Route of intestinal absorption and tissue distribution of iron contained in the novel phosphate binder ferric citrate.

BACKGROUND: Anemia of chronic kidney disease (CKD) is, in part, caused by hepcidin-mediated impaired iron absorption. However, phosphate binder, ferric citrate (FC) overcomes the CKD-induced impairment of iron absorption and increases serum iron, transferrin saturation, and iron stores and reduces erythropoietin requirements in CKD/ESRD patients. The mechanism and sites of intestinal absorption of iron contained in FC were explored here. METHODS: Eight-week old rats were randomized to sham-operated or 5/6 nephrectomized (CKD) groups and fed either regular rat chow or rat chow containing 4% FC for 6 weeks. They were then euthanized, and tissues were processed for histological and biochemical analysis using Prussian blue staining, Western blot analysis to quantify intestinal epithelial tight junction proteins and real-time PCR to measure Fatty Acid receptors 2 (FFA2) and 3 (FFA3) expressions. RESULTS: CKD rats exhibited hypertension, anemia, azotemia, and hyperphosphatemia. FC-treated CKD rats showed significant reductions in blood pressure, serum urea, phosphate and creatinine levels and higher serum iron and blood hemoglobin levels. This was associated with marked increase in iron content of the epithelial and subepithelial wall of the descending colon and modest iron deposits in the proximal tubular epithelial cells of their remnant kidneys. No significant difference was found in hepatic tissue iron content between untreated and FC-treated CKD or control groups. Distal colon's epithelial tight Junction proteins, Occludin, JAM-1 and ZO-1 were markedly reduced in the CKD groups. The FFA2 expression in the jejunum and FFA3 expression in the distal colon were significantly reduced in the CKD rats and markedly increased with FC administration. CONCLUSION: Iron contained in the phosphate binder, FC, is absorbed by the distal colon of the CKD animals via disrupted colonic epithelial barrier and upregulation of short chain fatty acid transporters.

Phosphate Binder, Ferric Citrate, Attenuates Anemia, Renal Dysfunction, Oxidative Stress, Inflammation, and Fibrosis in 5/6 Nephrectomized CKD Rats.

Chronic kidney disease (CKD) causes anemia and impairs intestinal iron absorption. However, use of the phosphate binder ferric citrate (FC) increases body iron stores and hemoglobin levels in CKD patients. By intensifying oxidative stress and inflammation iron overload resulting from excessive use of intravenous iron can accelerate CKD progression. The present study explored the route of absorption and tissue distribution of iron with FC administration and its effect on renal function, histology, and inflammatory, oxidative, and fibrosis pathways in CKD rats. Male Sprague Dawley rats were randomized to sham-operated control (CTL) group and 5/6 nephrectomized (CKD) groups fed either regular or 4% FC-supplemented diets for 6 weeks. Animals were then sacrificed, and blood and target tissues were harvested and processed. The untreated CKD rats exhibited anemia, hypertension, upregulation of renal tissue inflammatory, oxidative, and fibrotic pathways, impaired nuclear translocation, and downregulation of Nrf2's target gene products and depletion of colonic epithelial tight junction proteins. FC administration raised serum iron, improved anemia, attenuated hyperphosphatemia, partially improved renal function, reduced oxidative stress, inflammation, and fibrosis, and restored colonic epithelial zonula occludens-1 protein abundance. Tissue iron staining detected presence of iron in epithelial cells and subepithelium of colon and in renal proximal tubules. In conclusion ferric citrate administration resulted in modest amelioration of renal function and histology and partial improvements of fibrosis, inflammation, and oxidative stress in the kidney tissues of CKD rats.

The Phosphate Binder Ferric Citrate Alters the Gut Microbiome in Rats with Chronic Kidney Disease.

