Gut microbiota in renal physiology: focus on short-chain fatty acids and their receptors

Abstract

A number of recent studies have begun to explore a new and exciting area: the interaction between the gut microbiome and renal physiology. In particular, multiple studies have focused on the role of microbially produced short chain fatty acids, which are generally thought to promote health. This review will focus on what is known to date regarding the influence of the microbiome on renal function, with emphasis on the cell biology, physiology, and clinical implications of short chain fatty acids and short chain fatty acid receptors. It is clear that microbe-host interactions are an exciting and ever-expanding field, which has implications for how we view diseases such as hypertension, acute kidney injury, and chronic kidney disease. However, it is important to recognize that although the potential promise of this area is extremely enticing, we are only the very edge of this new field.

In recent years it has become increasingly clear that the physiology of a number of organisms—from humans^1,2 and mice^3,4 to Drosophila⁵ and Caenorhabditis elegans⁶—can be influenced by the gut microbiota. In particular, metabolites of the gut microbiota have been shown to play a role in influencing “host” physiology. These metabolites include those thought to have both positive and negative effects on host health. For example, in patients with chronic kidney disease, the gut microbiome produces uremic toxins, including indoles,⁷ ammonia,^8,9 and trimethylamine N-oxide.¹⁰ On the other hand, other metabolites—notably, short chain fatty acids (SCFAs)—are generally thought to promote health.^11–14 This review will emphasize the role of SCFA receptors with regards to the cell biology, physiology, and clinical relevance of renal SCFA signaling.

SCFAs: where do they come from, and what is the evidence?

The gut microbiota, sometimes referred to as the “forgotten organ,” are a significant component of the physiology of the host. The total wet weight of gut microbes in humans is estimated to be 175 g to 1.5 kg,^15,16 and the gut microbial population is a dynamic one whose composition and activity change over time. A significant body of research points to the fact that the gut microbial composition changes in disease (dysbiosis).^3,9,16–34 In addition, it is clear that the gut microbiota naturally undergoes dynamic changes during different stages of life^35,36 and in response to changes in habits of the host.^37,38 For example, dietary changes in the host lead to significant alterations in gut microbial composition^37,38 and subsequently to changes in microbial metabolite production.³⁹ One type of microbial metabolite, which we will focus on in this review, is SCFAs. SCFAs most commonly refers to the straight-chain 2-4 carbon variety (acetate, propionate, and butyrate). SCFAs are produced from dietary fiber: because most dietary fiber cannot be degraded by host enzymes in the upper gastrointestinal tract, this fiber instead travels intact to the colon and cecum, where it is broken down by the gut microbiota. As a byproduct of this fermentation process, gut microbes produce a number of metabolites, with SCFAs being a major product. Gut microbial production is so robust that the concentration of SCFAs in the colon lumen itself is ~100 mmol/l.¹⁵

SCFAs produced by the gut microbiota enter the circulation of the host via monocarboxylate transporters⁴⁰ as well as by diffusion. Although it is commonly stated that SCFAs found in the bloodstream of the host are produced by the gut microbiota, it is worth carefully considering the potential contribution of the gut microbiota toward plasma SCFA levels. Analysis of SCFAs in conventionally raised (with gut microbiota) versus germ-free animals (no gut microbiota) revealed that SCFA concentrations in the cecum are enhanced by over 100-fold by gut microbiota.⁴¹ Furthermore, a recent study reported that the most abundant microbial SCFA, acetate, is virtually undetectable in the plasma of germ-free mice.⁴² Additionally, it has been shown that serum SCFA levels in the host (conventional mice) correlate with the amount of fiber in the diet, again implicating gut microbiota as playing an important role in determining serum SCFA levels.⁴³ It has been demonstrated that suppressing microbial SCFA production using a low-fiber diet, or increasing microbial SCFA production using a high-fiber diet, caused drastic changes in not only cecal SCFAs, but also in serum SCFAs. For example, on a low-fiber diet serum SCFAs decreased by ~75% (from ~1.3 mmol/l to approximately 300 mmol/l), whereas on a high-fiber diet serum SCFAs levels are roughly doubled. Therefore, gut microbial production is responsible for setting circulating SCFA levels. In plasma, SCFA concentrations are between 0.1 mmol/l and 10 mmol/l.^22,24,43,44 Although acetate is generally reported to be the most abundant SCFA produced, the precise ratios of acetate:butyrate:propionate reported vary. For example, acetate:butyrate:propionate ratios have been reported to be 55:35:10⁴³ or 55:23:22,⁴⁵ but it is clear from the literature that these ratios change quite dramatically with dietary manipulation (i.e., 40:26:34, 43:20:37, 73:5:22, 78:17:5, etc.).^39,46

