*4.2. Animal Models of Early-Life Gut Microbiota-Targeted Therapy*

Here, we list in Table 2 a summary of studies documenting gut microbiota-targeted interventions in animal models of CKD of developmental origins and its comorbidities [101–110]. The therapeutic duration is during fetal and childhood stages. The literature review states that gut microbiota-targeted interventions used to prevent CKD and its comorbidities primarily include probiotics, prebiotics, and postbiotics.

As shown in Table 2, rats are the dominant species used by experiments, and hypertension is the most commonly studied CKD-related comorbidity. A variety of early-life insults can lead to structural and functional changes in the developing kidney by the so-called renal programming [6]. Unlike in humans, kidney development in rats continues up to postnatal week 1–2. According to DOHaD theory, adverse environmental insults during pregnancy and lactation period can interrupt kidney development, resulting in renal programming and adult kidney disease. Several models of renal programming have been used to examine gut microbiota-targeted interventions in CKD of developmental origins, such as maternal high-fructose diet [101,108], perinatal high-fat diet [102,107,109], perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure [103], maternal adenine-induced CKD [104], maternal TMAO and ADMA exposure [105], and maternal high-fructose diet and TCDD exposure [110].


**Table 2.** Summary of early-life gut microbiota-targeted therapies for CKD and its comorbidities.

Studies tabulated according to types of intervention, animal models, and age at evaluation. TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; CKD = chronic kidney disease; TMAO = trimethylamine-N-oxide; ADMA = asymmetric dimethylarginine; SD = Sprague-Dawley rat; DMB = 3,3-maternal dimethyl-1-butanol.

Taking the example of the maternal high-fructose diet model, high-fructose intake during pregnancy and lactation modified over 200 renal transcripts from nephrogenesis stage to adulthood [111]. Using whole-genome RNA next-generation sequencing (NGS), high-fructose-induced alterations of the renal transcriptome were reported in kidneys from 1-day-, 3-week-, and 3-month-old male offspring. NGS identified genes in arachidonic acid metabolism (*Cyp2c23*, *Hpgds*, *Ptgds* and *Ptges*) that contribute to renal programming and hypertension. Notably, this renal programming model has been used to examine the reprogramming effects of gut microbiota-targeted therapy on fructose-induced developmental programming [112]. Since the above-mentioned renal programming models have been established and linked to adverse renal outcomes in adult offspring, readers are referred to original references. There was only one study conducting an adenine-induced pediatric CKD model to determine the effects of probiotic resveratrol on CKD progression [106].

Review elsewhere showed that several probiotic microorganisms and prebiotics have benefits on adult CKD [81,82], while there was only very limited evidence regarding their role on CKD of developmental origins. Supplementation with *Lactobacillus casei rhamnosus* from pregnancy through lactation protected adult male rat progeny against hypertension programmed by a maternal high-fructose diet [101] or perinatal high-fat diet [102].

Additionally, inulin as a prebiotic has been examined for its protective effect in hypertension of developmental origins [101,102]. In a high-fat model [102], we previously demonstrated that inulin treatment protected against hypertension in adult rat offspring coinciding with alterations of the gut microbiota, particularly increasing the abundance of *Lactobacillus*, a well-known probiotic strain. Likewise, perinatal supplementing to rat dams with inulin protected adult offspring against maternal high-fructose diet-induced hypertension, which coincided with an increased plasma level of propionic acid [102].