In chronic kidney disease (CKD), the gut microbiome is altered and bacterial-derived uremic toxins promote systemic inflammation and cardiovascular disease. Ferric citrate complex is a dietary phosphate binder prescribed for patients with end-stage kidney disease to treat hyperphosphatemia and secondary hyperparathyroidism. Iron is an essential nutrient in both microbes and mammals. This study was undertaken to test the hypothesis that the large iron load administered with ferric citrate in CKD may significantly change the gut microbiome. Male Sprague-Dawley rats underwent 5/6 nephrectomy to induce CKD. Normal control and CKD rats were randomized to regular chow or a 4% ferric citrate diet for 6 weeks. Fecal and cecal microbial DNA was analyzed via 16S ribosomal RNA gene sequencing on the Illumina MiSeq system. CKD rats had lower abundances of Firmicutes and Lactobacillus compared with normal rats and had lower overall gut microbial diversity. CKD rats treated with ferric citrate had improved hemoglobin and creatinine clearance and amelioration of hyperphosphatemia and hypertension. Ferric citrate treatment increased bacterial diversity in CKD rats almost to levels observed in control rats. The tryptophanase-possessing families Verrucomicrobia, Clostridiaceae, and Enterobacteriaceae were increased by ferric citrate treatment. The uremic toxins indoxyl sulfate and p-cresyl sulfate were not increased with ferric citrate treatment. Verrucomicrobia was largely represented by Akkermansia muciniphila, which has important roles in mucin degradation and gut barrier integrity. In summary, ferric citrate therapy in CKD rats was associated with significant changes in the gut microbiome and beneficial kidney and blood pressure parameters.

Tetrahydrocurcumin Add-On therapy to losartan in a rat model of diabetic nephropathy decreases blood pressure and markers of kidney injury.

Tetrahydrocurcumin (THC), a principal metabolite of curcumin, was tested in a rat model of type 2 diabetes mellitus. THC was administered via daily oral gavage with the lipid carrier polyenylphosphatidylcholine (PPC) as add-on therapy to losartan (angiotensin receptor blocker) to examine effects on kidney oxidative stress and fibrosis. A combination of unilateral nephrectomy, high-fat diet and low-dose streptozotocin was used to induce diabetic nephropathy in male Sprague-Dawley rats. Animals with fasting blood glucose >200 mg/dL were randomized to PPC, losartan, THC + PPC or THC + PPC + losartan. Untreated chronic kidney disease (CKD) animals had proteinuria, decreased creatinine clearance, and evidence of kidney fibrosis on histology. THC + PPC + losartan treatment significantly lowered blood pressure concurrent with increased messenger RNA levels of antioxidant copper-zinc-superoxide dismutase and decreased protein kinase C-α, kidney injury molecule-1 and type I collagen in the kidneys; there was decreased albuminuria and a trend for increased creatinine clearance compared to untreated CKD rats. There was decreased fibrosis on kidney histology in PPC-only and THC-treated CKD rats. Plasma levels of kidney injury molecule-1 were decreased in THC + PPC + losartan animals. In summary, add-on THC to losartan therapy improved antioxidant levels and decreased fibrosis in the kidneys, and lowered blood pressure in diabetic CKD rats.

Insights Into the Mechanisms of Brain Endothelial Erythrophagocytosis.

The endothelial cells which form the inner cellular lining of the vasculature can act as non-professional phagocytes to ingest and remove emboli and aged/injured red blood cells (RBCs) from circulation. We previously demonstrated an erythrophagocytic phenotype of the brain endothelium for oxidatively stressed RBCs with subsequent migration of iron-rich RBCs and RBC degradation products across the brain endothelium in vivo and in vitro, in the absence of brain endothelium disruption. However, the mechanisms contributing to brain endothelial erythrophagocytosis are not well defined, and herein we elucidate the cellular mechanisms underlying brain endothelial erythrophagocytosis. Murine brain microvascular endothelial cells (bEnd.3 cells) were incubated with tert-butyl hydroperoxide (tBHP, oxidative stressor to induce RBC aging in vitro)- or PBS (control)-treated mouse RBCs. tBHP increased the reactive oxygen species (ROS) formation and phosphatidylserine exposure in RBCs, which were associated with robust brain endothelial erythrophagocytosis. TNFα treatment potentiated the brain endothelial erythrophagocytosis of tBHP-RBCs in vitro. Brain endothelial erythrophagocytosis was significantly reduced by RBC phosphatidylserine cloaking with annexin-V and with RBC-ROS and phosphatidylserine reduction with vitamin C. Brain endothelial erythrophagocytosis did not alter the bEnd.3 viability, and tBHP-RBCs were localized with early and late endosomes. Brain endothelial erythrophagocytosis increased the bEnd.3 total iron pool, abluminal iron levels without causing brain endothelial monolayer disruption, and ferroportin levels. In vivo, intravenous tBHP-RBC injection in aged (17-18 months old) male C57BL/6 mice significantly increased the Prussian blue-positive iron-rich lesion load compared with PBS-RBC-injected mice. In conclusion, RBC phosphatidylserine exposure and ROS are key mediators of brain endothelial erythrophagocytosis, a process which is associated with increased abluminal iron in vitro. tBHP-RBCs result in Prussian blue-positive iron-rich lesions in vivo. Brain endothelial erythrophagocytosis may provide a new route for RBC/RBC degradation product entry into the brain to produce iron-rich cerebral microhemorrhage-like lesions.

