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We conducted an anonymous survey in 9 of our university affiliated outpatient dialysis units to address the concern that many in-center hemodialysis patients may not feel comfortable sharing their experiences. Major goals of this study: Investigating level of patient satisfaction with their care; Evaluating the subjective perception of the level of understanding of patients regarding pertinent issues of their disease and its management; Identifying potential avenues for care improvement. Survey was conducted in English, paper-based, with answer choices to individual questions for patient satisfaction and education graded using a 5-point Likert scale. Regarding potential areas of improvement, patients were asked to choose as many areas as deemed appropriate. To ensure anonymity, the completed surveys were folded and dropped into a box. Overall, 253 out of 516 (49%) screened patients were eligible and completed the survey. Patients expressed favorable responses regarding satisfaction (mean rating 4 in each of 14 questions) and education (mean rating 4 in 8 questions, 3.5 in 2 questions) regarding hemodialysis. About 62% of overall study participants identified at least one area where they felt additional information would result in improvement of care. Our results indicate that patients undergoing outpatient hemodialysis were overall satisfied and had a good perceptive understanding about their health. Based on the patients' input, strategies focused on addressing pain and discomfort, privacy, providing information about palliative care/hospice, mental health resources, and the process of kidney transplantation may promote improvement in overall quality of care.

BACKGROUND: Emerging evidence supports the superiority of balanced crystalloids such as Lactated Ringer’s (LR) compared to normal saline but concerns for the development of hyperkalemia have limited its use. Although LR inherently contains potassium, there exists a paucity of evidence to suggest that LR could potentiate hyperkalemia. To address this, we evaluated the effect of LR on serum potassium in patients with reduced kidney function who are at risk of developing hyperkalemia. METHODS: We conducted a single-center, retrospective cohort-based observational clinical study that included 293 clinical encounters who were hospitalized with an estimated glomerular filtration rate (eGFR) of < 30 ml/min/1.73m(2), at the time of hospital admission. Subjects must have received a minimum of 500 ml of LR continuously during the admission. Only those with a minimum of one lab report within 24 hours prior to-, and post-LR administration that reported serum measurements of potassium, glucose, and bicarbonate levels were included. Other potential risk factors for developing hyperkalemia including medication, tube feeds, potassium supplements, and red blood cell transfusion during or within 24 hours after LR administration were recorded. RESULTS: Serum potassium prior to LR use was highly correlated and predictive of the serum potassium after LR use [P < 0.0001; Odds Ratio 6.77 (3.73 – 12.28)]. Sixteen encounters (5%) developed de-novo hyperkalemia following LR use. No significant positive correlation between the amount of LR administered and the development of hyperkalemia was found. CONCLUSIONS: LR use was not independently associated with the development of hyperkalemia in patients with reduced kidney function.

Serum soluble Fas (sFas) levels are associated with erythropoietin (Epo) hyporesponsiveness in patients with chronic kidney disease (CKD). Whether sFas could predict the need for erythropoiesis-stimulating agent (ESA) usage and its influence in erythropoiesis remain unclear. We evaluated the relation between sFas and ESA therapy in patients with CKD with anemia and its effect on erythropoiesis in vitro. First, we performed a retrospective cohort study with 77 anemic patients with nondialysis CKD. We performed in vitro experiments to investigate whether sFas could interfere with the behavior of hematopoietic stem cells (HSCs). HSCs were isolated from umbilical cord blood and incubated with recombinant sFas protein in a dose-dependent manner. Serum sFas positively correlated with Epo levels ( r = 0.30, P = 0.001) but negatively with hemoglobin ( r = −0.55, P < 0.001) and glomerular filtration rate ( r = −0.58, P < 0.001) in patients with CKD at baseline. Elevated sFas serum levels (4,316 ± 897 vs. 2,776 ± 749, P < 0.001) with lower estimated glomerular filtration rate (26.2 ± 10.1 vs. 33.5 ± 14.3, P = 0.01) and reduced hemoglobin concentration (11.1 ± 0.9 vs. 12.5 ± 1.2, P < 0.001) were identified in patients who required ESA therapy compared with patients with non-ESA. Afterward, we detected that the sFas level was slight correlated with a necessity of ESA therapy in patients with nondialysis CKD and anemia. In vitro assays demonstrated that the erythroid progenitor cell frequency negatively correlated with sFas concentration ( r = −0.72, P < 0.001). There was decreased erythroid colony formation in vitro when CD34+ HSCs were incubated with a higher concentration of sFas protein (1.56 ± 0.29, 4.33 ± 0.53, P < 0.001). Our findings suggest that sFas is a potential predictor for ESA therapy in patients with nondialysis CKD and that elevated sFas could affect erythropoiesis in vitro.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity. NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

