How renal toxins respond to renal function deterioration and oral toxic adsorbent in pH-controlled releasing capsule.

The number of patients with chronic kidney disease (CKD) is increasing. Oral toxin adsorbents may provide some value. Several uremic toxins, including indoxyl sulfate (IS), p-cresol (PCS), acrolein, per- and poly-fluoroalkyl substances (PFAS), and inflammation markers (interleukin 6 [IL-6] and tumor necrosis factor [TNF]-alpha) have been shown to be related to CKD progression. A total of 81 patients taking oral activated charcoal toxin adsorbents (AC-134), which were embedded in capsules that dissolved in the terminal ileum, three times a day for 1 month, were recruited. The renal function, hemoglobulin (Hb), inflammation markers, three PFAS (PFOA, PFOS, and PFNA), and acrolein were quantified. Compared with the baseline, an improved glomerular filtration rate (GFR) and significantly lower acrolein were noted. Furthermore, the CKD stage 4 and 5 group had significantly higher concentrations of IS, PCS, IL-6, and TNF but lower levels of Hb and PFAS compared with the CKD Stage 3 group at baseline and after the intervention. Hb was increased only in the CKD Stage 3 group after the trial (p = .032). Acrolein did not differ between the different CKD stage groups. Patients with improved GFR (responders) (about 77%) and nonresponders had similar baseline GFR. Responders had higher acrolein and PFOA levels throughout the study and a more significant reduction in acrolein, indicating a better digestion function. Both the higher PFOA and lower acrolein may be related to improved eGFR (and possibly to improvements in proteinuria, which we did not measure. Proteinuria is associated with PFAS loss in the urine), AC-134 showed the potential to improve the GFR and decrease acrolein, which might better indicate renal function change. Future studies are needed with longer follow-ups.

P-cresol and Indoxyl Sulfate Impair Osteogenic Differentiation by Triggering Mesenchymal Stem Cell Senescence.

Chronic kidney disease (CKD) patients obtained high levels of uremic toxins progressively develop several complications including bone fractures. Protein-bound uremic toxins especially p-cresol and indoxyl sulfate are hardly eliminated due to their high molecular weight. Thus, the abnormality of bone in CKD patient could be potentially resulted from the accumulation of uremic toxins. To determine whether protein-bound uremic toxins have an impact on osteogenesis, mesenchymal stem cells were treated with either p-cresol or indoxyl sulfate under in vitro osteogenic differentiation. The effects of uremic toxins on MSC-osteoblastic differentiation were investigated by evaluation of bone phenotype. The results demonstrated that p-cresol and indoxyl sulfate down-regulated the transcriptional level of collagen type I, deceased alkaline phosphatase activity, and impaired mineralization of MSC-osteoblastic cells. Furthermore, p-cresol and indoxyl sulfate gradually increased senescence-associated beta-galactosidase positive cells while upregulated the expression of p21 which participate in senescent process. Our findings clearly revealed that the presence of uremic toxins dose-dependently influenced a gradual deterioration of osteogenesis. The effects partially mediate through the activation of senescence-associated gene lead to the impairment of osteogenesis. Therefore, the management of cellular senescence triggered by uremic toxins could be considered as an alternative therapeutic approach to prevent bone abnormality in CKD patients.

Toxic Effects of Indoxyl Sulfate on Osteoclastogenesis and Osteoblastogenesis.

Uremic toxins, such as indoxyl sulfate (IS) and kynurenine, accumulate in the blood in the event of kidney failure and contribute to further bone damage. To maintain the homeostasis of the skeletal system, bone remodeling is a persistent process of bone formation and bone resorption that depends on a dynamic balance of osteoblasts and osteoclasts. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates the toxic effects of uremic toxins. IS is an endogenous AhR ligand and is metabolized from tryptophan. In osteoclastogenesis, IS affects the expression of the osteoclast precursor nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) through AhR signaling. It is possible to increase osteoclast differentiation with short-term and low-dose IS exposure and to decrease differentiation with long-term and/or high-dose IS exposure. Coincidentally, during osteoblastogenesis, through the AhR signaling pathway, IS inhibits the phosphorylation of ERK, and p38 reduces the expression of the transcription factor 2 (Runx2), disturbing osteoblastogenesis. The AhR antagonist resveratrol has a protective effect on the IS/AhR pathway. Therefore, it is necessary to understand the multifaceted role of AhR in CKD, as knowledge of these transcription signals could provide a safe and effective method to prevent and treat CKD mineral bone disease.

Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery.

OBJECTIVE: Due to the global increase in obesity rates and success of bariatric surgery in weight reduction, an increasing number of women now present pregnant with a previous bariatric procedure. This study investigates the extent of bariatric-associated metabolic and gut microbial alterations during pregnancy and their impact on fetal development. DESIGN: A parallel metabonomic (molecular phenotyping based on proton nuclear magnetic resonance spectroscopy) and gut bacterial (16S ribosomal RNA gene amplicon sequencing) profiling approach was used to determine maternal longitudinal phenotypes associated with malabsorptive/mixed (n=25) or restrictive (n=16) procedures, compared with women with similar early pregnancy body mass index but without bariatric surgery (n=70). Metabolic profiles of offspring at birth were also analysed. RESULTS: Previous malabsorptive, but not restrictive, procedures induced significant changes in maternal metabolic pathways involving branched-chain and aromatic amino acids with decreased circulation of leucine, isoleucine and isobutyrate, increased excretion of microbial-associated metabolites of protein putrefaction (phenylacetlyglutamine, p-cresol sulfate, indoxyl sulfate and p-hydroxyphenylacetate), and a shift in the gut microbiota. The urinary concentration of phenylacetylglutamine was significantly elevated in malabsorptive patients relative to controls (p=0.001) and was also elevated in urine of neonates born from these mothers (p=0.021). Furthermore, the maternal metabolic changes induced by malabsorptive surgery were associated with reduced maternal insulin resistance and fetal/birth weight. CONCLUSION: Metabolism is altered in pregnant women with a previous malabsorptive bariatric surgery. These alterations may be beneficial for maternal outcomes, but the effect of elevated levels of phenolic and indolic compounds on fetal and infant health should be investigated further.

Bowel Habits and the Association With Uremic Toxins in Non-Dialysis-Dependent Chronic Kidney Disease Patients.

OBJECTIVE: The aim of this study is to evaluate the association between bowel habits and microbial-derived uremic toxins p-cresyl sulfate (PCS) and indoxyl sulfate (IS) in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD). DESIGN AND METHODS: This is a cross-sectional analysis including 43 nondiabetic NDD-CKD patients (58% men; 59.0 ± 13.5 years; estimated glomerular filtration rate, 21.3 ± 7.9 mL/min/1.73 m2). Bowel habit was assessed by the Bristol Stool Scale (BSS <3, characterized by hard consistency of stools and/or low frequency of evacuation and BSS ≥3, representing a more regular bowel habit) and by the Rome III criteria. PCS and IS (serum, free and total; urinary, total) were determined by high-performance liquid chromatography. Dietary intake was assessed by the 3-day food records. RESULTS: The frequency of constipation assessed by BSS and Rome III criteria was 33% (n = 14/43) and 35% (n = 15/43), respectively. The BSS <3 exhibited higher PCS, independent of renal function and dietary protein-fiber ratio (ß [95% confidence interval {CI}]: serum, total PCS = 1.54 [1.06-2.23], P = .02; serum free PCS = 1.40 [1.00-1.97], P = .05; urinary PCS = 1.78 [1.10-2.90], P < .02). According to the Rome III criteria, a tendency for a higher serum total PCS (ß [95% CI]: 1.39 [0.95-2.03 µmol/L], P = .09) and a significantly higher urinary PCS (ß [95% CI]: 1.80 [1.11-2.94 µmol/24 h], P = .02) was found in constipated participants. No effect of a compromised bowel habit (Rome III criteria or BSS) was found on IS. CONCLUSION: Constipation may lead to production of PCS in nondiabetic NDD-CKD patients.

Serum Levels and Removal by Haemodialysis and Haemodiafiltration of Tryptophan-Derived Uremic Toxins in ESKD Patients.