Cell biology of SCFA signaling: how does it work?

Once absorbed into the bloodstream of the host, there are multiple ways in which SCFAs can have cellular effects. Here, we will discuss these in 2 major categories: G-protein coupled receptor (GPCR)–mediated (Figure 1) and non-GPCR–mediated effects. For the purpose of this review, we will pay particular attention to what is known of these mechanisms in the kidney.

Figure 1. One way in which the gut microbiota and the host communicate is via host receptors that recognize microbial metabolites.

Three receptors for short chain fatty acids have been reported in the kidney, all of which are 7-transmembrane G-protein–coupled receptors. Gpr41 and Gpr43 respond to formate, acetate, and butyrate (as well as a few other fatty acids), whereas Olfr78 rather strictly responds to acetate and propionate.

GPCR-mediated mechanisms

To date, 4 SCFA receptors have been described: Gpr41, Gpr43, Gpr109a, and Olfr78. Here, we will first introduce what is known about the ligands and activities of each receptor, and then will discuss what (if anything) has been reported about their expression in the kidney.

Gpr41/Gpr43

Gpr41 (free fatty acid receptor 3, Ffar3) and Gpr43 (free fatty acid receptor 2, Ffar2) are the most well studied of the SCFA GPCRs and were initially described as SCFA receptors in 2003 by 2 separate groups.^24,47 Both studies found that Gpr41 and Gpr43 are most responsive to propionate, although both receptors are activated by a number of other SCFA ligands. Among these, the strongest are acetate, butyrate, and isobutyrate, although they both also respond more weakly to other compounds with similar chemical structures.^24,47 Although the exact EC₅₀ values vary with both the assay and the species tested, Gpr41 and Gpr43 generally are reported to respond to acetate, propionate, and butyrate in the micromolar range, with propionate being the strongest ligand (Tables 1 and 2). In addition, beta-hydroxybutyrate has been reported, by different groups, to be either an agonist⁴⁸ or an antagonist⁴⁹ for Gpr41. Gpr41 couples to G_i, whereas Gpr43 can couple to both G_i and G_q.^24,47 It is noteworthy that the human and rat orthologs of Gpr41 respond similarly to SCFAs,⁴⁷ indicating that Gpr41 signaling is evolutionarily conserved.

Table 1.

Reported Gpr41 EC₅₀ values

Receptor	hGPR41	rGPR41	rGPR41	hGPR41	hGPR41
Method	GTPƴS binding	GTPƴS binding	Yeast	cAMP	Ca2+
Formate	7760 mmol/l	1000 mmol/l	> 10,000 mmol/l	No response	No response
Acetate	1020 mmol/l	393 mmol/l	422 mmol/l	1023 mmol/la	1072 mmol/la
Propionate	127 mmol/l	41 mmol/l	8 mmol/l	6 mmol/la	20 mmol/la
Butyrate	158 mmol/l	33 mmol/l	64 mmol/l	42 mmol/la	58 mmol/la
Citation (yr)	Brown et al.47 (2003)	Brown et al.47 (2003)	Brown et al.47 (2003)	Le Poul et al.24 (2003)	Le Poul et al.24 (2003)

Standard errors omitted for simplicity; additional (weaker) ligands omitted for simplicity.

^aLe Poul et al.²⁴ reported pEC50 values; values here were converted to EC50.

Table 2.