With iron at its core, the tetrapyrrole heme ring is a cardinal prosthetic group made up of many proteins that participate in a wide array of cellular functions and metabolism. Once released, due to its pro-oxidant properties, free heme in sufficient amounts can result in injurious effects to the kidney and other organs. Heme oxygenase-1 (HO-1) has evolved to promptly attend to such injurious potential by facilitating degradation of heme into equimolar amounts of carbon monoxide, iron, and biliverdin. HO-1 induction is a beneficial response to tissue injury in diverse animal models of diseases, including those that affect the kidney. These protective attributes are mainly due to: (i) prompt degradation of heme leading to restraining potential hazardous effects of free heme, and (ii) generation of byproducts that along with induction of ferritin have proven beneficial in a number of pathological conditions. This review will focus on describing clinical aspects of some of the conditions with the unifying end-result of increased heme burden and will discuss the molecular mechanisms that ensue to protect the kidneys.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are interconnected syndromes with significant attributable morbidity and mortality. The disturbing trend of increasing incidence and prevalence of these clinical disorders highlights the urgent need for better understanding of the underlying mechanisms that are involved in pathogenesis of these conditions. Lymphangiogenesis and its involvement in various inflammatory conditions is increasingly recognized while its role in AKI and CKD remains to be fully elucidated. Here, we studied lymphangiogenesis in three models of kidney injury. Our results demonstrate that the main ligands for lymphangiogenesis, VEGF-C and VEGF-D, are abundantly present in tubules at baseline conditions and the expression pattern of these ligands is significantly altered following injury. In addition, we show that both of these ligands increase in serum and urine post-injury and suggest that such increment may serve as novel urinary biomarkers of AKI as well as in progression of kidney disease. We also provide evidence that irrespective of the nature of initial insult, lymphangiogenic pathways are rapidly and robustly induced as evidenced by higher expression of lymphatic markers within the kidney.

Significance Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality, claiming the lives of over ∼4.5 × 105 individuals per year. Paradoxically, however, up to 98% of infected individuals survive the infection, establishing disease tolerance to malaria. We found that this host defense strategy, which does not target Plasmodium directly, relies on the capacity of renal proximal tubule epithelial cells to detoxify labile heme, a pathologic by-product of hemolysis that accumulates in plasma and urine during the blood stage of infection. This defense strategy prevents the onset of acute kidney injury, a clinical hallmark of severe malaria., Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium. However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Given the abundance of heme proteins (cytochromes) in the mitochondrion, it is evident that a meticulously orchestrated iron metabolism is essential for cardiac health. Here, we examined the functional significance of myocardial ferritin heavy chain (FtH) in a model of acute myocardial infarction. We report that FtH deletion did not alter either the mitochondrial regulatory and surveillance pathways (fission and fusion) or mitochondrial bioenergetics in response to injury. Furthermore, deletion of myocardial FtH did not affect cardiac function, assessed by measurement of left ventricular ejection fraction, on days 1, 7, and 21 post injury. To identify the modulated pathways providing cardiomyocyte protection coincident with FtH deletion, we performed unbiased transcriptomic analysis. We found that following injury, FtH deletion was associated with upregulation of several genes with anti-ferroptotic properties, including heme oxygenase-1 (HO-1) and the cystine/glutamate anti-porter (Slc7a11). These results suggested that HO-1 overexpression mitigates ferroptosis via upregulation of Slc7a11. Indeed, using transgenic mice with HO-1 overexpression, we demonstrate that overexpressed HO-1 is coupled with increased Slc7a11 expression. In conclusion, we demonstrate that following injury, myocardial FtH deletion leads to a compensatory upregulation in a number of anti-ferroptotic genes, including HO-1. Such HO-1 induction leads to overexpression of Slc7a11 and protects the heart against ischemia-reperfusion-mediated ferroptosis, preserves mitochondrial function, and overall function of the myocardium.

