Brush Border Research Articles

Elucidating the Proximal Tubule HNF4A Gene Regulatory Network in Human Kidney Organoids.

HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development. The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated. We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation. Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation. This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.

Agomelatine on cisplatin-induced nephrotoxicity via oxidative stress and apoptosis.

Drug-induced nephrotoxicity is the greatest deterrent to the use of cisplatin, which is a frequently used chemotherapeutic with proven effectiveness in cancer therapy. Agomelatine, which is used in the treatment of sleep disorders and depression, has gained attention in recent years with its antioxidative and anti-inflammatory effects. In this study, the effects of the synthetic melatonin agonist agomelatine on nephrotoxicity were investigated in a rat model of cisplatin-induced nephrotoxicity using biochemical, histological, and immunohistochemical methods. Thirty-two male rats were divided into 4 groups: 1. control group, 2. agomelatine group, 3. cisplatin group, 4. cisplatin + agomelatine group. In the cisplatin group, there were widespread atypical glomerular structures and vacuolization in tubular epithelial cells, necrotic tubules, deterioration of brush border structure in proximal tubules, and fibrotic areas characterized by diffuse polymorphonuclear leukocyte (PNL) and extensive collagen deposition in the interstitial spaces. However, in the cisplatin + agomelatine group, we observed a reduction in glomeruli of atypical structure and necrotic tubules, in PNL infiltration in interstitial spaces, and fibrotic areas compared to the cisplatin group. The cisplatin + agomelatine group showed lower malondialdehyde (MDA) serum creatinine, serum urea levels, and higher glutathione (GSH) levels compared to the cisplatin group. Immunohistochemical analyses revealed that the elevated NF-kβ/p65, 8-OHdG, and cleaved caspase-3 positivity in the cisplatin group had significantly decreased in the cisplatin + agomelatine group. In conclusion, agomelatine showed a nephroprotective effect against cisplatin-induced nephrotoxicity.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.

Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.

Mfsd2a-mediated lysolipid transport is important for renal recovery after acute kidney injury

Acute kidney injury (AKI) is a global public health concern with high mortality and morbidity. In ischemic–reperfusion injury (IRI), a main cause of AKI, the brush border membrane of S3 proximal tubules (PT) is lost to the tubular lumen. How injured tubules reconstitute lost membrane lipids during renal recovery is not known. Here, we identified Mfsd2a, a sodium-dependent lysophosphatidylcholine (LPC) transporter, to be expressed specifically in the basolateral membrane of S3 PT. Using an in vivo activity probe for Mfsd2a, transport activity was found to be specific to the S3 PT. Mice with haploinsufficiency of Mfsd2a exhibited delayed recovery of renal function after acute IRI, with depressed urine osmolality and elevated levels of histological markers of damage, fibrosis, and inflammation, findings corroborated by transcriptomic analysis. Lipidomics revealed a deficiency in docosahexaenoic acid (DHA) containing phospholipids in Mfsd2a haploinsufficiency. Treatment of Mfsd2a haploinsufficient mice with LPC-DHA improved renal function and reduced markers of injury, fibrosis, and inflammation. Additionally, LPC-DHA treatment restored S3 brush border membrane architecture and normalized DHA-containing phospholipid content. These findings indicate that Mfsd2a-mediated transport of LPC-DHA is limiting for renal recovery after AKI and suggest that LPC-DHA could be a promising dietary supplement for improving recovery following AKI.

Effect of fermented rapeseed meal on growth performance, nutrient digestibility, and intestinal health in growing pigs

