In this study, we demonstrated first that a LA-AAD exhibited a similar reno-protective effect as a LPD without the need of reducing total protein content. Indeed, the concomitant observation of kidney fibrosis, serum creatinine and urinary protein improvement provides support that reducing aromatic amino acids is efficient to metabolically mimic the action of a LPD. It is recognized from previous studies 7 that patients under a LPD are more prone to malnutrition. This experiment was not designed to explore the nutritional status in the different groups. Nevertheless, we failed to find any difference in weight in CKD groups, neither for the LPD group nor for the LA-AAD group.
In this study, we showed secondly that a specific restriction in the intake of aromatic amino acids lowered uremic toxin concentrations and mitigated inflammation that play major roles in the progression of renal damage. Surprisingly, in our CKD experimental model, LPD was able to decrease only to a limited extent the free uremic toxin concentrations, while total uremic toxin concentrations were not significantly different between each CKD diet groups. Black & al reported in a longitudinal study with 30 non-dialysis CKD patients (stage 3–4) a favorable effect of LPD to improve renal function and decrease uremic toxins concentrations after six months of nutritional intervention 6. However, they only found a significant effect on total PCS values, but neither on IS nor on IAA serum concentration. It is very likely that lower uremic toxin concentrations could have been reached using either very low protein diet (VLPD), or aromatic amino acid corresponding concentrations. Indeed, a recent study showed that VLPD was effective to beneficially modulate gut microbiota, improving intestinal permeability and reducing serum levels of total and free IS and PCS in CKD patients 20. We cannot exclude that in the present study, the composition of diet by itself could have altered intestinal microbiota and uremic toxins production. For example, the total amount of starch is higher in LDP and LA-AAD than in NPD. Starch has recently been described as a prebiotic that promotes proliferation of some gut bacteria such as Bifidobacteria and Lactobacilli, increases the production of metabolites including short-chain fatty acids, which confer a number of health-promoting benefits.21 In the present study, CKD mice exhibited a higher concentration of albuminemia without straightforward explanation. Only CKD mice fed with LA-AAD had a higher percentage of protein binding (%PB) of indoxyl sulfate (Data not shown). In the literature, albumin concentration (when in the normal range) did not influence the level of protein binding of uremic toxins, suggesting that other mechanism are involved such as post-translational modifications of plasma proteins (oxidation, carbamylation and glycosylation are for instance the most relevant processes)22. Also, we cannot eliminate that an increase of de novo albumin production which is probably less modified and binds better uremic toxins, could partially participate of the reduction of free fraction in CKD mice treated with LA-AAD. We can however not exclude that the reduction of free uremic toxins by LA-AAD could be, at least partially, a consequence of a structural changes in the proteins. Further studies are needed to explore this point.
Previous results, along with the present study, raise the issue that mechanisms, other than reduction in uremic toxin levels, could account for the positive effect of LPD. In an elegant experiment 23, Vaziri & al explored the impact of urea concentration on intestinal permeability in cultured CACO-2 cells. These results were consistent with the fact that the higher blood urea nitrogen, the higher the gut barrier was permeable, leading to endotoxemia, systemic inflammation and supposedly organ fibrosis. It is widely accepted that blood urea nitrogen is directly related to protein intake. Based on this assumption, we would have expected that the NPD but not LA-AAD group could improve renal function. However, with normoproteic intakes and blood urea nitrogen levels similar to the standard diet, the LA-AAD had comparable reno-protective effects as the LPD group, challenging this view.
Renal hemodynamics variation in response to protein feeding is a well-established process, and is nowadays being explored through the renal functional reserve concept 24. Several studies have shown that a high protein intake was associated with a higher intra-glomerular pressure, glomerular hyper-filtration and damage to glomerular structure 2,3. Hence, the benefit of a protein-restricted diet in patients with CKD, through this hemodynamic effect, is thought to promote preservation of kidney function. Once more, our results challenge this hypothesis, given that the LA-AAD group had a comparable protein intake to the standard diet group. Amino acids, either given through stomach tube or as an intravenous perfusion, have the same renal hemodynamic effects as an acute protein ingestion, leading to renal vasodilation and GFR rise 25–27. Different hypotheses have been discussed regarding the physiological mechanisms that may play a role in hemodynamic variations. First, amino acids could influence renal vasodilation through metabolic substrates that influence tubular sodium reabsorption or renal oxygen consumption. Secondly, humoral mechanisms have also been discussed, based on the notion that renal hemodynamic variations occurring during post-prandial state could be directly related to the release of a humoral mediator into the systemic circulation, acting on the kidney vasodilation and glomerular filtration rate. The third hypothesis regarding amino acid impact on renal hemodynamic variations focuses on intrinsic renal mechanisms, such as tubule-glomerular feedback and tubular transport 28. It may be possible that some specific amino acids, but not all, have significant effects on renal hemodynamics 25. In the renal proximal tubule, amino acids are co-transported with sodium, resulting in reabsorption of both through epithelial transporters. Basolateral transporters also play an important role to regulate intracellular concentration of different amino acids. Among others, the uniporter TAT1 (T-Type Amino Acid Transporter 1) is present in the small intestine and in renal proximal tubule epithelial cells. TAT1 acts as an aromatic amino acid efflux transporter and is important for the absorption of aromatic amino acids in the kidney and intestine 29,30. It could therefore be interesting to measure TAT1 expression in a future study, to assess whether or not these three specific diets could have modified TAT1 expression or function.
