Celiac disease (CD) is a multifactorial immune-mediated enteropathy distributed worldwide with a prevalence of approximately 1% [1], although significantly higher in children [2], that is triggered by environmental and genetic factors [3]. CD exhibits a wide spectrum of clinical, serological and histological manifestations. The environmental factor responsible for its onset and maintenance is gluten, a complex mixture of protein contained in grains, such as wheat, rye, oat and barley. Gluten includes two major protein types: gliadins and glutenins (hordeins and secalins in barley and rye, respectively) [4]. Gliadins and glutenins have a high proline and glutamine content and are resistant to gastrointestinal enzymatic proteolysis. Therefore, large and potentially immunogenic peptides are prevalent on the intestinal mucosal surface [5].

Until now, the only available treatment for CD has been lifelong adherence to a gluten-free diet (GFD) ^[6][18]. For many years, clinicians and researchers discussed the sensitivity, specificity and accuracy of the many available serological tests, such as TGA-IgA, EMA-IgA and intestinal fatty acid binding protein (I-FABP) or citrulline ^[7][19], employed to monitor the response to GFD without reaching a conclusive consensus on a particular test. It is also true that the adherence to GFD by performing repeated intestinal biopsies is not feasible or practical ^[8][9][10][20,21,22].

2. Monitoring Adherence to GFD

Adherence to a strict GFD is the only treatment available for CD patients to date. Therefore, following diagnosis of CD, it is important to monitor adherence to GFD to prevent intestinal symptoms and minor damage ^[11][28]. However, gluten is ubiquitous, food cross contaminations are possible, food labeling is sometimes inadequate and all of these issues make it difficult for CD patients to strictly adhere to GFD ^[12][29]. It has been reported that most CD patients consume measurable amounts of gluten, although they are on a GFD, and that there is a discordance between gluten detection and villous atrophy, as consumption of as much as 10 mg of gluten daily seems to be tolerated by CD patients ^[13][30], whereas higher amounts (i.e., 200 mg/day) trigger symptoms and sustain intestinal damage ^[14][31]. To date, the most commonly used method to assess adherence to GFD consists in dietary questionnaires, serological tests or the evaluation of clinical symptoms, although none of these methods can directly and accurately estimate adherence. In fact, patient self-reporting appeared to be subjective and inaccurate in terms of the level of adherence to GFD or the lack of reporting the intentional or unintentional consumption of gluten ^[11][15][28,32]. The evaluation of serum levels of CD-specific antibodies plays a crucial role in monitoring adherence to a GFD diet, and numerous studies report that high levels of TGA-IgA or DGP-IgG may suggest non-adherence to the diet ^[16][17][33,34]. However, this method has limitations, as the normalization of antibody titers is lengthy, and it cannot identify sporadic episodes of occasional gluten exposure ^[11][28]. Small intestine biopsy is the most accurate method for diagnosing CD and its severity ^[18][35] and could be used to monitor adherence to GFD. However, this procedure is invasive, expensive and not particularly practical for this purpose ^[19][36]. Finally, the persistence of gastrointestinal symptoms after prolonged GFD may not be the evidence of non-adherence to the diet, as patients under strict GFD showed more symptoms than healthy subjects ^[20][37]. Moreover, bacterial overgrowth in the small intestine, irritable bowel disease or refractory CD can develop in subjects on a GFD, demonstrating the inefficacy of symptom monitoring ^[11][28]. For all of these reasons, the discovery of novel molecules able to function as biomarkers of GFD is particularly relevant and urgently needed. We think that GIPs and miRNAs could fulfill this role and that they can be used as complimentary biomarkers to monitor GFD in CD patients.