To explore the effects of fermented rapeseed meal (FRSM) on growth performance and intestinal health, a total of 30 growing pigs were randomly allotted to three treatments consisting of corn-soybean meal diet (CSD), rapeseed meal diet (RSD), and fermented rapeseed meal diet (FRSD). Results showed that compared with RSD, FRSD feeding increased the average daily gain and final body weight in pigs (P < 0.01). Compared with RSD feeding, FRSD feeding elevated the apparent digestibility of crude protein, acid detergent fiber, and ether extract in pigs (P < 0.01). Moreover, the FRSD group exhibited greater apparent ileal digestibility of His, Thr, Lys, and Ser than the RSD group (P < 0.01). The digestible energy, metabolic energy, and nitrogen utilization were higher in the FRSD and CSD groups than in the RSD group (P < 0.01). As compared to the RSD, FRSD feeding decreased the serum concentration of leptin but significantly increased the concentrations of immunoglobulin (Ig) A, IgG, ghrelin, and enzyme activities of amylase, lipase, and trypsin in the pancreas (P < 0.05). Interestingly, the villus height, the ratio of villus height to crypt depth, and the activities of brush border enzymes (e.g., maltase and sucrase) in the small intestine were higher in the CSD and FRSD groups than in the RSD group (P < 0.05). As compared to the RSD, the FRSD feeding not only increased the expression level of the occludin in the small intestinal epithelium (P < 0.05) but also elevated the expression levels of claudin-1, MUC1, and PepT1 genes in the duodenum, and elevated the expression levels of SGLT1 and CAT1 genes in the jejunum (P < 0.05). Importantly, FRSD feeding significantly decreased the abundance of Escherichia coli, but increased the abundance of Lactobacillus and the content of butyrate in the cecum and colon (P < 0.05). These results indicated that compared with rapeseed meal, fermented rapeseed meal exhibited a positive effect on improving the growth performance and intestinal health in growing pigs, and the results may also help develop novel protein sources for animal nutrition and the feed industry.

Distribution of nephrons in the head kidney of three species of Sebastes (Teleostei: Scorpaenidae).

Nephrons are generally not distributed in the head kidney of teleost. Nonetheless, in this study, the nephron structure was identified in the head kidney of three species of Sebastes (Sebastes inermis, Sebastes schlegelii and Sebastes thompsoni). The kidney is Y shaped, with the development in the head kidney. The nephron structure was confirmed in the head kidney and body kidney. In addition, the nephron consisted of renal corpuscles and tubules, and the renal corpuscle consisted of the Bowman's capsule and glomerulus. Histologically, previous studies reported that the nephron structure is similar to that of other marine teleost. The renal tubule is a simple columnar epithelial layer with microvilli and cilia on the free surface, which is observed as a brush border. The Rrk (relative area ratio of kidney to body surface) was 5.14%, 7.58% and 5.17% in S. inermis, S. schlegelii and S. thompsoni, respectively. The Gar (glomerular area ratio of the head kidney) was higher in the central area than in the peripheral area, and species, which showed significant difference (P < 0.05), were in the following order: S. thompsoni (1.60%) > S. schlegelii (0.90%) > S. inermis (0.66%).

Brush border enzyme hydrolysis and glycaemic effects of isomaltulose compared to other saccharides in dogs.

Digestible carbohydrates differ in glycaemic response, therewith having the potential to influence metabolic conditions such as insulin resistance and diabetes mellitus. Isomaltulose has been proven to lower the glycaemic response in humans, which to date has not been studied in dogs. Therefore, the aim of the present study was to characterise the digestibility, as well as the physiological effects of isomaltulose in dogs, in comparison to other saccharides. To this end, three studies were performed. Study 1 was an in vitro study, evaluating the small intestinal hydrolysis of isomaltulose compared to other relevant carbohydrate sources. Three of these saccharides, having close and low-moderate degrees of hydrolysis by brush border enzymes, were also evaluated in vivo for their glycaemic effects by measuring plasma levels of glucose, insulin and glucagon-like peptide 1 (GLP-1) 0-180 min after administration of a single dosage after an overnight fast (i.e., isomaltulose, sucrose and maltodextrin in a 3 × 3 Latin-square design, in 9 dogs, Study 2). To understand if digestive enzymes, underlying glycaemic responses for isomaltulose and sucrose can be upregulated, we exposed dogs to these saccharides for 2 weeks and repeated the measurements after an overnight fast in 18 dogs (Study 3). Isomaltulose was hydrolysed by intestinal enzyme preparation from all three dogs, but the degrading activity was low (e.g., 3.95 ± 1.03 times lower vs. sucrose), indicating a slower rate of hydrolysis. Isomaltulose had a low glycaemic response, in line with in vitro data. In vitro hydrolysis of sucrose was comparable or even higher than maltodextrin in contrast to the more pronounced glycaemic response to maltodextrin observed in vivo. The numerically higher blood glucose response to sucrose after continuous consumption, might indicate an adaptive response. In conclusion, the current work provides valuable insights into the digestion physiology of various saccharides in dogs. Further investigations on related benefits are thus warranted.

Distinct Organotypic Platforms Modulate Rainbow Trout (Oncorhynchus mykiss) Intestinal Cell Differentiation In Vitro.