Reported Gpr43 EC₅₀ values

Receptor	hGPR43	hGPR43	hGPR43	hGPR43
Method	FLIPR	GTPƴS binding	cAMP	Ca2+
Formate	> 1300 mmol/l	5640 mmol/l	2455 mmol/la	10233 mmol/la
Acetate	52 mmol/l	431 mmol/l	35 mmol/la	102 mmol/la
Propionate	31 mmol/l	290 mmol/l	14 mmol/la	79 mmol/la
Butyrate	100 mmol/l	371 mmol/l	28 mmol/la	339 mmol/la
Citation (yr)	Brown et al.47 (2003)	Brown et al.47 (2003)	Le Poul et al.24 (2003)	Le Poul et al.24 (2003)

Standard errors omitted for simplicity; additional (weaker) ligands omitted for simplicity.

^aLe Poul et al.²⁴ reported pEC50 values; values here were converted to EC50.

Subsequent work has outlined a number of roles for Gpr41 and Gpr43 in physiology—perhaps most prominently, Gpr41 has a role in metabolism,⁵⁰ whereas Gpr43 plays a role in immune responses.^22,24 Within the kidney, Gpr41 and Gpr43 have both been reported to be expressed in whole kidney and in the renal artery by reverse transcriptase-polymerase chain reaction.⁵¹

OR51E2/Olfr78

OR51E2 was initially deorphanized in 2009 by 2 groups: one reported that OR51E2 responded to propionate,⁵² whereas the other reported that OR51E2 was a receptor for β-ionone as well as several androgens.⁵³ A subsequent study by a separate group in 2013⁵¹ confirmed that OR51E2 (and its murine ortholog, Olfr78) responded to propionate as well as acetate, but did not detect any response of Olfr78 or OR51E2 to β-ionone. In 2015, it was reported⁵⁴ that Olfr78 responded to acetate, propionate, and lactate (OR51E2 was not examined in this study).

Although OR51E2/Olfr78 have been reported to respond to other compounds (as described above), for the purpose of this review we will focus on the role of OR51E2/Olfr78 as an SCFA receptor. It is intriguing to note that there are very few examples of “functional” murine-human orthologs for ORs, that is, orthologs that respond to the same ligand. Unfortunately, the large number of similar OR genes in both mice (1000)⁵⁵ and humans (300)⁵⁶ makes it difficult to bioinformatically assign ortholog pairs with certainty. However, Olfr78 rather uniquely has a clear human ortholog (OR51E2) based on sequence similarity, and we know that these 2 receptors both recognize SCFAs. In fact, this OR is extremely well conserved among mammalian species and has a clear ortholog in mice, rats, rabbits, elephants, horses, and 5 species of primates,⁵⁷ further implying that the role of this receptor is evolutionarily important and conserved. It should be noted that in contrast to Gpr41/43, OR51E2/Olfr78 responds to acetate and propionate but not to butyrate and has a much higher EC₅₀ than does Gpr41/43. The reported ligands of Olfr78 and OR51E2 are summarized in Table 3.

Table 3.

Reported Olfr78/hOR51E2 EC₅₀ values

Receptor	hOR51E2	hOR51E2	Olfr78 (mouse)	hOR51E2	Olfr78 (mouse)
Method	Ca2+	cAMP/luciferase	cAMP/luciferase	cAMP/luciferase	cAMP/luciferase
Formate			No response
Acetate			2.35 mmol/l	2.93 mmol/l	2.01 mmol/l
Propionate		~0.1 mmol/lb	0.92 mmol/l	2.16 mmol/l	0.63 mmol/l
Butyrate			No response
β-iononea	~2–3 mmol/la,b		No response	No response
Lactate					4.04
Citation (yr)	Neuhaus et al.53 (2009)	Saito et al.52 (2009)	Pluznick et al.51 (2013)	Pluznick et al.51 (2013)	Chang et al.54 (2015)

Standard errors omitted for simplicity; boxes left blank indicate that the ligand was not tested.

^aNeuhaus et al.⁵³ also reported that OR51E2 responded to several androgens.

^bEC50 not reported (value estimated based on published data).

