Part Three Postbiotics And Kidney Disease

Diet-Induced Kidney Injury

Postbiotics have been studied in preclinical, but not in clinical diet-induced hypertension and kidney disease. A postbiotic derived from sonicated Lactobacillus paracasei isolated from Egyptian cheese was administered to adult Wistar albino male rats fed with a high-fat diet for 9 weeks and compared to atorvastatin treated or placebo to assess the impact on weight and lipids [86]. The postbiotic contained several enzymes, including proteases, lipases, and antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), and displayed antibacterial activity against pathogenic bacteria. Both the postbiotic and atorvastatin reduced total serum lipids, serum triglycerides, and total serum cholesterol and prevented an increase in body weight. However, the overall body weight of atorvastatin- and postbiotic-treated rats was lower than that of rats fed on a normal diet, which may be a cause for concern. In this regard, there was evidence for atorvastatin toxicity such as increased liver enzymes and bilirubin levels, whereas this was not observed for the postbiotic treatment. A high-fat diet results in kidney disease characterized by an increase in serum creatinine, uric acid, and urea levels, and, surprisingly, this was exacerbated by atorvastatin. In postbiotic-treated rats instead, serum creatinine, uric acid, and urea levels remained in the normal range and were lower than in high-fat diet rats with or without atorvastatin treatment [86]. Although the molecular mechanisms or underlying kidney pathophysiology were not explored, the postbiotic treatment appeared to have a protective effect from high-fat diet-induced kidney dysfunction.

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A potential postbiotic that has also been studied in a preclinical diet-induced hypertension and kidney injury model is GABA salt. For preclinical studies, GABA-salt was prepared by culturing Lactobacillus brevis BJ20 for 24 h and then further fermented with 40% (w/w) refined seawater salt for 6 h and then filtered and spray dried to yield GABA-salt. GABA-salt contained 0.76% ± 0.01% GABA, as well as other unmeasured postbiotics components [87]. Based on the information provided in the manuscript, no purification step appears to have been performed to eliminate other bacterial metabolites. Oral GABA-salt and salt treatment were compared in a murine model of hypertension induced by a high salt and high cholesterol diet. Replacing salt for GABA-salt attenuated the diet-induced increase in serum creatinine and urea, blood pressure, intima-media thickness, and other changes in the aorta, such as M1 polarization (CD86 expression), cell death (TUNEL staining) and TNF-α and inducible nitric oxide synthase (NOS) levels. Furthermore, GABA-salt-induced changes are consistent with the in vivo observations in cultured macrophages, endothelial cells, and vascular smooth muscle cells. While the authors hypothesized that any impact of GABA-salt was due to the GABA content, the GABA-salt preparation would be expected to contain other bacterial products, i.e., to be a postbiotic, and from the experiments performed, it remains unclear which individual or combination of postbiotic components was responsible for the observed effect. Indeed, it was not tested whether GABA itself added to control salt resulted in the same effects as the postbiotic GABA salt.

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GABA-salt studies followed a prior description of fermented milk containing GABA by using Lactobacillus casei strain Shirota and Lactococcus lactis YIT 2027 which lowered blood pressure in humans and rats [88,89]. The Lb. casei strain hydrolyzes milk protein into glutamic acid and the Lc. lactic converts glutamic acid into GABA (GABA 10–12 mg/dL). However, from the manuscripts, it is unclear whether the fermented milk contained probiotics (i.e., live bacteria) and/or postbiotics (i.e., dead bacteria and their products) [89]. The human study was a pilot study in which 39 mildly hypertensive patients received d ly GABGABA-fermented or placebo (non-fermented milk) for 12-12 weeksystolic blood pressure decreased by 17.4 ± 4.3 mmHg in the intervention group and this was reported to b statistically significantly different from the placebo group, in which blood pressure also d released [89]. In SHR hypertensive rats, a diet containing freeze-dried GABA fermented m k (100 g/kg, final concentration of GABA 0.1 g/kg) prevented the progressive increase in systolic blood pressure observed between 7 and 10 weeks of age [88].