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.

Features of nutrition in exocrine pancreatic insufficiency and metabolic syndrome

The review presents literature data on the importance of nutritional goals, which include ensuring sufficient intake of macro- and micronutrients, reducing indigestion, the degree of malabsorption, and other risk factors in order to prevent or treat malnutrition associated with exocrine pancreatic insufficiency. Nutritional status and nutritional interventions depend on this. Attention is drawn to the indication of different nutritional options depending on the severity of the course of exocrine insufficiency and the presence of diabetes (10–15% need oral nutritional supplements, 5% — enteral nutrition through a tube, and about 1% — parenteral nutrition). The European Society for Clinical Nutrition and Metabolism (ESPEN) Guidelines for Nutritional Screening are presented. The question of the impact of chronic pancreatitis and exocrine pancreatic insufficiency on digestion with the development of maldigestion and malabsorption and the importance of these factors for the quality of life is discussed. The author reviewed in detail the metabolism of carbohydrates, proteins, and fats in enzyme pancreatic insufficiency and the importance of this knowledge in providing nutritional recommendations. The digestion of lipids in the lumen of the small intestine depends on pancreatic lipase and cofactors such as colipase and bile acids. Lipases of the stomach and salivary glands play a minor role in the digestion of triglycerides and cannot compensate for the insufficient fat digestion. There are no enzyme systems for digesting triglycerides in the brush border membrane. Therefore, lipid digestion is reduced due to insufficient secretion of lipase and a decrease in the concentration of bile acids in the lumen. Attention is paid to research methods that determine the state of fat, lean mass, muscle state, bone health, and resting energy expenditure, provide an opportunity to conduct a detailed assessment of current and usual food consumption, which promotes successful treatment.

The effect of acetaminophen on the structural and functional state of the large intestine and intestinal microflora

Objective. To assess the condition of the microbial-tissue complex of the large intestine when hepatotoxic concentrations of acetaminophen are administrated to the body of animals.Materials and methods. The experiments were performed on 24 white outbred rats weighing 180–220 g, which were divided into three groups. The control group received 2% starch solution enterally, the first experimental group was enterally administered with acetaminophen in starch solution at a dose of 1500 mg per kilogram of body weight, five times a day with one day interval; and the second experimental group was administered with acetaminophen at a dose of 2500 mg per kilogram of body weight the same way. Free amino acids and their nitrogen-containing metabolites were determined by high-performance liquid chromatography in samples of the microbial-tissue complex (MTC) of the large intestine previously frozen at -70 °C. For microbiological study, the aseptically isolated MTC was immediately sent to a microbiological laboratory for identification of the content of the main representatives of the intestinal microflora. Samples of the wall of the ascending colon were subjected to histological and electron microscopic examination. The hepatotoxic effect of acetaminophen was evaluated by registering the activity of enzymes and the content of total bilirubin in blood plasma.Results. Enteral administration of hepatotoxic amounts of acetaminophen to rats increases the concentrations of free amino acids and their nitrogen-containing derivatives in the microbial-tissue complex of the large intestine. At the same time, the concentrations of essential amino acids are significantly increased. Morphological changes in the cells of the epithelium of the large intestine and the structure of mitochondria have been shown.Conclusions. Toxic doses of acetaminophen have a significant impact on the indicators of the microbial-tissue complex of the large intestine. There are changes in biochemical parameters of amino acid metabolism: increased levels of substitutable amino acids and changes in the colonocytes (size and shape of mitochondria, the height of the brush border, the volume of the goblet cells), indicating decreased ability of the cells to use amino acids to support the functioning of the Krebs cycle. This is also confirmed by significantly increased concentrations of essential amino acids, which are mainly used for protein synthesis. The dysbiosis caused by acetaminophen further contributes to the damage to the large intestine. The negative effect of acetaminophen is confirmed by the dose-dependent changes we found in the microbial-tissue complex.

A Novel Peptide Prevents Enterotoxin- and Inflammation-Induced Intestinal Fluid Secretion by Stimulating Sodium-Hydrogen Exchanger 3 Activity

Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine invitro and invivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in invitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine invivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

Rapamycin treatment induces tubular proteinuria: role of megalin-mediated protein reabsorption.