Spectroscopic and deep learning-based approaches to identify and quantify cerebral microhemorrhages.

Cerebral microhemorrhages (CMHs) are associated with cerebrovascular disease, cognitive impairment, and normal aging. One method to study CMHs is to analyze histological sections (5-40 µm) stained with Prussian blue. Currently, users manually and subjectively identify and quantify Prussian blue-stained regions of interest, which is prone to inter-individual variability and can lead to significant delays in data analysis. To improve this labor-intensive process, we developed and compared three digital pathology approaches to identify and quantify CMHs from Prussian blue-stained brain sections: (1) ratiometric analysis of RGB pixel values, (2) phasor analysis of RGB images, and (3) deep learning using a mask region-based convolutional neural network. We applied these approaches to a preclinical mouse model of inflammation-induced CMHs. One-hundred CMHs were imaged using a 20 × objective and RGB color camera. To determine the ground truth, four users independently annotated Prussian blue-labeled CMHs. The deep learning and ratiometric approaches performed better than the phasor analysis approach compared to the ground truth. The deep learning approach had the most precision of the three methods. The ratiometric approach has the most versatility and maintained accuracy, albeit with less precision. Our data suggest that implementing these methods to analyze CMH images can drastically increase the processing speed while maintaining precision and accuracy.

Rapamycin treatment correlates changes in primary cilia expression with cell cycle regulation in epithelial cells.

Primary cilia are sensory organelles that regulate cell cycle and signaling pathways. In addition to its association with cancer, dysfunction of primary cilia is responsible for the pathogenesis of polycystic kidney disease (PKD) and other ciliopathies. Because the association between cilia formation or length and cell cycle or division is poorly understood, we here evaluated their correlation in this study. Using Spectral Karyotyping (SKY) technique, we showed that PKD and the cancer/tumorigenic epithelial cells PC3, DU145, and NL20-TA were associated with abnormal ploidy. We also showed that PKD and the cancer epithelia were highly proliferative. Importantly, the cancer epithelial cells had a reduction in the presence and/or length of primary cilia relative to the normal kidney (NK) cells. We then used rapamycin to restore the expression and length of primary cilia in these cells. Our subsequent analyses indicated that both the presence and length of primary cilia were inversely correlated with cell proliferation. Collectively, our data suggest that restoring the presence and/or length of primary cilia may serve as a novel approach to inhibit cancer cell proliferation.

Chronic Kidney Disease Increases Cerebral Microbleeds in Mouse and Man.

Brain microbleeds are increased in chronic kidney disease (CKD) and their presence increases risk of cognitive decline and stroke. We examined the interaction between CKD and brain microhemorrhages (the neuropathological substrate of microbleeds) in mouse and cell culture models and studied progression of microbleed burden on serial brain imaging from humans. Mouse studies: Two CKD models were investigated: adenine-induced tubulointerstitial nephritis and surgical 5/6 nephrectomy. Cell culture studies: bEnd.3 mouse brain endothelial cells were grown to confluence, and monolayer integrity was measured after exposure to 5-15% human uremic serum or increasing concentrations of urea. Human studies: Progression of brain microbleeds was evaluated on serial MRI from control, pre-dialysis CKD, and dialysis patients. Microhemorrhages were increased 2-2.5-fold in mice with CKD independent of higher blood pressure in the 5/6 nephrectomy model. IgG staining was increased in CKD animals, consistent with increased blood-brain barrier permeability. Incubation of bEnd.3 cells with uremic serum or elevated urea produced a dose-dependent drop in trans-endothelial electrical resistance. Elevated urea induced actin cytoskeleton derangements and decreased claudin-5 expression. In human subjects, prevalence of microbleeds was 50% in both CKD cohorts compared with 10% in age-matched controls. More patients in the dialysis cohort had increased microbleeds on follow-up MRI after 1.5 years. CKD disrupts the blood-brain barrier and increases brain microhemorrhages in mice and microbleeds in humans. Elevated urea alters the actin cytoskeleton and tight junction proteins in cultured endothelial cells, suggesting that these mechanisms explain (at least in part) the microhemorrhages and microbleeds observed in the animal and human studies.