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Postbiotics in Nephrotoxic AKI

Postbiotics have been studied in preclinical, but not in clinical AKI. In preclinical studies, AKI was induced by cisplatin administration to mice and the severity of the injury was magnified by the oral administration of salt from 1 h before cisplatin up to 48 h post-induction of AKI, with a 72 h readout [90]. GABA-salt was prepared in the hypertension studies [87] with slight modifications involving further fermentation of Lactobacillus. plantarum BJ21, to yield GABA-salt, and two other compounds that were termed lactoGABA-salt and postbiotic-GABA-salt. The manuscript does not provide further details on the differential processes performed/used to generate the three compounds or their full composition other than indicating that they contained 93, 112, and 97 mg/g GABA and that 108, 175, and 109 mg/g were neither sodium chloride nor GABA, respectively. The sodium chloride content of the parent seawater salt was not reported. The results are n very clearly expressed, but it appears that salt administration increased the severity of c platin-induced AKI as assessed by serum creatinine and urea, histological injury,d e recession of inflammatory mediators, while the different GABA-salt was protective, the results for lacto-GABA-salt and postbiotic-GABA-salt being unclear [90].

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A Roadmap towards Postbiotic Therapy for Kidney Disease

The ultimate aim of preclinical research is to develop products that could improve t diagnosis and treatment of diseases. Postbiotics are not well-known and postbiotics research is still in its early stages and has not yet led to products for clinical use. Indeed, as r recently as July 2022 postbiotics were still erroneously defined as therapeutic strategies that t get downstream signaling pathways of the microbiome [91]. We suggest a roadmap for the clinical development of postbiotics in the field of kidney disease (Figure 4). First, it would be necessary to identify the key bacterial strains that play an important role in the development and progression of kidney disease and define the mechanisms underlying their beneficial function as it might be that not all the bacterial components might be involved. Recognizing w ch fragments have a functional role and which do not would be of extreme importance in the development of postbiotics. A postbiotic approach would overcome limitations f ed with probiotics-related products, this mainly their instability. Furthermore, it is necessary to characterize the optimal procedures of manufacturing, formulation,d ministration, and in vitro experiments required to establish the optimal concentration r ge and to identify early any safety issues. In vitro studies should address any positive or negative impact of the postbiotic on cultured mammal cells, including the human gut, kidney, and immune cells, as well as on complex bacterial communities, using technologies such as the Dynamic Gastrointestinal Simulator (SIMGI®) [92]. Tubular cells are the key k ney cell types to study, given that they represent most of the kidney mass and have an array of transporters and receptors, although glomerular and endothelial cells may an o be of interest. Subsequently, in vivo, ex,periments with animal models of kidney ury are required. Given the short timelines, preclinical AKI models may be of interest. Moreover, some of them result in features of CKD within a reasonable time frame (the so-called AKI-to-CKD transition), so insight into chronicity may be gained from these studies. However, no AKI therapy identified in preclinical models has ever reached routine clinical use, pointing to potentially large differences in pathophysiology and/or timing of the intervention between preclinical models and human disease. In this regard, models representing clinical situations in which the invention may be applied prophylactically in the clinic may offer advantages (e.g., kidney ischemia during surgery, and exposure to nephrotoxic chemotherapeutic agents). To be effective, postbiotics should be prepared in such a way that it makes them resistant to degradation whilst reaching the targeted segment of the gastrointestinal tract. Manufacturing procedures should ensure low variability of the finished product and scalability to produce large batches. In this regard, several postbiotic possibilities are available, ranging from bacterial lysates to conditioned cultured medium to extracellular vesicles as recently demonstrated for O. formigenes [93]. Moreover, more c plex pospostdocsnsisting of diverse bacteria encoding oxalate degradation pathways m be explored [72,94]. Finally, RCTs in patients with kidney diseases should be planned. Short-term trials should establish their safety in humans and explore biomarkers of their b logical activity (e.g., oxaluria in patients with hyperoxaluria). Larger efficacy trials focusing on broader endpoints will be more difficult to design and fund but are required to gather the dataset necessary for approval by the medicine’s regulatory agencies. In this regard, PH1 is a rare disease that has severe, potentially life-threatening complications, and treatment of PH1 represents both an unmet clinical need and an attractive target from the human and regulatory points of view. However, being a severe condition in which oxalic acid is produced endogenously, PH1 may be less amenable to interventions acting at the level of the gut lumen. A shortcut to postbiotics as a drug treatment would be to c sider them as nutritional supplements or medical nutrition. This would increase the f sibility factor for clinical studies but would decrease the trustworthiness of any claim o health benefit. O. formigenes postbiotics may be suitable for assessment as nutritional supplements in hyperoxaluria of enteric origin. This condition is more common and less severe than PH1, facilitating clinical trials or nutritional research; the via of entry of oxalic acid in the body is by ingestion, similar to the postbiotics, which may increase efficacy and finally; there is a subset of health-conscious but ill-advised persons with hyperoxaluria causing kidney injury resulting from fashionable trends or diets (e.g., juicing) who would may also be prone to taking supplements such as O. formigenes postbiotics, especially if this would allow them to pursue their diets more safely. Another potential scenario of O. formigenes postbiotics use is the prevention of antibiotic-associated urolithiasis, especially if long-term antibiotics are needed, as while on antibiotics, attempts at colonization by O. formigenes probiotics are expected to be futile [95]. Once on the market, post-registration studies will monitor the long-lasting safety and efficacy effects. In the case of postbiotics for enteric hyperoxaluria, the whole process from research to marketing and post-marketing experience may allow us to learn enough about the intervention to re-design it for more severe conditions such as PH1.