Introduction: Rapamycin is an immunosuppressor that acts by inhibiting the serine/threonine kinase mechanistic target of rapamycin complex 1. Therapeutic use of rapamycin is limited by its adverse effects. Proteinuria is an important marker of kidney damage and a risk factor for kidney diseases progression and has been reported in patients and animal models treated with rapamycin. However, the mechanism underlying proteinuria induced by rapamycin is still an open matter. In this work, we investigated the effects of rapamycin on parameters of renal function and structure and on protein handling by proximal tubule epithelial cells (PTECs). Methods: Healthy BALB/c mice were treated with 1.5mg/kg rapamycin by oral gavage for 1, 3, or 7days. At the end of each treatment, the animals were kept in metabolic cages and renal function and structural parameters were analyzed. LLC-PK1 cell line was used as a model of PTECs to test specific effect of rapamycin. Results: Rapamycin treatment did not change parameters of glomerular structure and function. Conversely, there was a transient increase in 24-h proteinuria, urinary protein to creatinine ratio (UPCr), and albuminuria in the groups treated with rapamycin. In accordance with these findings, rapamycin treatment decreased albumin-fluorescein isothiocyanate uptake in the renal cortex. This effect was associated with reduced brush border expression and impaired subcellular distribution of megalin in PTECs. The effect of rapamycin seems to be specific for albumin endocytosis machinery because it did not modify renal sodium handling or (Na++K+)ATPase activity in BALB/c mice and in the LLC-PK1 cell line. A positive Pearson correlation was found between megalin expression and albumin uptake while an inverse correlation was shown between albumin uptake and UPCr or 24-h proteinuria. Despite its effect on albumin handling in PTECs, rapamycin treatment did not induce tubular injury measured by interstitial space and collagen deposition. Conclusion: These findings suggest that proteinuria induced by rapamycin could have a tubular rather than a glomerular origin. This effect involves a specific change in protein endocytosis machinery. Our results open new perspectives on understanding the undesired effect of proteinuria generated by rapamycin.

Regulatory mechanisms of glucose absorption in the mouse proximal small intestine during fasting and feeding

Fasting is known to alter the function of various organs and the mechanisms of glucose metabolism, which affect health outcomes and slow aging. However, it remains unclear how fasting and feeding affects glucose absorption function in the small intestine. We studied the effects of the fasting and feeding on glucose-induced short-circuit current (Isc) in vitro using an Ussing chamber technique. Glucose-induced Isc by SGLT1 was observed in the ileum, but little or no Isc was observed in the jejunum in ad libitum-fed mice. However, in mice fasted for 24–48 h, in addition to the ileum, robust glucose-induced Isc was observed over time in the jejunum. The expression of SGLT1 in the brush border membranes was significantly decreased in the jejunum under fed conditions compared to 48 h fasting, as analyzed by western blotting. Additionally, when mice were fed a 60% high glucose diet for 3 days, the increase in glucose-induced Isc was observed only in the ileum, and totally suppressed in the jejunum. An increase in Na+ permeability between epithelial cells was concomitantly observed in the jejunum of fasted mice. Transepithelial glucose flux was assessed using a non-metabolizable glucose analog, 14C-methyl α-d-glucopyranoside glucose (MGP). Regardless of whether fed or fasted, no glucose diffusion mechanism was observed. Fasting increased the SGLT1-mediated MGP flux in the jejunum. In conclusion, segment-dependent up- and down-regulation mechanisms during fasting and feeding are important for efficient glucose absorption once the fast is broken. Additionally, these mechanisms may play a crucial role in the small intestine's ability to autoregulate glucose absorption, preventing acute hyperglycemia when large amounts of glucose are ingested.

Specific Blood Plasma Circulating miRs Are Associated with the Physiological Impact of Total Fish Meal Replacement with Soybean Meal in Diets for Rainbow Trout (Oncorhynchus mykiss).

High dietary SBM content is known to induce important physiological alterations, hampering its use as a major FM alternative. Rainbow trout (Oncorhynchus mykiss) juveniles were fed two experimental diets during 9 weeks: (i) a FM diet containing 12% FM; and (ii) a vegetable meal (VM) diet totally devoid of FM and based on SBM (26%). Fish fed the VM diet did not show reduced growth performance when compared with fish fed the FM diet. Nevertheless, fish fed the VM diet had an increased viscerosomatic index, lower apparent fat digestibility, higher aminopeptidase enzyme activity and number of villi fusions, and lower α-amylase enzyme activity and brush border integrity. Small RNA-Seq analysis identified six miRs (omy-miR-730a-5p, omy-miR-135c-5p, omy-miR-93a-3p, omy-miR-152-5p, omy-miR-133a-5p, and omy-miR-196a-3p) with higher expression in blood plasma from fish fed the VM diet. Bioinformatic prediction of target mRNAs identified several overrepresented biological processes known to be associated with high dietary SBM content (e.g., lipid metabolism, epithelial integrity disruption, and bile acid status). The present research work increases our understanding of how SBM dietary content has a physiological impact in farmed fish and suggests circulating miRs might be suitable, integrative, and less invasive biomarkers in fish.