The process by which a LA-AAD may delay renal function decline remains unclear. Tryptophan is the precursor of indoxyl sulfate, but also of serotonin and kynurenin. It has been shown that plasma tryptophan levels are decreased during CKD, proportionately to the stage of disease 31. Accordingly, several tryptophan metabolites are also decreased in CKD patients, such as melatonin and 5-methoxytryptophan, related to a lower tryptophan hydroxylase-1 (TRP-1) expression, its main regulatory enzyme 32. Conversely, some tryptophan metabolites are increased in CKD patients, among others IS, kynurenines, kynurenic acid and quinolonic acid 31. Recent studies have involved tryptophan metabolites in the modulation of inflammation and fibrosis 33,34, and anti-fibrotic therapy targeting these tryptophan metabolic by-products are currently under investigation 35. Further experimental studies are however needed to explore whether tryptophan metabolism pathways are involved in the development of kidney fibrosis.
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase which participates in several key cell functions such as cellular protein synthesis and cell growth. The mTOR pathway has recently been studied in kidney pathology, as it is involved in renal hypertrophy and urinary excretion 36. In a 5/6 nephrectomized rat model, Ohkawa & al demonstrated that LPD and the use of mTOR inhibitor (rapamycin) had similar reno-protective effects, decreasing proteinuria and improving kidney histological damage 37. Chang & al demonstrated in a bone marrow-derived mesenchymal stem cell (BM-MSC) model that 5-methoxytryptophan (5-MTP), a recently discovered tryptophan metabolite, had the ability to inhibit pro-inflammatory mediators and protects BM-MSC against stress-induced senescence. Moreover, they showed that beneficial effects of 5-MTP was mediated through FoxO3a and mTOR pathway 38. Because mTOR activity is regulated by aminoacid intake, it would be interesting to explore the hypothesis that qualitative modulation of aminoacid intake could influence mTOR pathway. It could be interesting, as a second step, to explore mTOR pathway and tryptophan metabolite concentrations in mice fed a LA-AAD. Likewise, we did not analyze intestinal microbiota composition of our different groups. It might be interesting to explore the abundance and diversity of the gut microbiome, according to each specific diet.
An additional issue to consider is whether such a LA-AAD could be deleterious to the body. Indeed, tryptophan metabolism has been studied for decades by psychiatrists, as it is the precursor of serotonin, the so-called “happiness neurotransmitter”. Several animal and human experiments have been carried out to test the hypothesis of tryptophan supplementation beneficial effect on depressive symptoms, with thus far ambivalent results 39. However, the present LA-AAD was not completely deprived in tyrosine, tryptophan and phenylalanine, and intake levels were similar to the LPD. Tryptophan and phenylalanine are essential amino acids, meaning they cannot be synthesized de novo by the human organism, and thus they must be supplied in diet. Conversely, tyrosine is considered as a conditionally essential amino acid, which means its synthesis can be limited under certain pathological conditions. Minimal dietary amounts of each aminoacid have been well established and any attempt to define a LA-AAD in human should comply with these requirements. However, current protein intakes in Western countries do bring essential aminoacids far above the minimal requirements.
In conclusion the present study in CKD mice suggests that an experimental LA-AAD (i.e. low tyrosine, tryptophan and phenylalanine intake) has beneficial systemic effects, mimicking those of a LPD. Indeed, a LA-AAD appears efficient to delay renal function decline and prevent kidney fibrosis, without exposing to malnutrition. Significant differences between the two groups (LPD and LA-AAD) were noted such as energy intake, urea level or even inflammatory biomarkers (such as MCP-1) which could be deleterious in the long run and therefore require further studies. Future studies will be needed to determine if the specific restriction of foodstuffs containing large amounts of AAAs ( e.g.: animal meats (beef, fish), eggs, lentils,…) could improve kidney function in humans.