Objective— Calcific aortic valve disease is a prominent finding in elderly and in patients with chronic kidney disease. We investigated the potential role of iron metabolism in the pathogenesis of calcific aortic valve disease. Approach and Results— Cultured valvular interstitial cells of stenotic aortic valve with calcification from patients undergoing valve replacement exhibited significant susceptibility to mineralization/osteoblastic transdifferentiation in response to phosphate. This process was abrogated by iron via induction of H-ferritin as reflected by lowering ALP and osteocalcin secretion and preventing extracellular calcium deposition. Cellular phosphate uptake and accumulation of lysosomal phosphate were decreased. Accordingly, expression of phosphate transporters Pit1 and Pit2 were repressed. Translocation of ferritin into lysosomes occurred with high phosphate-binding capacity. Importantly, ferritin reduced nuclear accumulation of RUNX2 (Runt-related transcription factor 2), and as a reciprocal effect, it enhanced nuclear localization of transcription factor Sox9 (SRY [sex-determining region Y]-box 9). Pyrophosphate generation was also increased via upregulation of ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase-2). 3 H-1, 2-dithiole-3-thione mimicked these beneficial effects in valvular interstitial cell via induction of H-ferritin. Ferroxidase activity of H-ferritin was essential for this function, as ceruloplasmin exhibited similar inhibitory functions. Histological analysis of stenotic aortic valve revealed high expression of H-ferritin without iron accumulation and its relative dominance over ALP in noncalcified regions. Increased expression of H-ferritin accompanied by elevation of TNF-α (tumor necrosis factor-α) and IL-1β (interleukin-1β) levels, inducers of H-ferritin, corroborates the essential role of ferritin/ferroxidase via attenuating inflammation in calcific aortic valve disease. Conclusions— Our results indicate that H-ferritin is a stratagem in mitigating valvular mineralization/osteoblastic differentiation. Utilization of 3H-1, 2-dithiole-3-thione to induce ferritin expression may prove a novel therapeutic potential in valvular mineralization.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that catalyzes the breakdown of heme to biliverdin, carbon monoxide, and iron. The beneficial effects of HO-1 expression are not merely due to degradation of the pro-oxidant heme but are also credited to the by-products that have potent, protective effects, including antioxidant, anti-inflammatory, and prosurvival properties. This is well reflected in the preclinical animal models of injury in both renal and nonrenal settings. However, excessive accumulation of the by-products can be deleterious and lead to mitochondrial toxicity and oxidative stress. Therefore, use of the HO system in alleviating injury merits a targeted approach. Based on the higher susceptibility of the proximal tubule segment of the nephron to injury, we generated transgenic mice using cre-lox technology to enable manipulation of HO-1 (deletion or overexpression) in a cell-specific manner. We demonstrate the validity and feasibility of these mice by breeding them with proximal tubule-specific Cre transgenic mice. Similar to previous reports using chemical modulators and global transgenic mice, we demonstrate that whereas deletion of HO-1, specifically in the proximal tubules, aggravates structural and functional damage during cisplatin nephrotoxicity, selective overexpression of HO-1 in proximal tubules is protective. At the cellular level, cleaved caspase-3 expression, a marker of apoptosis, and p38 signaling were modulated by HO-1. Use of these transgenic mice will aid in the evaluation of the effects of cell-specific HO-1 expression in response to injury and assist in the generation of targeted approaches that will enhance recovery with reduced, unwarranted adverse effects.

Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D3 analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using β‐glycerophosphate with activated vitamin D3, or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D3 analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast‐like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H‐1,2‐Dithiole‐3‐thione was able to inhibit the SMC transition into osteoblast‐like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D3 caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D3 and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.