Tryptophan is an essential dietary amino acid that originates uremic toxins that contribute to end-stage kidney disease (ESKD) patient outcomes. We evaluated serum levels and removal during haemodialysis and haemodiafiltration of tryptophan and tryptophan-derived uremic toxins, indoxyl sulfate (IS) and indole acetic acid (IAA), in ESKD patients in different dialysis treatment settings. This prospective multicentre study in four European dialysis centres enrolled 78 patients with ESKD. Blood and spent dialysate samples obtained during dialysis were analysed with high-performance liquid chromatography to assess uremic solutes, their reduction ratio (RR) and total removed solute (TRS). Mean free serum tryptophan and IS concentrations increased, and concentration of IAA decreased over pre-dialysis levels (67%, 49%, -0.8%, respectively) during the first hour of dialysis. While mean serum total urea, IS and IAA concentrations decreased during dialysis (-72%, -39%, -43%, respectively), serum tryptophan levels increased, resulting in negative RR (-8%) towards the end of the dialysis session (p < 0.001), despite remarkable Trp losses in dialysate. RR and TRS values based on serum (total, free) and dialysate solute concentrations were lower for conventional low-flux dialysis (p < 0.001). High-efficiency haemodiafiltration resulted in 80% higher Trp losses than conventional low-flux dialysis, despite similar neutral Trp RR values. In conclusion, serum Trp concentrations and RR behave differently from uremic solutes IS, IAA and urea and Trp RR did not reflect dialysis Trp losses. Conventional low-flux dialysis may not adequately clear Trp-related uremic toxins while high efficiency haemodiafiltration increased Trp losses.

Specific Urinary Metabolites in Malignant Melanoma.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

Generation, clearance, toxicity, and monitoring possibilities of unaccounted uremic toxins for improved dialysis prescriptions.

Current dialysis-dosing calculations provide an incomplete assessment of blood purification. They exclude clearances of protein-bound uremic toxins (PB-UTs), such as polyamines, p-cresol sulfate, and indoxyl sulfate, relying solely on the clearance of urea as a surrogate for all molecules accumulating in patients with end-stage renal disease (ESRD). PB-UTs clear differently in dialysis but also during normal renal function. The kidney clears PB toxins via the process of secretion, whereas it clears urea through filtration. Herein, we review the clearance, accumulation, and toxicity of various UTs. We also suggest possible methods for their monitoring toward the ultimate goal of a more comprehensive dialysis prescription. A more inclusive dialysis prescription would retain the kidney-filtration surrogate, urea, and consider at least one PB toxin as a surrogate for UTs cleared through cellular secretion. A more comprehensive assessment of UTs that includes both secretion and filtration is expected to result in a better understanding of ESRD toxicity and consequently, to reduce ESRD mortality.

Uremic Toxins Activates Na/K-ATPase Oxidant Amplification Loop Causing Phenotypic Changes in Adipocytes in In Vitro Models.

BACKGROUND: Oxidant stress plays a key role in the development of chronic kidney disease (CKD). Experimental CKD leads to accumulation of uremic toxins (UT) in the circulation resulting in increased ROS production, which in turn, is known to activate the Na/K-ATPase/ROS amplification loop. Studies in a murine model of obesity have shown that increased oxidative stress in plasma is due to increased ROS and cytokine production from dysfunctional adipocytes. Therefore, we hypothesized that adipocytes exposed to UTs will activate the Na/K-ATPase oxidant amplification loop causing redox imbalance and phenotypic alterations in adipocytes. We also aimed to demonstrate that the Na/K-ATPase signaling antagonist, pNaKtide, attenuates these pathophysiological consequences. METHODS: In the first set of experiments, 3T3-L1 murine pre-adipocytes were treated with varying concentrations of UTs, indoxyl sulfate (IS) (50, 100 and 250 µM) and p-cresol (50, 100 and 200 µM), with or without pNaKtide (0.7 µM) for five days in adipogenic media, followed by Oil Red O staining to study adipogenesis. RT-PCR analysis was performed to study expression of adipogenic, apoptotic and inflammatory markers, while DHE staining evaluated the superoxide levels in UT treated cells. In a second set of experiments, visceral fat was obtained from the West Virginian population. MSCs were isolated and cultured in adipogenic media for 14 days, which was treated with indoxyl sulfate (0, 25, 50 and 100 µM) with or without pNaKtide (1 µM). MSC-derived adipocytes were evaluated for morphological and molecular analysis of the above markers. RESULTS: Our results demonstrated that 3T3-L1 cells and MSCs-derived adipocytes, treated with UTs, exhibited a significant decrease in adipogenesis and apoptosis through activation of the Na/K-ATPase/ROS amplification loop. The treatment with pNaKtide in 3T3-L1 cells and MSC-derived adipocytes negated the effects of UTs and restored cellular redox in adipocytes. We noted a varying effect of pNaKtide, in adipocytes treated with UTs, on inflammatory markers, adipogenic marker and superoxide levels in 3T3-L1 cells and MSC-derived adipocytes. CONCLUSIONS: This study demonstrates for the first time that the Na/K-ATPase/ROS amplification loop activated by elevated levels of UTs has varying effect on phenotypic alterations in adipocytes in various in vitro models. Thus, we propose that, if proven in humans, inhibition of Na/K-ATPase amplification of oxidant stress in CKD patients may ultimately be a novel way to combat adipocyte dysfunction and metabolic imbalance in these patients.

