Title: Targeting the Gut for Early Diagnosis, Prevention, and Cure of Diabetic Kidney Disease: Is the Phenyl Sulfate Story Another Step Forward?
Abstract: Commentary on Kikuchi K, Saigusa D, Kanemitsu Y, et al. Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease. Nat Commun. 2019;10(1):1835. Commentary on Kikuchi K, Saigusa D, Kanemitsu Y, et al. Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease. Nat Commun. 2019;10(1):1835. Diabetic kidney disease (DKD) is the leading microvascular complication of both type 1 and type 2 diabetes mellitus (DM) and a major cause of end-stage kidney disease.1Umanath K. Lewis J.B. Update on diabetic nephropathy: core curriculum 2018.Am J Kidney Dis. 2018; 71: 884-895Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar Recent progress in DM pharmacologic treatment, such as the introduction of new antihyperglycemic agents like SGLT2 inhibitors,1Umanath K. Lewis J.B. Update on diabetic nephropathy: core curriculum 2018.Am J Kidney Dis. 2018; 71: 884-895Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 2Alicic R.Z. Johnson E.J. Tuttle K.R. SGLT2 inhibition for the prevention and treatment of diabetic kidney disease: a review.Am J Kidney Dis. 2018; 72: 267-277Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar has led to important advances in the effort to retard the progression of chronic kidney disease (CKD),3Perkovic V. Jardine M.J. Neal B. et al.Canagliflozin and renal outcomes in type 2 diabetes and nephropathy.N Engl J Med. 2019; 380: 2295-2306Crossref PubMed Scopus (2619) Google Scholar giving new impetus to the research in the field. However, the current gold standard for diagnosis (early or late) and follow-up of microvascular kidney injury in DKD is still urinary albumin excretion.4American Diabetes AssociationMicrovascular complications and foot care: standards of medical care in diabetes-2018.Diabetes Care. 2018; 41: S105-S118Crossref PubMed Scopus (234) Google Scholar Unfortunately, urinary albumin excretion does not fully meet the requirements for an early marker of kidney injury in DKD because it fails to identify patients with DKD with normal albuminuria levels, may be transient, does not necessarily reflect permanent damage, appears after histologic damage is already established,4American Diabetes AssociationMicrovascular complications and foot care: standards of medical care in diabetes-2018.Diabetes Care. 2018; 41: S105-S118Crossref PubMed Scopus (234) Google Scholar and is not always predictive of the risk for future kidney damage and DKD progression.5Papadopoulou-Marketou N. Kanaka-Gantenbein C. Marketos N. Chrousos G.P. Papassotiriou I. Biomarkers of diabetic nephropathy: a 2017 update.Crit Rev Clin Lab Sci. 2017; 54: 326-342Crossref PubMed Scopus (64) Google Scholar Thus, a strong unfulfilled need persists for early markers that are able to identify both the very initial phases of kidney damage in DKD and the patients at highest risk for developing progressive DKD. A recent study by Kikuchi et al6Kikuchi K. Saigusa D. Kanemitsu Y. et al.Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease.Nat Commun. 2019; 10: 1835Crossref PubMed Scopus (111) Google Scholar could represent a relevant advance because it aimed to investigate 4 key aspects of phenyl sulfate (PS) in the context of DKD: as a marker for early diagnosis, as a pathogenic factor for kidney damage and albuminuria, as a predictor of progression risk, and as a therapeutic target to reduce albuminuria. To this purpose, the authors performed a series of elegant and conceptually well-founded analyses in different experimental models and clinical settings (transgenic rats, diabetic mice, and diabetic patients) that together suggest a major role for PS as both a marker and a mediator of kidney injury in DKD. PS is produced by the metabolism of the amino acid l-tyrosine by intestinal bacteria and is considered a gut microbiota–derived uremic toxin.7Sabatino A. Regolisti G. Brusasco I. Cabassi A. Morabito S. Fiaccadori E. Alterations of intestinal barrier and microbiota in chronic kidney disease.Nephrol Dial Transplant. 2015; 30: 924-933Crossref PubMed Scopus (128) Google Scholar Initially, a precursor of PS, namely phenol, is synthesized in the gut from dietary l-tyrosine by microbes expressing the enzyme tyrosine phenol-lyase (TPL; Fig 1). After absorption, phenol is metabolized into PS by the liver, with PS subsequently secreted by the proximal tubular cells through the action of SLCO4C1, the only organic acid transporter polypeptide in human kidney.8Mikkaichi T. Suzuki T. Onogawa T. et al.Isolation and characterization of a digoxin transporter and its rat homologue expressed in the kidney.Proc Natl Acad Sci U S A. 2004; 101: 3569-3574Crossref PubMed Scopus (254) Google Scholar The key role of the gut microbiota–derived uremic toxins in the pathophysiology of CKD, DKD, and their kidney and cardiovascular complications has been the subject of in-depth studies over the past few years.7Sabatino A. Regolisti G. Brusasco I. Cabassi A. Morabito S. Fiaccadori E. Alterations of intestinal barrier and microbiota in chronic kidney disease.Nephrol Dial Transplant. 