are several key findings in this report. First, we observed in subjects with CKD, that TMAO levels are not only elevated compared to non-CKD subjects, but importantly, portend poorer overall survival. Second, we observed that within the non-CKD cohort, higher levels of TMAO portend poorer survival both within the cohort of low levels as well as high levels of cystatin C (stratified at median levels). Interestingly, the prognostic value for the highest TMAO quartile in predicting future mortality risk in this cohort remained robust even after adjustment for traditional risk factors. Third, extending to animal models studies, dietary exposure of either choline or TMAO lead to the development of renal tubulointerstitial fibrosis and early measures of dysfunction (elevated cystatin C). These studies thus suggest both a causal relationship and clinical relevance of dietary choline-induced, gut microbiota-mediated, TMAO formation in CKD development and progression.

Trimethylamine N-oxide is a low molecular weight compound that is easily filtered by the kidney, and effectively removed by hemodialysis¹⁶. Considered a nitrogenous waste product whose levels rise with diminished renal function, elevated TMAO levels have been reported in small cohorts (n<20) of subjects with either end-stage renal disease, or CKD, where levels were shown to correlate with both serum urea and creatinine¹⁸. Detailed animal and human experiments on the renal clearance of methylamines such as TMA and TMAO have been performed, and confirming the kidneys as the primary elimination route²⁴. Interestingly, the urinary clearances of both TMA and TMAO are higher than the glomerular filtration rate, and TMAO clearance also decreases with increasing dose, which suggests that saturable renal tubular secretion occurs²⁵. The majority of a dose of TMA is also excreted in the urine, with varying proportions in the forms of TMA and TMAO being dependent on the dose level²⁶. Urinary TMAO levels are reported to rise with episodes of kidney graft dysfunction in renal transplant recipients, suggesting an intrinsic accumulation of TMAO (presumably as an osmolyte like urea) that is released during damage of the renal medullary cells^27–30. The mechanistic link between elevated TMAO and adverse prognosis in CKD and even in the setting of subclinical renal insufficiency (elevated cystatin C) in non-CKD patients observed in our study is therefore consistent with the heightened risk of developing CVD in the CKD population.

Our results from animal studies showed for the first time a direct mechanistic link between dietary choline, or dietary TMAO, and progressive renal dysfunction, even in the C57BL/6J mouse model that is known to be relatively resistant to renal injury³¹. Indeed, exposure to either a high choline diet or a diet supplemented directly with TMAO both led to increased levels of the early kidney injury marker KIM-1, and enhanced phosphorylation of Smad3, an important regulator of renal fibrosis³². A more prolonged exposure to either the high choline diet or the TMAO supplemented diet both led to increased plasma levels of cystatin C, a sensitive indicator of renal functional impairment. Importantly, the plasma TMAO levels observed within the animal models on either choline or TMAO supplemented diets were well within the range of TMAO levels observed among subjects with CKD. Further, examining TMAO levels as a continuous variable, a dose-dependent relationship between plasma TMAO levels and mortality risk was observed (log[TMAO]: adjusted HR=1.41, 95%CI 1.23–1.61, p<0.001 for CKD cohort; adjusted HR=1.21, 95%CI 1.07–1.36, p<0.01 for non-CKD cohort). While there is the appearance of a threshold level of TMAO that is associated with increased risk with the predicted outcomes using quartile-based analyses in the current sized study, the biological data collected in the animal model studies (scatter plots comparing TMAO levels versus the CKD-related phenotypes) shows a continuous dose dependent relationship between TMAO levels and renal tubulointerstitial fibrosis (pSMAD3, and various measures of collagen or fibrosis), and renal functional impairment (cystatin C). Interestingly, a recent untargeted metabolomic study from the Framingham Heart Study among subjects with normal renal function identified elevated choline and TMAO levels at baseline were each associated with an increased future risk of developing CKD ³³. Our animal model findings therefore provide a potential mechanistic rationale for the Framingham observational data, and collectively, further link elevated systemic TMAO levels with increased susceptibility for development of CKD.

The prospects that exposure to specific dietary nutrients like choline, phosphatidylcholine (lecithin) and L-carnitine via gut microbiota may impact susceptibility to the development and progression of both CKD and CVD has important potential public health implications. Randomized nutritional intervention studies in CKD patients to date have not explored a potential role for choline, phosphatidylcholine, L-carnitine or TMAO (which can be abundant in certain types of fish) in disease progression. Similarly, epidemiological studies are rather limited on the topic of diet and CKD risks, even though a recommended renal diet is typically low in protein intake. Dietary management of CKD patients represents a challenge, and much less is known about nutritional factors that might predispose to enhanced risk for development of CKD or its progression. Interestingly, in a sub-study (n=3,296) amongst women who had urine microalbumin levels available from the Nurses Health Study, two or more servings of red meat (primary dietary source of L-carnitine) per week were directly associated with enhanced risk for development of microalbuminuria (OR: 1.51; 95% CI: 1.01 to 2.26)³⁴. Further investigations in dietary predisposition to CKD development and progression are warranted. Based upon the present studies, a diet monitored by following TMAO levels and designed to limit TMAO precursors (low in red meat, meats, liver, egg yolk, and high fat dairy products) and TMAO itself (certain fish) would be an attractive diet to test to see if it limited rate of CKD progression. However, it is important to note that choline is an essential nutrient; therefore, its total elimination from the diet is ill advised, and could result in development of a deficiency state³⁵.

Collectively, the present data indicate a dietary-induced, intestinal microbiota-dependent mechanism may contribute to both progressive renal fibrosis and dysfunction, and mortality risks, among subjects with CKD. They also build upon the recent body of evidence demonstrating a mechanistic link between gut microbiota-associated metabolic dysregulation and cardiovascular risk in humans^8–12. The discovery of the metaorganismal pathway involved in TMAO generation thus affords a unique opportunity to systematically investigate the potential contributions of discrete participants in the overall diet → microbe → host enzyme pathways for TMAO formation and development and progression of cardio-renal dysfunction, thereby offering insights into modulation of such pathways. It is interesting that in both animal models and patients with established CKD, pre- and probiotic intervention studies have been performed, with reports of changes in gut microbiota composition and activity. For example, lactobacillus acidophilus or bifidobacterium have been reported to reduce inflammatory signaling associated with the microbiota-derived metabolites that accumulate in CKD^36–39, in addition to modestly improving renal function^40, 41. Similarly, prebiotic compound use to interrupt pathways that lead to gut microbiota derived uremic toxins such as indoxyl sulfate and p-cresyl sulfate has shown some efficacy in both human and animal trials of CKD^5, 42. Further studies are warranted to see whether dietary interventions, or disruption of the meta-organismal pathway involving TMAO production, may retard development of CKD, progression of renal functional impairment among subjects with CKD, and adverse CVD event risks in subjects with CKD.

Conclusion

The gut microbiota-dependent product, TMAO, is associated with both higher risk of progressive renal fibrosis and functional impairment, and poorer long-term survival.

Nonstandard Abbreviations and Acronyms

References