Cytokine Profile and Enzymes of Tubular Nephrothelium in the Urine of Children with Congenital Megaureter

The objective: to determine the concentration of transforming growth factor β1 (TGF-β1), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α), activity levels of tubular nephrothelium enzymes total N-acetyl-β-D-glucosaminidase (NAG), its thermostable NAG isoenzyme B (NAG-B), β-galactosidase (β-Gal), γ-glutamyl transpeptidase (GGT) and neutral α-glucosidase (Nα-Gl) as cytokine-enzymological risk criteria for the development of nephrofibrosis in the urine of children with congenital nonrefluxing megaureter (NRMU) and refluxing megaureter (RMU) with unilateral and bilateral localization of the pathological process. Materials and methods. 174 patients with congenital MU were examined, who were divided into two groups. Group 1 included 131 children with NRMU, group 2 – 43 children with RMU aged from 6 months to 15 years. The control group included 25 practically healthy children. There were 97 patients with unilateral NRMU (group 1a), with bilateral – 34 persons (group 1b); there were 16 patients with unilateral RMU (group 2a), with bilateral – 27 (group 2b). Cytokine and enzyme indicators were studied in the morning portion of urine before reconstructive treatment. The levels of cytokines were determined by the ELISA method. Enzyme activity of urine was determined by the colorimetric method. Statistical processing of data was performed after analyzing the conformity of their distribution to the norm parameters. Results. It was found that the levels of TGF-β1, IL-1β and TNF-α, as well as the activity levels of NAG, NAG-B, β-Gal, GGT and Nα-Gl in the urine of patients with both congenital NRMU and congenital RMU are significantly increased compared to similar indicators in the control group (p&lt;0.001). In patients with congenital RMU, compared to similar average indicators of patients with congenital NRMU, there is a significant increase of all cytokine and enzyme parameters (р&lt;0.05–0.001), which indicates the probability of ischemic-hypoxic, as well as fibrotic damages in the kidney parenchyma in children with MU, especially in patients with RMU. A significant increase in the cytokine levels and enzyme activity levels were established in patients with NRMU and RMU with a unilateral process compared to similar control data (p&lt;0.001. A statistically significant increase of TNF-α, the activity of NAG-B and β-Gal in patients with bilateral NRMU, the concentration of IL-1β, TNF-α and activity levels of NAG, NAG-B, β-Gal, GGT and Nα-Gl in patients with bilateral RMU compared with similar data in groups of patients with unilateral process was found (p&lt;0.05–0.001). Conclusions. Congenital MU (NRMU, RMU) negatively affects the function of the tubular nephrothelium, which is manifested by an increased level of profibrogenic cytokine TGF-β1 and pro-inflammatory cytokines IL-1β and TNF-α in the urine of children, as well as the activity levels of lysosomal enzymes NAG, NAG-B, β-Gal and brush border enzymes GGT and Nα-Gl, which have renal specific properties. This indicates a high probability of ischemic processes in the kidney parenchyma and the risk of nephrofibrosis development in these patients, especially in patients with RMU with bilateral localization of the pathological process.

New perspective into possible mechanism in growth promotion of potassium diformate (KDF) on the juvenile grass carp (Ctenopharyngodon idella)