Resveratrol can modulate gut microbiota composition, undergo biotransformation to activate metabolites via the intestinal microbiota, affect gut barrier function, modify the *Firmicutes* to *Bacteroidetes* (F/B) ratio, and reverse the gut microbial dysbiosis [113–116]. With a prebiotic effect for gut microbes, increasing evidence supports the beneficial effects of resveratrol on many diseases, including CKD [117,118]. One study revealed that perinatal resveratrol therapy could protect adult offspring against hypertension and CKD of developmental origins [119]. Studies using a maternal TCDD exposure rat model showed TCDD-induced renal hypertrophy and hypertension in adult progeny, and both are key features of early CKD. TCDD-induced hypertension is associated with activation of AHR signaling, induction of TH17-dependent renal inflammation, and alterations of gut microbiota compositions [103]. Conversely, the induction of AHR- and TH17-mediated renal inflammation could be counterbalanced by perinatal resveratrol supplementation. The beneficial effects of resveratrol are associated with reshaping the gut microbiome by augmenting microbes that can inhibit TH17 responses and reduce the F/B ratio, a microbial marker of hypertension [14]. In a maternal CKD model, adult offspring developed renal hypertrophy and hypertension [104]. Perinatal resveratrol therapy protected hypertension, coinciding with the restoration of microbial richness and diversity and an increase in *Lactobacillus* and *Bifidobacterium* [104]. Similar to TMAO, asymmetric dimethylarginine (ADMA) is a well-known uremic toxin [120]. Another study using a maternal TMAO plus ADMA exposure model demonstrated that adult offspring born to dams exposed to uremic toxins had renal dysfunction and hypertension [105]. Conversely, maternal treatment with resveratrol rescued hypertension induced by TMAO plus ADMA exposure, accompanied by increased butyrate-producing microbes and fecal butyric acid level.

Of note is that the low bioavailability of resveratrol diminishes its efficacy and clinical translation [121]. Accordingly, we produced resveratrol butyrate ester (RBE) via the esterification of resveratrol with the SCFA butyrate to improve the efficacy [122]. Using a pediatric CKD model [85], we recently found low-dose RBE (25 mg/L) is as effective as resveratrol (50 mg/L) in protecting against hypertension and renal dysfunction. The beneficial effects of RBE include regulation of SCFA receptors, decreased AHR signaling, and increased abundance of the beneficial microbes *Blautia* and *Enterococcus*.

Although there are many prebiotic foods, only garlic oil has shown beneficial effects against high-fat diet-induced hypertension in adult progeny [106]. These effects include increased α-diversity, increased plasma levels of acetic acid, butyric acid, and propionic acid, and increased beneficial bacteria *Lactobacillus* and *Bifidobacterium*.

In addition to probiotics and prebiotics, postbiotics is another gut microbiota-targeted therapy. Postbiotics include various components, such as microbial cell fractions, extracellular polysaccharides, functional proteins, cell lysates, extracellular vesicles, cell-wall-derived muropeptides, etc. [94]. Nevertheless, very limited information exists regarding the use of postbiotics in CKD. Acetate supplementation within gestation and lactation was reported to protect offspring against high-fructose-diet-induced hypertension, a major complication of CKD [108]. However, its protective effects on other complications of CKD are still waiting for clarification. Another example of postbiotic use in hypertension of developmental origins is conjugated linoleic acid [109]. Linoleic acid is a gut microbial metabolite derived from dietary polyunsaturated fatty acids (PUFA) [123]. Several gut microbes have been identified as producing PUFA-derived intermediate metabolites [124]. Administration of PUFA-derived bacterial metabolites such as linoleic acid has been shown to provoke anti-obesity and anti-inflammatory effects [125]. However, unlike probiotics and prebiotics [126,127], currently there is a lack of a clear definition for postbiotics. Considering the complex nature of postbiotics [94], a clear definition is important for future research from a regulatory perspective.

Moreover, there are other microbiota-related therapies applied for preventing CKD and its comorbidities. Microbe-dependent TMA and TMAO formation can be inhibited by 3,3-dimethyl-1-butanol (DMB), a structural analogue of choline [128]. Recently, two studies reported that maternal oral administration of DMB protected hypertension in adult rat progeny exposed to a maternal high-fructose diet [87] or high-fructose diet plus TCDD exposure [110]. This was accompanied by affecting the metabolic pathway of TMA-TMAO and reshaping gut microbiota [110].

As far as the multifaceted relationship between the gut and kidney, there might be other potential approaches by which the gut microbiota might prevent CKD and its associated complications. For example, RAS blockers are currently the most common therapies used for renoprotection and antihypertension [129]. Considering drug-mediated alterations in the gut microbiota compositions can have beneficial effects on the host [130], a greater understanding of mechanisms driving drug–gut microbiota interactions might aid in guiding the development of microbiota-targeted pharmacological interventions. Together, early microbiota-targeted therapies, in the long term, may enable the capacity to prevent the development of CKD and its comorbidities in a desired favorable direction. However, there is an urgent need to identify and fill the knowledge gaps on gut microbiotatargeted therapies between established CKD and CKD of developmental origins.