2015; 30: 924-933Crossref PubMed Scopus (128) Google Scholar, 9Niewczas M.A. Sirich T.L. Mathew A.V. et al.Uremic solutes and risk of end-stage renal disease in type 2 diabetes: metabolomic study.Kidney Int. 2014; 85: 1214-1224Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Cosola C. Rocchetti M.T. Sabatino A. Fiaccadori E. Di Iorio B.R. Gesualdo L. Microbiota issue in CKD: how promising are gut-targeted approaches?.J Nephrol. 2019; 32: 27-37Crossref PubMed Scopus (36) Google Scholar The most well studied among these uremic solutes are indoxyl sulfate (IS), p-cresyl sulfate (PCS), and trimethylamine N-oxide (TMAO), all produced from proteolytic fermentation in the context of deranged gut microbiota typical of patients with CKD ("intestinal dysbiosis").7Sabatino A. Regolisti G. Brusasco I. Cabassi A. Morabito S. Fiaccadori E. Alterations of intestinal barrier and microbiota in chronic kidney disease.Nephrol Dial Transplant. 2015; 30: 924-933Crossref PubMed Scopus (128) Google Scholar IS and PCS levels have been found to be elevated in patients with type 2 DM who progressed to end-stage kidney disease.9Niewczas M.A. Sirich T.L. Mathew A.V. et al.Uremic solutes and risk of end-stage renal disease in type 2 diabetes: metabolomic study.Kidney Int. 2014; 85: 1214-1224Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11Sabatino A. Regolisti G. Cosola C. Gesualdo L. Fiaccadori E. Intestinal microbiota in type 2 diabetes and chronic kidney disease.Curr Diab Rep. 2017; 17: 16-25Crossref PubMed Scopus (100) Google Scholar These uremic solutes may inhibit endothelial repair after injury and exert direct vascular damage, thus worsening the risk for cardiovascular complications7Sabatino A. Regolisti G. Brusasco I. Cabassi A. Morabito S. Fiaccadori E. Alterations of intestinal barrier and microbiota in chronic kidney disease.Nephrol Dial Transplant. 2015; 30: 924-933Crossref PubMed Scopus (128) Google Scholar (Fig 1). The study by Kikuchi et al clearly showed that in experimental models of DM, plasma PS levels are increased and may directly induce podocyte damage and albuminuria. In addition, they explored the role of PS as an early diagnostic marker and a prognostic indicator by showing that PS levels were statistically significantly correlated (albeit weakly) with both basal albuminuria and 2-year progression of DKD in a cohort of diabetic patients with moderately increased albuminuria. Finally, the authors were able to demonstrate that pharmacologic inhibition of TPL, which lowers the synthesis of the PS precursor phenol, may reduce albuminuria in diabetic mice. Several models were used to identify the compound, study its effects on albuminuria and DKD, and test whether it could become a therapeutic target. Initially, Kikuchi et al administered streptozotocin to induce DM in wild-type (WT) rats and those engineered to express the human SLCO4C1 transporter. Despite no difference in kidney function, WT rats had higher proteinuria than their transgenic counterparts. By the application of untargeted metabolomics, an approach allowing the detection of novel and potentially relevant plasma metabolites,10Cosola C. Rocchetti M.T. Sabatino A. Fiaccadori E. Di Iorio B.R. Gesualdo L. Microbiota issue in CKD: how promising are gut-targeted approaches?.J Nephrol. 2019; 32: 27-37Crossref PubMed Scopus (36) Google Scholar PS was identified as a specific substrate of the SLCO4C1 receptor. Of note, SLCO4C, which is the proximal tubular cell transporter responsible for the excretion of many gut-derived uremic toxins, including PS, is an organic acid transporter polypeptide not normally expressed in rodents.12Toyohara T. Suzuki T. Morimoto R. et al.SLCO4C1 transporter eliminates uremic toxins and attenuates hypertension and renal inflammation.J Am Soc Nephrol. 2009; 20: 2546-2555Crossref PubMed Scopus (106) Google Scholar Its overexpression has been related to increased excretion of uremic solutes in the setting of kidney failure, in parallel with blunting hypertension and inflammation.12Toyohara T. Suzuki T. Morimoto R. et al.SLCO4C1 transporter eliminates uremic toxins and attenuates hypertension and renal inflammation.J Am Soc Nephrol. 