The present study was mainly designed to evaluate the effect of potassium diformate (KDF) on growth performance, digestion and absorption capacity and intestinal microbes of juvenile grass carp (Ctenopharyngodon idella). Six experimental diets with KDF 0.0, 1.5, 3.0, 4.5, 6.0 and 7.5 g/kg were respectively fed to 1620 fish (5.73 ± 0.01 g) for 70 days. Results indicated that dietary KDF addition improved growth performance, intestinal growth and development, digestive enzymes and brush border enzymes activity. Additionally, mRNA levels of most amino acid transporters (AATs) and peptide transporter 1 (PepT1), and protein expressions of SLC7A1, SLC1A5 and SLC15A1 in intestine were up-regulated with addition of 3.0 g/kg KDF, which might be related to the activated TOR signaling pathway, as evident by the up-regulated mRNA levels of TOR and S6K1, the increased protein expression of phosphorylation TORSer2448, as well as the down-regulated mRNA level of 4EBP1. Furthermore, the 16S rRNA sequencing revealed that low dose of KDF addition enriched the abundances of Fusobacteria and Cetobacterium, and reduced the abundances of Proteobacteria and Ralstonia, which might be related to the reduced pH of intestine. In a word, this study using grass carp as a model provided novel evidence on the positive role and possible mechanisms of KDF in promoting fish growth.

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment.

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.

Dietary glutamine enhances growth performance and gut integrity of Totoaba macdonaldi juveniles fed low fishmeal diets but has limited synergetic effects in combination with a prebiotic

Fishmeal (FM) replacement using cost-effective blends of complementary ingredients is commonly used in carnivorous marine fish diets for economic and sustainable purposes. The use of functional compounds such as prebiotics or functional ingredients is a nutritional strategy in low FM diets to maximize growth and feed utilization, as well as reducing enteropathies caused by plant proteins, in particular soybean meal (SBM) that can induce enteritis in Totoaba macdonaldi juveniles. Therefore, this study was conducted to evaluate the effects of glutamine (Gln) and agavin (AGA) as a novel prebiotic or a combination of both on growth parameters, digestive capacity, whole-body proximate composition, gut health, and sustainable and economic indexes in totoaba juvenile fed low FM (17%) diet for 56 days. Juvenile totoaba with an initial weight of 168 ± 4 g were fed one of four isoproteic and isolipidic diets formulated with poultry by-product meal and FM in a 2:1 ratio and 24% of SBM. For the control basal diet (SBM), no Gln or AGA was added. The other three experimental diets consisted of the basal diet with either 1.5% glutamine (GLN), 1% prebiotic agavin (AGA), or 1.5% glutamine and 1% agavin (Gln + AGA) supplementation. Fish fed the Gln diet significantly improved growth, feed utilization and increased trypsin, total alkaline proteases, and L-aminopeptidase activities. Additionally, an enhanced brush border height (BBH) in the distal intestine was observed in the Gln group, suggesting improved intestinal health compared to the rest of the treatments. However, no significant synergistic effect was observed in fed fish the Gln + AGA diet. Interestingly, no signs of enteritis among the fish fed the four experimental diets was observed. These results suggest that fish with an initial size of 168 g are less susceptible to the adverse effects of SBM compared to previous studies. The inclusion of 1.5% dietary glutamine is recommended in totoaba diets with low FM containing 24% of SBM. Since the fish fed the diet supplemented with glutamine + agavin resulted in intermediate values for the parameters evaluated, it is recommended to evaluate different levels of agavin in combination with glutamine or perform longer bioassays using immunological or environmental challenges to aid in elucidating potential benefits of these functional compounds and their synergistic effects.

Maternal acrylamide exposure changes intestinal epithelium, immunolocalization of leptin and ghrelin and their receptors, and gut barrier in weaned offspring

Acrylamide (ACR) is an amide formed as a byproduct in many heat-processed starchy-rich foods. In utero ACR exposure has been associated with restricted fetal growth, but its effects of postnatal functional development of small intestine is completely unknown. The current study investigated the time- and segment-dependent effects of prenatal ACR exposure on morphological and functional development of small intestine in weaned rat offspring. Four groups of pregnant female Wistar rats were exposed to ACR (3 mg/kg b.w./day) for 0, 5, 10 and 15 days during pregnancy. Basal intestinal morphology, immunolocalization of gut hormones responsible for food intake and proteins of intestinal barrier, activity of the intestinal brush border disaccharidases, apoptosis and proliferation in intestinal mucosa were analyzed in offspring at weaning (postnatal day 21). The results showed that in utero ACR exposure disturbs offspring gut structural and functional postnatal development in a time- and segment-depended manner and even a short prenatal exposure to ACR resulted in changes in intestinal morphology, immunolocalization of leptin and ghrelin and their receptors, barrier function, activity of gut enzymes and upregulation of apoptosis and proliferation. In conclusion, prenatal ACR exposure disturbed the proper postnatal development of small intestine.