3. Detection of GIPs in Feces

Due to resistance of GIPs to gastrointestinal digestion and the potential immunogenic reactions of TCD4⁺ cells in CD patients, the clinical usefulness of fecal GIPs quantification has been recently evaluated as a marker of dietary transgression/adherence to GFD ^{[21][22][23][24][25][26]}[12,41,42,43,44,45] or to detect duodenal mucosal damage in CD patients ^[27][46]. Comino and colleagues were the first to detect gluten-derived peptides in feces of patients with CD or other intestinal pathologies ^[21][12]. They found that the recovery of immunotoxic fractions in feces could indicate that gluten has passed through the digestive tract because a significant portion of the ingested gluten peptides was excreted. Therefore, gluten has been consumed and could be measurable in feces. Additionally, the concentration of 33-mer peptide was determined using in vitro simulated gastrointestinal digestion. Their results indicate that more than 30% of the gliadin-reactive peptides remained undigested, even after hydrolysis during digestion. The variations in the absorption of intact gliadin-reactive peptides between individuals could explain the diversity of the microbial population in the gastrointestinal tract, which might impact the peptide concentration in feces ^[21][28][12,47]. In a further comprehensive study, the authors evaluated the efficacy of fecal GIPs to support the diagnosis of the disease and monitor adherence to GFD in a cohort of CD children. Their analysis of fecal GIPs resulted in two main findings: the direct confirmation of gluten intake days before CD diagnosis and a substantial decrease in gluten consumption after diagnosis of CD and the beginning of GFD ^[29][48]. Prior prospective, nonrandomized, and multicenter studies performed by the same group revealed the effectiveness of fecal GIPs in 188 CD patients who were on a GFD for at least 1 year versus 84 healthy controls. Fecal GIPs quantification by measurement of serological anti-tissue transglutaminase IgA (TGA-IgA) and anti-deamidated gliadin peptide IgG (DGP-IgG) antibodies was performed simultaneously. The authors found a significant association between age and GIPs in feces with an increasing dietary transgression with advancing age and with gender in certain age groups: 39.2% in subjects ≥13 years old and 60% in men ≥13 years old ^[15][32]. With respect to the traditional dietary questionnaire or serological methods, their analyses showed a low GFD adherence rate among patients on an established GFD using GIPs analysis. These findings suggest that the fecal GIPs analysis can be an accurate method that enables a direct and quantitative assessment of gluten exposure soon after ingestion. However, a combination of GIPs tests with serological tests did not show a good correlation and remained a complementary technique. It has been recently reported that GIPs quantification in feces indicates dietary transgressions in patients on long-term GFD according to dietary reports. Costa and colleagues followed-up CD adult patients who were on a GFD for at least two years. They found that in 67 out of 74 (90.5%) fecal samples, both ELISA and point-of-care tests (PoCTs) were concordant (either positive or negative) ^[30][49]. Integration of GIPs quantification in feces with conventional strategies could better determine adherence to GFD both in children and in the adult population. Altogether, the more gluten consumed, the higher the detectable concentration of GIPs in feces of CD patients on a GFD; however, the presence of several gastrointestinal factors that could influence GIPs recovery cannot be ruled out and needs to be further investigated.

4. Detection of GIPs in Urines

Similar to fecal GIPs, the presence of GIPs in urine has been investigated in recent years to evaluate the potential toxic effects of gluten peptides contained in food accidentally ingested by CD patients ^[31][8]. Urinary miRNAs can provide a possible alternative to currently available biomarkers. In fact, the significance of miRNA in DN has been explored in several studies. The first evidence that the miRNA profile is altered in urinary exosomes from type 2 DN patients (14 miRNAs upregulated and 2 miRNAs downregulated) was provided a few years ago ^[32][60]. Principal component analysis revealed that differential urinary exosomal miRNA expression is different in patients with microalbuminuria compared to normo-albuminuric patients. The increased expression of miRNA-320c, which is indirectly involved in TGF-β signaling via targeting of TSP-1, may represent a novel candidate biomarker for early progression of disease.Ruiz-Carnicer and colleagues investigated the clinical utility of GIPs as biomarkers to monitor adherence to GFD and the relationships between the detectable levels of GIPs in urine and the degree of damage to the intestinal mucosa ^[33][52]. About 24% of CD patients on GFD still had mucosal damage, and 94% of them had detectable urinary GIPs. In contrast, 97% of patients without duodenal damage had no detectable GIPs. The authors demonstrated the high sensitivity (94%) and negative predictive value (97%) of GIPs measurements in relation to duodenal biopsy results. In addition, the authors demonstrated that in the group of neo-diagnosed CD patients, 82% had measurable amounts of GIPs in their urine.