How's your microbiota? Let's check your urine.

In this issue of Blood, Weber and colleagues demonstrate that in the first 10 days following allogeneic hematopoietic transplantation, urinary 3-indoxyl sulfate is a biomarker of intestinal microbiota health and predicts reduced intestinal graft-versus-host disease (GVHD) and treatment-related mortality, as well as improved overall survival.

Low urinary indoxyl sulfate levels early after transplantation reflect a disrupted microbiome and are associated with poor outcome.

Indole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation.

Alterations of urinary metabolite profile in model diabetic nephropathy.

Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology.

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 µmol/L to 11.8 ± 2.8 µmol/L in all post-transplant samples and to 3.5 ± 3 µmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

Cardiorenal syndrome: the emerging role of protein-bound uremic toxins.

Cardiorenal syndrome is a condition in which a complex interrelationship between cardiac dysfunction and renal dysfunction exists. Despite advances in treatment of both cardiovascular and kidney disease, cardiorenal syndrome remains a major global health problem. Characteristic of the pathophysiology of cardiorenal syndrome is bidirectional cross-talk; mediators/substances activated by the disease state of 1 organ can play a role in worsening dysfunction of the other by exerting their biologically harmful effects, leading to the progression of the syndrome. Accumulation of uremic toxins is a hallmark of renal excretory dysfunction. Removal of some toxins by conventional dialysis is particularly problematic because of their high protein binding. In this review, we demonstrate that protein-bound uremic toxins may play an important role in progression of cardiovascular disease in the setting of chronic kidney disease. The highly protein-bound uremic toxin indoxyl sulfate has emerged as a potent toxin adversely affecting both the kidney and heart. Direct cardiac effects of this toxin have been recently demonstrated both in vitro and in vivo. Specifically, potent fibrogenic and prohypertrophic effects, as well as oxidative stress-inducing effects, appear to play a central role in both renal and cardiac pathology. Many of these adverse effects can be suppressed by use of a gut adsorbent, AST-120. Potential mechanisms underlying indoxyl sulfate-induced cardiorenal fibrosis are discussed. Future research and clinical implications conclude this review.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni.

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

Metabonomics evaluation of urine from rats administered with phorate under long-term and low-level exposure by ultra-performance liquid chromatography-mass spectrometry.