Ferric Citrate Attenuates Cardiac Hypertrophy and Fibrosis in a Rat Model of Chronic Kidney Disease.

INTRODUCTION: Chronic kidney disease (CKD) promotes hypertrophy and fibrosis in heart, and increases the risk of cardiovascular mortality. Ferric citrate is a dietary phosphate binder used to control hyperphosphatemia in CKD patients. It has been shown to raise iron stores, improve anemia and secondary hyperparathyroidism, and decrease vascular calcification in CKD patients. The present study was done to explore the effects and mechanism of actions of ferric citrate on cardiac hypertrophy and fibrosis. MATERIALS AND METHODS: Male SD rats were randomized to CKD (5/6 nephrectomized) and sham-operated control groups. CKD rats were fed regular diet or a diet containing 4% ferric citrate. After 8 weeks, hemoglobin, renal function and cardiovascular endpoints including blood pressure, heart/body weight ratio, serum N-terminal prohormone of brain natriuretic peptide (NT-proBNP), cardiac histology and markers of hypertrophy, fibrosis and inflammation were assessed. RESULTS: Compared to the controls, untreated CKD group exhibited hypertension, elevated serum urea, creatinine, phosphate, and NT-proBNP concentrations, anemia, cardiomegaly ,cardiac hypertrophy and fibrosis. Treatment with ferric citrate significantly increased hemoglobin and serum iron concentrations, reduced serum phosphate and NT-proBNP levels and ameliorated hypertension, heart/body weight ratio, cardiac hypertrophy, fibrosis and inflammation. In addition, ferric citrate administration reduced the size of cardiomyocytes and expressions of myocardin, transforming growth factor-ß, interleukin-6 and monocyte chemotactic protein 1. CONCLUSIONS: Treatment with ferric citrate attenuated renal failure and cardiovascular abnormalities including myocardial hypertrophy and fibrosis in CKD rats.

The impact of Catechol-O-methyl transferase knockdown on the cell proliferation of hormone-responsive cancers.

Estrogen (E2) plays a central role in the development and progression of hormone-responsive cancers. Estrogen metabolites exhibit either stimulatory or inhibitory roles on breast and prostate cells. The catechol metabolite 4-hydroxyestradiol (4-OHE2) enhances cell proliferation, while 2-methoxyestradiol (2 ME) possesses anticancer activity. The major metabolizing enzyme responsible for detoxifying the deleterious metabolite 4-OHE2 and forming the anticancer metabolite 2 ME is Catechol-O-Methyl Transferase (COMT). The current work investigated the relationship between the expression level of COMT and the cell proliferation of hormone-responsive cancers. The results showed that COMT silencing enhanced the cell proliferation of ER-α positive cancer cells MCF-7 and PC-3 but not the cells that lack ER-α expression as MDA-MB231 and DU-145. The data generated from our study provides a better understanding of the effect of COMT on critical signaling pathways involved in the development and progression of breast cancer (BC) and prostate cancer (PC) including ER-α, p21cip1, p27kip1, NF-κB (P65) and CYP19A1. These findings suggest that COMT enzyme plays a tumor suppressor role in hormone receptor-positive tumors which opens the door for future studies to validate COMT expression as a novel biomarker for the prediction of cancer aggressiveness and treatment efficacy.

Association of Serum Paraoxonase/Arylesterase Activity With All-Cause Mortality in Maintenance Hemodialysis Patients.

CONTEXT: In end-stage renal disease (ESRD), serum high-density lipoprotein cholesterol (HDL-C) level is not an accurate predictor of mortality, partly because it does not necessarily correlate with indices of HDL function. Paraoxonase (PON) is a major enzyme constituent of HDL and a key component of HDL antioxidant activity. Apolipoprotein A-I (Apo A-1) is the core HDL structural protein that plays a major role in various aspects of HDL function. OBJECTIVE: We sought to examine PON activity and Apo A-I levels in patients with ESRD vs healthy controls. DESIGN AND SETTING: PON/arylesterase activity was measured in 499 patients with maintenance hemodialysis (MHD) and 24 healthy controls with similar distributions of age, sex, and race/ethnicity. Serum acrolein-modified Apo A-I was measured in 30 patients with MHD and 10 healthy controls. MAIN OUTCOME MEASURES: Multilevel Cox models were used to assess associations among PON activity, Apo A-I, and HDL-C levels with 12-month all-cause mortality. RESULTS: PON activity was significantly lower in patients with MHD vs controls. Furthermore, acrolein-modified Apo A-I levels were higher in patients with MHD vs controls. In fully adjusted models, high PON activity was associated with lower 12-month mortality, whereas no difference of mortality risk was observed across HDL-C levels. The combination of high PON and low Apo A-I compared with low PON and low Apo A-I was associated with lower mortality risk. CONCLUSIONS: In patients with MHD, PON activity had a stronger association with 12-month mortality than HDL-C. Future studies are needed to examine the role of these markers as potential diagnostic and therapeutic tools in ESRD.