Within the kidney, Olfr78 has been localized by use of an lacZ reporter construct, which found that Olfr78 is expressed in the renal afferent arteriole as well as in a subset of large renal vessels. OR51E2 has not been localized in kidney on the protein level; however, RNA for OR51E2 is found in tissues that are consistent with the LacZ localization of Olfr78 (kidney as well as heart, skeletal muscle, etc.).⁵⁸

Gpr109a

Gpr109a has also been reported as an SCFA receptor that responds to butyrate but not to acetate or propionate.⁵⁹ The reported EC₅₀ for butyrate is ~1 mmol/l. Gpr109a is arguably better known as a receptor for niacin and additionally has been reported to be a receptor for beta-D-hydroxybutyrate.^59–61 Gpr109a was reported to be expressed (at a relatively low level) in the kidney by an reverse transcriptase-polymerase chain reaction Taqman array screen.⁶²

SCFA GPCRs in the kidney

As for expression of these 4 GPCRs in the kidney, to date Gpr41, Gpr43, and Olfr78 have all been reported to be expressed in the kidney by reverse transcriptase-polymerase chain reaction.⁵¹ Gpr41 and Gpr43 were localized to major blood vessels (including the renal artery), but otherwise the specific localization of these receptors within the kidney has not been reported. Olfr78 was localized in the kidney with an lacZ reporter mouse (in which the native promoter of Olfr78 is used to drive expression of an lacZ reporter gene) and was found to localize to the renal afferent arteriole as well as to large renal arteries. Outside the kidney, Olfr78 has been found in vascular smooth muscle cells in a variety of vascular resistance beds.

Non-GPCR mechanisms

Although this review is focused on GPCR-mediated SCFA signaling, it would be remiss to not briefly discuss other aspects of cell biology related to SCFAs. For example, SCFAs can be transported across membranes by transporters: in the colon, SCFAs are transported across the apical membrane by SLC5A8, a sodium-coupled monocarboxylate transporter^40,63–65; however, there is evidence that other transporters may be involved as well.⁶⁶ Potential candidates include SCFA-permeant ion channels,⁶⁷ organic anion transporters (i.e., mOat2, which has been found in the kidney⁶⁸), or monocarboxylate transporter 1, which transports lactate and butyrate.⁶⁹ In future work it will be important to understand how SCFA transporters contribute to the renal effects of SCFAs.

In addition to interactions with receptors and transporters, SCFAs can affect cell biology through additional pathways, including effects on cell proliferation, apoptosis, and histone acetylation. Propionate and butyrate both are known to inhibit cell proliferation^14,70,71 and to induce apoptosis.^72–74 Butyrate, in particular,⁷⁵ is a strong inhibitor of histone deacetylases, and thus by altering histone acetylation, SCFAs can alter gene expression.^76–80 SCFA-mediated inhibition of HDACs is thought to be independent of GPCRs,⁸¹ although it has been suggested that Gpr41 may play a role.⁸²

Physiology of SCFA signaling: what does it do?

Here, we will discuss reported links between the microbiota and microbial SCFAs, and 3 different aspects of renal pathophysiology: blood pressure regulation, acute kidney injury, and chronic kidney disease (CKD). This section will attempt to summarize the chief findings to date for each of these areas.