5. Urinary miRNAs as Biomarkers of Celiac Disease and adherence to GFD

Liquid biopsies, such as miRNAs circulating in the human serum/plasma (i.e., circulating miRNAs), have recently emerged as affordable and reliable biomarkers in many diseases ^[34][35][36][76,77,78], including CD ^[37][79]. This year, aour group reported the discovery of novel circulating miRNAs as biomarkers for the diagnosis of CD and adherence to GFD by pediatric patients ^[38][23]. One of the aims of this study was to find reliable biomarkers able to avoid intestinal biopsies in children, especially those with serological levels of TGA-IgA <10 × the upper limit of normal, who generally undergo gastroduodenoscopy ^[39][17]. These scholarsWe found that a panel of circulating miRNAs can predict not only CD but that they are also able to return to the levels observed in control subjects after a strict GFD. ItWe demonstrated the reliability and stability of these biomarkers (in particular, of miR-192-5p, miR-215-5p and miR-125b-5p either alone or in combination) and their potential in the clinical practice to help gastroenterologists in diagnosing and treating CD.

TheOur group also hypothesized a link between circulating and fecal miRNAs in intestinal diseases and the presence of an intricate network of interactions also involving the gut microbiota ^[40][80]. This is corroborated by a couple of seminal studies previously reported by others ^[41][42][81,82]. However, circulating and fecal miRNAs can represent only a face of the medal, as urinary miRNAs can complete this multifaceted picture.

IWe think that in the near future, urinary miRNAs could provide complimentary information not only to help clinicians to diagnose CD but possibly to assess adherence to GFD or the presence of comorbidities in CD patients.

6. Conclusions and Future Directions

We have discussed herein many papers dealing with the use of GIn tPs as biomarkers for monitoring adherence to GFD by CD patients. In the last decade, GIPs have been demonstrated to be reliable and stable biomarkers in many studies, although fecal GIPs could not significantly discriminate adult CD patients with persistent villous atrophy and those who recovered after a GFD for two years ^[43][83].

More recently, miRNAs have emerged as innovative diagnostic tools (i.e., liquid biopsies) that can be employed as potential biomarkers, as they have already been employed to diagnose many diseases, including CD. Larger cohorts are required to validate these results, as well as, in some cases, a confirmatory intestinal biopsy.

Considerable efforts have been made to find diagnostic methods to assess the presence of GIPs in feces and urine in order to evaluate the amount of ingested gluten or to monitor deviations from a GFD (i.e., adherence to GFD). the only non-invasive and direct methods reported in the literature in the last few years rely on the determination of GIPs either in feces or urine, although some of these determinations have been demonstrated to be dose- and time-dependent and only partially correlated to the interindividual kinetics of gluten processing and elimination ^[44][50].

TWe support the study of urinary miRNAs not only to evaluates their ability as biomarkers of adherence to GFD in CD patients but also as a tool to monitor the status of the underlying tissues (i.e., duodenum, glomerulus and kidney), eventually corroborated by intestinal biopsies. Moreover, urine samples are more readily acceptable to patients compared to fecal samples due to the simplicity of collection and their transferability to the clinician.

Finally, further studies are needed to evaluate whether urinary miRNAs can predict the presence of intestinal/renal permeability and the concomitant presence of other comorbidities.