The purpose of this study was to investigate the toxic effect of long-term and low-level exposure to phorate using a metabonomics approach based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Male Wistar rats were given phorate daily in drinking water at low doses of 0.05, 0.15 or 0.45 mg kg⁻¹ body weight (BW) for 24 weeks consecutively. Rats in the control group were given an equivalent volume of drinking water. Compared with the control group, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), urea nitrogen (BUN) and creatinine (CR) were increased in the middle- and high-dose groups whereas albumin (ALB) and cholinesterase (CHE) were decreased. Urine metabonomics profiles were analyzed by UPLC-MS. Compared with the control group, 12 metabolites were significantly changed in phorate-treated groups. In the negative mode, metabolite intensities of uric acid, suberic acid and citric acid were significantly decreased in the middle- and high-dose groups, whereas indoxyl sulfic acid (indican) and cholic acid were increased. In the positive mode, uric acid, creatinine, kynurenic acid and xanthurenic acid were significantly decreased in the middle- and high-dose groups, but 7-methylguanine (N7G) was increased. In both negative and positive modes, diethylthiophosphate (DETP) was significantly increased, which was considered as a biomarker of exposure to phorate. In conclusion, long-term and low-level exposure to phorate can cause disturbances in energy-related metabolism, liver and kidney function, the antioxidant system, and DNA damage. Moreover, more information can be provided on the evaluation of toxicity of phorate using metabonomics combined with clinical chemistry.

Indigofera cryptantha-induced pigmenturia in cattle in South Africa.

Two field cases of reddish-black pigmenturia occurred where cattle grazed on an established Cenchrus ciliaris (blue buffalo grass) pasture in South Africa. The pasture was noticeably invaded by Indigofera cryptantha, which was heavily grazed. Apart from the discolored urine, no other clinical abnormalities were detected. Urinalysis revealed hemoglobinuria, proteinuria and an alkaline pH. When the animals were immediately removed from the infested pasture, they made an uneventful recovery. However, a bull died when one of the herds could not be removed from the I. cryptantha-infested pasture. Macroscopically, the kidneys were dark red in color and the urinary bladder contained the dark pigmented urine. Microscopically, the renal tubules contained eosinophilic, granular pigment casts in the lumen. In addition, many renal tubular epithelial cells were attenuated with granular cytoplasm and were detached from the basement membranes. Chemical analysis was performed on dried, milled plant material and two urine samples collected during the field investigations. Qualitative UPLC-UV-qTOF/MS analysis revealed the presence of indican (indoxyl-ß-glucoside) in the stems, leaves and pods of I. cryptantha and indoxyl sulfate was identified, and confirmed with an analytical standard, in the urine samples. It is proposed that following ingestion of I. cryptantha, indican will be hydrolysed in the liver to indoxyl and conjugated with sulfate. Indoxyl sulfate will then be excreted in relatively high concentrations in the urine. In the alkaline urine, two indoxyl molecules might dimerize to form leucoindigo with subsequent oxidation to indigo, thus, contributing to the dark pigmentation of the urine. It is also possible that indoxyl sulfate contributed to the renal failure and death of the bull. Although I. suffruticosa-induced hemoglobinuria has been described in Brazil, this is the first report of I. cryptantha-induced pigmenturia in cattle in South Africa.

Intrarenal metabolomic investigation of chronic kidney disease and its TGF-ß1 mechanism in induced-adenine rats using UPLC Q-TOF/HSMS/MS(E).

Chronic kidney disease (CKD) is becoming a worldwide public health problem. In this study, a kidney metabonomics method based on the ultra performance liquid chromatography/high-sensitivity mass spectrometry with MS(E) data collection technique was undertaken to explore the excretion pattern of low molecular mass metabolites in rat model of adenine-induced chronic renal failure (CRF). Coupled with blood biochemistry and kidney histopathology results, the significant difference in metabolic profiling between the adenine-induced CRF group and the control group by using pattern recognition analysis indicated that changes in global tissue metabolites were occurred. Some significantly changed metabolites like fatty acids, p-cresol sulfate, and indoxyl sulfate have been identified. The results showed that the most important CRF-related metabolites were polyunsaturated fatty acids, indoxyl sulfate, and p-cresyl sulfate. Indoxyl sulfate and p-cresyl sulfate (uremic toxins) were significantly increased in CRF rats. Indoxyl sulfate and p-cresyl sulfate stimulate progressive tubulointerstitial fibrosis by increasing the expression of transforming growth factor-ß1 (TGF-ß1). These biochemical changes in tissue metabolites are related to the perturbations of fatty acid metabolism and amino metabolism, which may be helpful to further understand the TGF-ß1 mechanisms of tubulointerstitial fibrosis. This work shows that the metabonomics method is a valuable tool for studying the essence of CKD.