Blood pressure regulation

Work on the microbiota and blood pressure regulation was encouraged by the realization that Olfr78 is expressed in cell types associated with blood pressure regulation.⁵¹ Olfr78 was localized to 2 specific subsets of renal blood vessels: (i) very large renal blood vessels (i.e., the renal artery and major branches of the renal artery) as well as (ii) the renal afferent arteriole. Intriguingly, Olfr78 was not found in intermediately sized vessels. Outside the kidney, Olfr78 was found in peripheral vascular beds in a variety of tissues (including muscle, skin, etc.). By polymerase chain reaction, Gpr41 and Gpr43 were shown to be expressed in the renal artery and in other large vessels (aorta, iliac artery). Subsequent experiments revealed that SCFAs act through both Olfr78 and Gpr41 to modulate blood pressure regulation, using 2 different pathways. First, SCFAs act via Olfr78 in the renal afferent arteriole to increase renin release, thereby contributing to basal renin levels (Olfr78 null mice have lowered plasma renin) and baseline blood pressure (Olfr78 null mice are hypotensive). However, SCFAs also act in the peripheral vasculature to acutely alter blood pressure. SCFAs act via Gpr41 to lower blood pressure (presumably via vasodilation, as SCFAs had been previously shown to dilate vessels ex vivo^83–86), whereas Olfr78 counters this response. Although it seems counterintuitive to have 2 different GPCRs responding to the same ligand but modulating blood pressure in opposing ways (that is, to have Gpr41 promoting hypotension and Olfr78 promoting hypertension), it has been suggested^51,87 that the “logic” behind this system is related to the very different EC₅₀s of these 2 receptors (Table 1 vs. Table 3). Gpr41 is much more likely to be active at lower, even basal levels of SCFAs, and thus would primarily drive the vasodilatory response seen with SCFAs. However, as SCFAs rise appreciably, Olfr78 (which has a much higher EC₅₀) would eventually be activated, thus serving as a brake on this pathway to prevent an inappropriate level of hypotension. In subsequent basic science studies, hypertension has been associated with changes in the microbiota^88–90 as well as in SCFAs.^88–90 However, it is clear that we still have much to learn in this area. Although one study⁹⁰ reported that rats that had altered microbiota and increased blood pressure also had an increase in plasma acetate, other studies reported that butyrate-producing bacteria⁸⁸ or both acetate and butyrate-producing bacteria⁸⁹ tended to decrease in a hypertension model. In the future, it will be important to show that changes in SCFA-producing bacteria truly translate to changes in plasma levels of SCFAs, and to what degree.

It is also intriguing to note that there are hints in the clinical literature that there may indeed be a connection between blood pressure regulation and microbial SCFAs. For example, it has been reported that high-fiber diets^91,92 and probiotic use⁹³ (both of which should increase microbial SCFA production) are associated with lowered blood pressure. In fact, dietary fiber not only increases SCFA production, but can dramatically alter the acetate:butyrate:propionate ratio.³⁹ In one study, which fed rats a diet with a daily fiber intake of 0, 1, 2, or 4 g/d, the acetate:butyrate:propionate ratio changed from 65:12:23 (0 g) to 40:26:34 (4 g)⁴⁶ when dietary fiber was increased. Given the fact that Gpr41 (which lowers blood pressure) and Olfr78 (which increases blood pressure) have different affinities for the different SCFAs (i.e., Gpr41 but not Olfr78 responds to butyrate), one can imagine scenarios in which the particular choice of dietary fiber may help to further promote the hypotensive benefits of SCFAs. In addition, a study of > 4000 individuals reported that lower urinary formate (a microbial SCFA) correlates with increased blood pressure.⁹⁴ Thus, although the majority of the clinical data to date are correlative, these data nevertheless underscore the need for further study in this new area. Indeed, there is an intriguing connection between the microbiota and blood pressure regulation, which likely involves SCFAs at least in part. However, it is clear that more studies must be done to truly understand this connection, let alone to investigate if and how we can manipulate it for potential benefit.

Acute kidney injury

Another area of renal (patho)physiology in which microbial SCFAs have been implicated is acute kidney injury.³⁰ In a mouse model of renal ischemia-reperfusion injury, it was found that treatment with acetate, propionate, or butyrate was able to reduce kidney injury (measured by plasma creatinine) after ischemia-reperfusion injury.¹² Intriguingly, a similar effect was also seen by treating mice with acetate-producing bacteria. The authors discuss potential roles for either SCFA GPCRs or for HDAC in ameliorating ischemia-reperfusion injury, although they state that this most likely occurs through “modulation of epigenetic processes.” Considering the difficulty of these studies, these are exciting and impressive findings. However, going forward it will be critical to reproduce these findings while (i) using, as a control, a closely related microbiota that does not produce high amounts of SCFAs, (ii) measuring the increase in plasma SCFAs as well as fecal SCFAs, and (iii) examining the underlying mechanism. In addition, it will be important to optimize conditions for colonization of the “new” microbiota. Therefore, although there is still much work to be done, this is an exciting new area with obvious clinical implications. It is also interesting to note that in ischemia-reperfusion models of other tissues, SCFAs¹¹ or SCFA-producing bacteria⁹⁵ have also been shown to be protective, implying that the underlying protective mechanism may be common across tissues.