References

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87. Son, M.; Oh, S.; Lee, H.S.; Choi, J.; Lee, B.-J.; Park, J.-H.; Park, C.H.; Son, K.H.; Byun, K. Gamma-Aminobutyric Acid-Salt Attenuated High Cholesterol/High Salt Diet-Induced Hypertension in Mice. Korean J. Physiol. Pharmacol. 2021, 25, 27–38. [CrossRef]

88. Hayakawa, K.; Kimura, M.; Kasaha, K.; Matsumoto, K.; Sansawa, H.; Yamori, Y. Effect of a Gamma-Aminobutyric Acid-Enriched Dairy Product on the Blood Pressure of Spontaneously Hypertensive and Normotensive Wistar-Kyoto Rats. Br. J. Nutr. 2004, 92, 411–417. [CrossRef]

89. Inoue, K.; Shirai, T.; Ochiai, H.; Kasao, M.; Hayakawa, K.; Kimura, M.; Sansawa, H. Blood-Pressure-Lowering Effect of a Novel Fermented Milk Containing Gamma-Aminobutyric Acid (GABA) in Mild Hypertensives. Eur. J. Clin. Nutr. 2003, 57, 490–495. [CrossRef]

90. Lee, H.; Ji, S.Y.; Hwangbo, H.; Kim, M.Y.; Kim, D.H.; Park, B.S.; Park, J.-H.; Lee, B.-J.; Kim, G.-Y.; Jeon, Y.-J.; et al. Protective Effect of Gamma-Aminobutyric Acid against Aggravation of Renal Injury Caused by High Salt Intake in Cisplatin-Induced Nephrotoxicity. Int. J. Mol. Sci. 2022, 23, 502. [CrossRef]

91. Shankaranarayanan, D.; Raj, D.S. Gut Microbiome and Kidney Disease: Reconciling Optimism and Skepticism. Clin. J. Am. Soc. Nephrol. 2022. online ahead of print. [CrossRef]

92. Aguilera-Correa, J.-J.; Madrazo-Clemente, P.; Martínez-Cuesta, M.D.C.; Peláez, C.; Ortiz, A.; Sánchez-Niño, M.D.; Esteban, J.; Requena, T. Lyso-Gb3 Modulates the Gut Microbiota and Decreases Butyrate Production. Sci. Rep. 2019, 9, 12010. [CrossRef]

93. Chamberlain, C.A.; Hatch, M.; Garrett, T.J. Extracellular Vesicle Analysis by Paper Spray Ionization Mass Spectrometry. Metabolites 2021, 11, 308. [CrossRef]

94. Chamberlain, C.A.; Hatch, M.; Garrett, T.J. Oxalobacter Formigenes Produces Metabolites and Lipids Undetectable in Oxalotrophic Bifidobacterium animalis. Metabolomics 2020, 16, 122. [CrossRef] [PubMed]

95. Joshi, S.; Goldfarb, D.S. The Use of Antibiotics and Risk of Kidney Stones. Curr. Opin. Nephrol. Hypertens. 2019, 28, 311–315. [CrossRef] [PubMed]

Chiara Favero 1 , Laura Giordano 2 , Silvia Maria Mihaila 2 , Rosalinde Masereeuw 2 , Alberto Ortiz 1,3,4, and Maria Dolores Sanchez-Niño 1,3,5,

1 Department of Nephrology and Hypertension, IIS-Fundacion Jimenez Diaz UAM, 28049 Madrid, Spain

2 Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands

3 Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) 2040, 28049 Madrid, Spain

4 Departamento de Medicina, Facultad de Medicina, Universidad Autónoma de Madrid, 28049 Madrid, Spain

5 Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, 28049 Madrid, Spain