Effects of end-stage renal disease and dialysis modalities on blood ammonia level.

INTRODUCTION: Uremia results in a characteristic breath odor (uremic fetor) which is largely due to its high ammonia content. Earlier studies have shown a strong correlation between breath ammonia and blood urea levels and a 10-fold reduction in breath ammonia after hemodialysis in patients with chronic kidney disease. Potential sources of breath ammonia include: (i) local ammonia production from hydrolysis of urea in the oropharyngeal and respiratory tracts by bacterial flora, and (ii) release of circulating blood ammonia by the lungs. While the effects of uremia and hemodialysis on breath ammonia are well known their effects on blood ammonia are unknown and were explored here. METHODS: Blood samples were obtained from 23 hemodialysis patients (immediately before and after dialysis), 14 peritoneal dialysis patients, and 10 healthy controls. Blood levels of ammonia, creatinine, urea, and electrolytes were measured. FINDINGS: No significant difference was found in baseline blood ammonia between hemodialysis, peritoneal dialysis and control groups. Hemodialysis procedure led to a significant reduction in urea concentration (P < 0.001) which was paradoxically accompanied by a modest but significant (P < 0.05) rise in blood ammonia level in 10 of the 23 patients studied. Change in blood ammonia pre- and post-hemodialysis correlated with change in serum bicarbonate levels (r = 0.61, P < 0.01). On subgroup analysis of patients who had a rise in blood ammonia levels after dialysis, there was a strong correlation with drop in mean arterial pressure (r = 0.88, P < 0.01). The nadir intradialytic systolic blood pressure trended lower in the hemodialysis patients who had a rise in blood ammonia compared to the patients who manifested a fall in blood ammonia (124 ± 8 vs. 136 ± 6 mmHg respectively, P = 0.27). DISCUSSION: Fall in blood urea following hemodialysis in ESRD patients was paradoxically accompanied by a modest rise in blood ammonia levels in 43% of the patients studied, contrasting prior reported effects of hemodialysis on breath ammonia. In this subgroup of patients, changes in blood ammonia during hemodialysis correlated with rise in blood bicarbonate and fall in mean arterial blood pressure.

Meta-analysis of the association of 4 angiotensinogen polymorphisms with essential hypertension: a role beyond M235T?

Angiotensinogen (AGT) gene polymorphisms have been linked to increased risk of hypertension, but the data remain controversial. In this study we review the most commonly investigated polymorphisms at the AGT locus (other than M235T) and provide summary estimates regarding their association with essential hypertension, while addressing heterogeneity, as well as publication biases. Data on 26 818 subjects from 46 studies for the 4 most-studied AGT variants (T174M in exon 2 and 3 promoter variants: A-6G, A-20C, and G-217A) were meta-analyzed. Statistically significant associations with hypertension were identified for the T174M (odds ratio [OR]: 1.19; 95% CI: 1.07 to 1.33; P=0.002) and G-217A (OR: 1.37; 95% CI: 1.17 to 1.59; P=0.00006) polymorphisms. A dual but consistent effect was observed for the -20C allele, which was associated with a decreased risk of hypertension in populations of mixed and European ancestries (OR: 0.64; 95% CI: 0.44 to 0.92; P=0.02 and OR: 0.77; 95% CI: 0.65 to 0.91; P=0.003, respectively), but with a 24% increase in the odds of hypertension in Asian subjects (OR: 1.24; 95% CI: 1.04 to 1.48; P=0.02). No association of the A-6G variant with hypertension was detected. Current studies support the notion that single variants at the AGT might modulate the risk of hypertension but indicate caution in interpreting these results because of the putative presence of publication bias and gene-environment interactions.