Chronic kidney disease

Finally, microbiota have also been implicated in studies of chronic kidney disease.^{2,7,10,30,96–104} Multiple studies (reviewed in Ramezani et al.⁷ and Ramezani and Raj¹⁰⁵) have reported a correlation between dysbiosis and CKD; that is, patients with CKD have altered microbiota as compared with control groups.¹⁰³ Several known uremic toxins are produced by the microbiota (e.g., p-cresol sulfate, indoxyl sulfate, ammonia, trimethylamine N-oxide^{8,9,105–108}), and most studies of the microbiota and CKD have focused on these toxins, with only a few references to SCFAs.¹⁰⁷ Because this review is focused on SCFAs we will not cover the general literature on uremic toxins in the depth that it deserves; for a comprehensive review, please refer to the article by Ramezani and Raj.¹⁰⁵ Regarding SCFAs, it has been shown that patients with CKD have an expansion of the microbiota that produce uremic toxins, and a contraction of microbiota that produce SCFAs.¹⁰⁷ Of note, recent studies have highlighted the benefits of a high-fiber diet (which can act as a prebiotic for SCFAs) in CKD.^98,109 In 2015, a metaanalysis of controlled feeding trials (143 participants in total) found that increased dietary fiber intake tended to reduce serum urea and creatinine.¹¹⁰ This is particularly noteworthy in light of the fact that CKD patients are often instructed not to consume a high-fiber diet (because of concerns regarding plasma potassium and phosphorus).

Clinical implications of SCFA signaling: what can we do?

Clearly, this entire field is still in its infancy, and many more studies need to be done to better understand these pathways and their potential implications. However, it is tempting to speculate that there may be opportunities in the future to take advantage of microbial signaling pathways for clinical benefit, on several possible fronts. First, understanding how and why dysbiosis correlates with disease may give us novel and unexpected insights into pathophysiology. Second, understanding whether dysbiosis may contribute to or perpetuate disease may give us new opportunities to intervene for clinical benefit. Clearly, the host genome is a major risk determinant for a number of different diseases (i.e., hypertension); however, unlike the host genome, the microbial genome present in a host can be modified. For example, by using prebiotics (foods that stimulate the growth or activity of certain microbes), probiotics (live bacteria), synbiotics (combinations of pre- and probiotics), or antibiotics, one can modify the content of the gut microbiota itself, and thus change the metabolites produced. But, there are a dizzying number of microbes to consider: which are the most crucial to “correct”? (Here, we must be careful to consider not only changes in genus, which are often what is reported because of sequencing limitations, but changes in species.) If we do correct them, would this necessarily result in a sustained improvement of an established phenotype? Clearly, much more needs to be done in this area, but there are encouraging hints in the literature; for example, an intriguing case report¹¹¹ outlined a case in which antibiotics appeared to alleviate hypertension. Finally, regardless of whether we are able to develop clinical therapeutics, it is crucial that we better understand how these pathways already may be influencing human health: many patients (both with and without disease) are exposed to antibiotics, prebiotics (i.e., fiber), and probiotics (i.e., yogurt) on a regular basis. Are these exposures (re)shaping their microbiome, and/or altering their risk for disease? It is critical that more research be done, both in animal models as well as in human populations in order to understand the impact of these prevalent modifiers of microbiota on health, and whether and how they might be manipulated for health benefit.

In summary, the role of the gut microbiome in renal physiology and pathophysiology is an exciting new area that has exploded in recent years (53 of the citations for this review were published in 2013–2016). Although it is clear that changes in gut microbiota correlate with disease, we do not yet know whether we can purposefully manipulate microbiota in order to alter the course of disease (or whether they, or we, are simply along for the ride). In coming years, careful studies will be needed to explore and uncover the exciting potential in this new area.

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