2009; 20: 2546-2555Crossref PubMed Scopus (106) Google Scholar In the diabetic rats studied by Kikuchi et al, PS levels were reduced in the blood of transgenic animals while remaining high in WT rats. Thus, the authors were able to mitigate some of the difficulties extrapolating results from rodents to humans by developing a useful model to evaluate the human kidney–specific elimination of metabolites and uremic toxins in DKD. Concerning the role of PS in kidney damage, it is well known that DKD involves a cellular injury, specifically targeting podocyte integrity and function.13Bose M. Almas S. Prabhakar S. Wnt signaling and podocyte dysfunction in diabetic nephropathy.J Investig Med. 2017; 65: 1093-1101Crossref PubMed Scopus (58) Google Scholar Using a different animal model in which db/db mice were treated with PS, Kikushi et al performed histologic examinations and found an association between PS levels and podocyte hypertrophy, foot-process effacement, and glomerular basement membrane thickening, confirming previous findings.13Bose M. Almas S. Prabhakar S. Wnt signaling and podocyte dysfunction in diabetic nephropathy.J Investig Med. 2017; 65: 1093-1101Crossref PubMed Scopus (58) Google Scholar They also observed associations with inflammatory activation and fibrosis. In vitro experiments confirmed the toxicity of PS in differentiated human podocytes at concentrations down to 30 μmol/L; this toxicity was characterized by a reduction in glutathione levels that rendered cells vulnerable to oxidative stress and mitochondrial dysfunction. The potential clinical relevance of the study to patients with DKD was suggested by the relationship between baseline PS level and the presence of albuminuria in a cohort of 362 diabetic patients. PS levels were correlated with 2-year risk for DKD progression in patients with moderately increased albuminuria, suggesting that PS may be associated with microvascular DKD complications. Finally, the authors tested a pharmacologic approach in db/db mice. The TPL enzyme is not present in eukaryotes, so this microbial-specific enzymatic pathway could be targeted using the TPL inhibitors 2-aza-tyrosine or l-meta-tyrosine. Both inhibitors were able to reduce PS levels. Interestingly, creatinine and PCS levels also declined after 2-aza-tyrosine treatment, suggesting a renoprotective effect. In addition, due to homology between TPL and an enzyme involved in IS synthesis, a reduction in IS levels was seen with both inhibitors. No major changes in microbial composition were identified, and PS levels rebounded after treatment cessation. Many previous studies focused on the negative effects of the well-known gut microbiota–derived uremic toxins, namely IS, PCS, and TMAO, which induce inflammation and oxidative stress, endothelial and kidney damage, and metabolic derangements.14Cosola C. Rocchetti M.T. Cupisti A. Gesualdo L. Microbiota metabolites: pivotal players of cardiovascular damage in chronic kidney disease.Pharmacol Res. 2018; 130: 132-142Crossref PubMed Scopus (60) Google Scholar The association between these toxins and CKD progression or increased cardiovascular risk found confirmation at the population level.7Sabatino A. Regolisti G. Brusasco I. Cabassi A. Morabito S. Fiaccadori E. Alterations of intestinal barrier and microbiota in chronic kidney disease.Nephrol Dial Transplant. 2015; 30: 924-933Crossref PubMed Scopus (128) Google Scholar, 14Cosola C. Rocchetti M.T. Cupisti A. Gesualdo L. Microbiota metabolites: pivotal players of cardiovascular damage in chronic kidney disease.Pharmacol Res. 2018; 130: 132-142Crossref PubMed Scopus (60) Google Scholar Although PS was previously identified using metabolomic approaches in the spectrum of gut-derived molecules and/or uremic toxicity,15Wikoff W.R. Anfora A.T. Liu J. et al.Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites.Proc Natl Acad Sci U S A. 2009; 106: 3698-3703Crossref PubMed Scopus (1788) Google Scholar no prior study examined its biological actions in the context of kidney disease.16Vanholder R. Pletinck A. Schepers E. Glorieux G. Biochemical and clinical impact of organic uremic retention solutes: a comprehensive update.Toxins (Basel). 2018; 10: 1-57Crossref Scopus (171) Google Scholar This study clarifies the toxic effects of PS on the kidney at molecular, histologic, and organismal levels and also highlights the association of this gut-derived uremic toxin with clinical outcomes in a cohort of diabetic patients. The gut microbiome has emerged as a pivotal actor and therapeutic target in a variety of diseases, now including CKD.7Sabatino A. Regolisti G. Brusasco I. Cabassi A. Morabito S. Fiaccadori E. Alterations of intestinal barrier and microbiota in chronic kidney disease.Nephrol Dial Transplant. 2015; 30: 924-933Crossref PubMed Scopus (128) Google Scholar, 14Cosola C. Rocchetti M.T. Cupisti A. Gesualdo L. Microbiota metabolites: pivotal players of cardiovascular damage in chronic kidney disease.Pharmacol Res. 2018; 130: 132-142Crossref PubMed Scopus (60) Google Scholar, 17Edamatsu T. Fujieda A. Itoh Y. Phenyl sulfate, indoxyl sulfate and p-cresyl sulfate decrease glutathione level to render cells vulnerable to oxidative stress in renal tubular cells.PLoS One. 2018; 13e0193342Crossref PubMed Scopus (35) Google Scholar The notion of "drugging the microbiome" was recently developed as a proof of concept in the context of atherosclerosis by using an approach based on selective inhibition of the microbial pathway generating TMAO.18Wang Z. Roberts A.B. Buffa J.A. et al.Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis.Cell. 2015; 163: 1585-1595Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar The work of Kikuchi et al further expands this proof of concept, demonstrating that inhibiting the PS synthetic pathway results in reductions in PS and creatinine levels in a mouse model, suggesting potential renoprotection without significantly altering microbial taxonomic balance. Many studies have previously investigated the possibility of manipulating gut microbiota composition and function by means of diet; probiotic, prebiotic, synbiotic, or postbiotic supplementation; or antibiotic treatments.14Cosola C. Rocchetti M.T. Cupisti A. Gesualdo L. Microbiota metabolites: pivotal players of cardiovascular damage in chronic kidney disease.Pharmacol Res. 2018; 130: 132-142Crossref PubMed Scopus (60) Google Scholar Pharmacologic inhibition of a bacterial metabolic pathway that may beneficially affect the kidneys is a novel concept that could drive the research on microbiota manipulation to a higher and more specific level of intervention. The most important implication of this study for the clinical nephrologist is represented by the broad potential of PS, which could represent not only an early DKD biomarker with prognostic significance, but also a pathogenic factor with potential for specific pharmacologic therapies. Due to the limitations of urinary albumin excretion, the need for new early biomarkers in DKD led to the identification of a variety of potential early biomarkers.5Papadopoulou-Marketou N. Kanaka-Gantenbein C. Marketos N. Chrousos G.P. Papassotiriou I. Biomarkers of diabetic nephropathy: a 2017 update.Crit Rev Clin Lab Sci. 2017; 54: 326-342Crossref PubMed Scopus (64) Google Scholar, 9Niewczas M.A. Sirich T.L. Mathew A.V. et al.Uremic solutes and risk of end-stage renal disease in type 2 diabetes: metabolomic study.Kidney Int. 2014; 85: 1214-1224Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Abbiss H. Maker G.L. Trengove R.D. Metabolomics approaches for the diagnosis and understanding of kidney diseases.Metabolites. 2019; 9: 1-22Crossref Scopus (45) Google Scholar However, some of these are only markers of risk for DKD occurrence in diabetic patients and are not involved in the causative pathway leading to kidney damage.20Macisaac R.J. Ekinci E.I. Jerums G. Markers of and risk factors for the development and progression of diabetic kidney disease.Am J Kidney Dis. 2014; 63: S39-S62Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar By contrast, Kikuchi et al identified PS as a marker correlated with kidney cellular and histologic damage at a very early stage, when kidney function is still normal, making it a promising candidate for the identification of diabetic patients at risk for developing DKD. The second important finding is clarification of the molecular mechanisms mediated by PS that lead to kidney injury and the possibility of pharmacologically targeting its upstream production in the gut, complementing the classical nutritional approach based on controlled intake of tyrosine precursors in the food.10Cosola C. Rocchetti M.T. Sabatino A. Fiaccadori E. Di Iorio B.R. Gesualdo L. Microbiota issue in CKD: how promising are gut-targeted approaches?.J Nephrol. 2019; 32: 27-37Crossref PubMed Scopus (36) Google Scholar In conclusion, the study is novel and important because it identifies the involvement of PS in podocyte injury, with a distinct mechanism beyond the well-known metabolic and hemodynamic determinants of damage in DKD1Umanath K. Lewis J.B. Update on diabetic nephropathy: core curriculum 2018.Am J Kidney Dis. 2018; 71: 884-895Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar and because it suggests a potential pharmacologic treatment to selectively target microbial (and not human) metabolic pathways. The promising results of metabolomics studies in DKD and atherosclerosis represent a first step toward a concept of "precision medicine" applied to gut microbiota modulation. However, in this respect, long-term studies to identify possible side effects affecting the host or the microbiota are needed. Enrico Fiaccadori, MD, PhD, Carmela Cosola, PhD, and Alice Sabatino, MSc. The authors declare that they have no relevant financial interests. We thank Francesco Peyronel, MD, for creating Figure 1. Received June 1, 2019, in response to an invitation from the journal. Direct editorial input from an Associate Editor and a Deputy Editor. Accepted in revised form July 1, 2019.