Background and Purpose Calcification of heart valves is a frequent pathological finding in chronic kidney disease and in elderly patients. Hydrogen sulfide (H2S) may exert anti‐calcific actions. Here we investigated H2S as an inhibitor of valvular calcification and to identify its targets in the pathogenesis. Experimental Approach Effects of H2S on osteoblastic transdifferentiation of valvular interstitial cells (VIC) isolated from samples of human aortic valves were studied using immunohistochemistry and western blots. We also assessed H2S on valvular calcification in apolipoprotein E‐deficient (ApoE−/−) mice. Key Results In human VIC, H2S from donor compounds (NaSH, Na2S, GYY4137, AP67, and AP72) inhibited mineralization/osteoblastic transdifferentiation, dose‐dependently in response to phosphate. Accumulation of calcium in the extracellular matrix and expression of osteocalcin and alkaline phosphatase was also inhibited. RUNX2 was not translocated to the nucleus and phosphate uptake was decreased. Pyrophosphate generation was increased via up‐regulating ENPP2 and ANK1. Lowering endogenous production of H2S by concomitant silencing of cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS) favoured VIC calcification. analysis of human specimens revealed higher Expression of CSE in aorta stenosis valves with calcification (AS) was higher than in valves of aortic insufficiency (AI). In contrast, tissue H2S generation was lower in AS valves compared to AI valves. Valvular calcification in ApoE−/− mice on a high‐fat diet was inhibited by H2S. Conclusions and Implications The endogenous CSE‐CBS/H2S system exerts anti‐calcification effects in heart valves providing a novel therapeutic approach to prevent hardening of valves. Linked Articles This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc

The incidence of atrial fibrillation is increased during hemodialysis (HD); however, the effects of hemodiafiltration (HDF) on atrial arrhythmias have not been evaluated. The prolongation of the P wave and P dispersion (Pd) can predict atrial arrhythmias. Data from 30 patients receiving HDF over a period of 3 months were collected; the same group of patients was then evaluated during treatment with conventional HD for at least another 3 months. Electrolyte values were obtained, and surface electrocardiograms (ECG), echocardiography, and Holter ECGs were performed. The duration of the P wave and Pd increased significantly during HD. The left atrial diameter decreased significantly only during HDF. During HDF, the left atrial cross diameter measured at the beginning of the session was positively correlated with the incidence of supraventricular premature beats (p = 0.011, r = 0.4556). The decrease in left atrial diameter during HDF was negatively correlated with the incidence of supraventricular premature beats (p = 0.016, r = −0.43). During HDF, the changes in sodium and Pd were significantly positively correlated (p

Iron is at the forefront of a number of pivotal biological processes due to its ability to readily accept and donate electrons. However, this property may also catalyze the generation of free radicals with ensuing cellular and tissue toxicity. Accordingly, throughout evolution numerous pathways and proteins have evolved to minimize the potential hazardous effects of iron cations and yet allow for readily available iron cations in a wide variety of fundamental metabolic processes. One of the extensively studied proteins in the context of systemic and cellular iron metabolisms is ferritin. While clinicians utilize serum ferritin to monitor body iron stores and inflammation, it is important to note that the vast majority of ferritin is located intracellularly. Intracellular ferritin is made of two different subunits (heavy and light chain) and plays an imperative role as a safe iron depot. In the past couple of decades our understanding of ferritin biology has remarkably improved. Additionally, a significant body of evidence has emerged describing the significance of the kidney in iron trafficking and homeostasis. Here, we briefly discuss some of the most important findings that relate to the role of iron and ferritin heavy chain in the context of kidney-related diseases and, in particular, vascular calcification, which is a frequent complication of chronic kidney disease.

Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule-specific FtH-knockout mice (FtH(PT-/-) mice). FtH(PT-/-) mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtH(PT-/-) mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase-associated lipocalin, hemopexin, and transferrin were increased in FtH(PT-/-) mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI.

Mitochondria are both a source and target of the actions of reactive oxygen species and possess a complex system of inter-related antioxidants that control redox signaling and protect against oxidative stress. Interestingly, the antioxidant enzyme heme oxygenase-1 (HO-1) is not present in the mitochondria despite the fact that the organelle is the site of heme synthesis and contains multiple heme proteins. Detoxification of heme is an important protective mechanism since the reaction of heme with hydrogen peroxide generates pro-oxidant ferryl species capable of propagating oxidative stress and ultimately cell death. We therefore hypothesized that a mitochondrially localized HO-1 would be cytoprotective. To test this, we generated a mitochondria-targeted HO-1 cell line by transfecting HEK293 cells with a plasmid construct containing the manganese superoxide dismutase mitochondria leader sequence fused to HO-1 cDNA (Mito-HO-1). Nontargeted HO-1-overexpressing cells were generated by transfecting HO-1 cDNA (HO-1) or empty vector (Vector). Mitochondrial localization of HO-1 with increased HO activity in the mitochondrial fraction of Mito-HO-1 cells was observed, but a significant decrease in the expression of heme-containing proteins occurred in these cells. Both cytosolic HO-1- and Mito-HO-1-expressing cells were protected against hypoxia-dependent cell death and loss of mitochondrial membrane potential, but these effects were more pronounced with Mito-HO-1. Furthermore, decrement in production of tricarboxylic acid cycle intermediates following hypoxia was significantly mitigated in Mito-HO-1 cells. These data suggest that specific mitochondrially targeted HO-1 under acute pathological conditions may have beneficial effects, but the selective advantage of long-term expression is constrained by a negative impact on the synthesis of heme-containing mitochondrial proteins.

Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies.

LB

Although short-term outcomes in kidney transplantation have improved dramatically, long-term survival remains a major challenge. A key component of long-term, chronic allograft injury in solid organ transplants is arteriosclerosis characterized by vascular neointimal hyperplasia and inflammation. Establishing a model of this disorder would provide a unique tool not only to identify mechanisms of disease but also to test potential therapeutics for late graft injury. To this end, we utilized a mouse orthotopic renal transplant model in which C57BL/6J (H-2b) recipients were given either a kidney allograft from a completely mismatched Balb/cJ mouse (H-2d) or an isograft from a littermate. A unilateral nephrectomy was performed at the time of transplant followed by a contralateral nephrectomy on post-transplant day 7. Recipients were treated with daily cyclosporine subcutaneously for 14 days and then studied 8 and 12 weeks post transplantation. Renal function was significantly worse in allograft compared with isograft recipients. Moreover, the allografts had significantly more advanced tubulointerstitial fibrosis and profound vascular disease characterized by perivascular leukocytic infiltration and neointimal hyperplasia affecting the intrarenal blood vessels. Thus, we describe a feasible and reproducible murine model of intrarenal transplant arteriosclerosis that is useful to study allograft vasculopathy.

Many different genes or mediators have been implicated in promoting the development of vasculitis, although little is known regarding the mechanisms that normally act to suppress lesion formation. Endothelial nitric oxide synthase (eNOS) has been shown to inhibit vascular inflammation in many different model systems, but its roles in the pathogenesis of vasculitis have not been elucidated. This study was undertaken to determine the functions of eNOS in the initiation and progression of vasculitic lesion formation.MRL/MpJ-Fas(lpr) mice lacking the gene for eNOS (Nos3(-/-) ) were generated and comprehensively evaluated and compared to controls with regard to the development of autoimmune disease, including vasculitic lesion formation and glomerulonephritis.Nos3(-/-) MRL/MpJ-Fas(lpr) mice exhibited accelerated onset and increased incidence of renal vasculitis compared to Nos3(+/+) controls. In contrast, no significant differences in severity of glomerulonephritis were observed between groups. Vasculitis was also observed in other organs of eNOS-deficient mice, including in the lungs of several of these animals. Ultrastructural analyses of renal lesions revealed the presence of electron-dense deposits in affected arteries, and IgG, IgA, and C3 deposition was observed in some vessels in the kidneys of Nos3(-/-) mice. In addition, Nos3(-/-) MRL/MpJ-Fas(lp) mice showed increased levels of circulating IgG-IgA immune complexes at 20 weeks of age, compared to Nos3(+/+) MRL/MpJ-Fas(lpr) and Nos3(-/-) C57BL/6 mice.These findings strongly indicate that eNOS serves as a negative regulator of vasculitis in MRL/MpJ-Fas(lpr) mice and further suggest that NO produced by this enzyme may be critical for inhibiting lesion formation and vascular damage in human vasculitic diseases.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation, but whether such control occurs in vivo is not known. To enable such an analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities, and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome conformation capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene, providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases.

Transforming growth factor-β (TGF-β) is a multifunctional regulatory cytokine that is implicated in a variety of kidney diseases, including diabetic nephropathy and chronic transplant rejection, where it promotes stimulation of the extracellular matrix deposition, cell proliferation, and migration. TGF-β exerts its biological functions largely via its downstream complex signaling molecules, Smad proteins. Paradoxically, TGF-β also is essential for normal homeostasis and suppression of inflammation through mechanisms that are yet to be fully elucidated. One feasible mechanism by which TGF-β may exert its beneficial properties is through induction of heme oxygenase-1 (HO-1). Induction of this redox-sensitive enzyme is known to be cytoprotective through its potent antioxidant, anti-inflammatory, and anti-apoptotic properties in different conditions including several kidney diseases. In this overview, recent advances in our understanding of the role of TGF-β in kidney disease, its molecular regulation of HO-1 expression, and the potential role of HO-1 induction as a therapeutic modality in TGF-β-mediated kidney diseases are highlighted.

Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is involved in the pathogenesis of vascular calcification. Hydrogen sulfide (H(2)S) is a gas endogenously produced by cystathionine γ-lyase in VSMC. Here we determined whether H(2)S plays a role in phosphate-induced osteoblastic transformation and mineralization of VSMC. Hydrogen sulfide was found to inhibit calcium deposition in the extracellular matrix and to suppress the induction of the genes involved in osteoblastic transformation of VSMC: alkaline phosphatase, osteocalcin, and Cbfa1. Moreover, phosphate uptake and phosphate-triggered upregulation of the sodium-dependent phosphate cotransporter (Pit-1) were also prevented by H(2)S. Reduction of endogenous production of H(2)S by inhibition of cystathionine γ-lyase activity resulted in increased osteoblastic transformation and mineralization. Low plasma levels of H(2)S, associated with decreased cystathionine γ-lyase enzyme activity, were found in patients with chronic kidney disease receiving hemodialysis. Thus, H(2)S is a potent inhibitor of phosphate-induced calcification and osteoblastic differentiation of VSMC. This mechanism might contribute to accelerated vascular calcification in chronic kidney disease.

Sepsis is a severe and dysregulated inflammatory response to infection characterized by end-organ dysfunction distant from the primary site of infection. Development of acute kidney injury (AKI) during sepsis increases patient morbidity, predicts higher mortality, has a significant effect on multiple organ functions, is associated with an increased length of stay in the intensive care unit, and hence consumes considerable healthcare resources. When compared with AKI of nonseptic origin, septic AKI is characterized by a distinct pathophysiology and therefore requires a different approach. Despite impressive advances in several fields of medicine, the pathophysiology, diagnostic procedures, and appropriate therapeutic interventions in sepsis are still highly debatable. Numerous immunomodulatory agents showing promise in preclinical studies fail to reduce the overwhelmingly high mortality rate of sepsis and provoke AKI when compared with other critically ill patients. Major impediments to progress in understanding, early diagnosis, and application of appropriate therapeutic modalities in sepsis-induced AKI include limited histopathologic information, few animal models that closely mimic human sepsis, and a relative shortage of specific diagnostic tools. Here we discuss the most recent advances in understanding the fundamental mechanisms of sepsis-induced AKI, characteristics of relevant animal models available, and potential therapies.

Multipotent mesenchymal stem cells (MSC) have become a popular and promising therapeutic approach in many clinical conditions. MSC are beneficial in animal models of acute kidney injury (AKI), by mediating differentiation-independent paracrine properties, and have prompted ongoing clinical trials to evaluate the safety and efficacy of MSC. Heme oxygenase-1 (HO-1) is induced in response to stress including AKI and has important anti-apoptotic, anti-inflammatory, and proangiogenic properties in these settings. We therefore examined whether HO-1 plays a role in the beneficial effects of MSC in AKI. We isolated MSC from bone marrow of age-matched HO-1+/+and HO-1−/−mice. Our studies indicate that while differentiation of MSC into osteo- and adipocytic lineages did not differ between cells isolated from HO-1+/+and HO-1−/−mice, MSC from HO-1−/−mice had significantly lower angiogenic potential. Moreover, HO-1−/−MSC demonstrated reduced expression and secretion of several important growth and proangiogenic factors (stromal cell-derived factor-1, vascular endothelial growth factor-A, and hepatocyte growth factor) compared with MSC derived from HO-1+/+mice. In addition, conditioned medium of HO-1+/+MSC rescued functional and morphological changes associated with cisplatin-induced AKI, while the HO-1−/−-conditioned medium was ineffectual. Our studies indicate that HO-1 plays an important role in MSC-mediated protection. The results expand understanding of the renoprotective effects of MSC and may provide novel strategies to better utilize MSC in various disease models.

Hemochromatosis is a known cause of osteoporosis, and iron overload has deleterious effects on bone. Although iron overload and its association with osteoporosis has long been recognized, the pathogenesis and exact role of iron have been undefined. Bone is an active tissue with constant remodeling capacity. Osteoblast (OB) development and maturation are under the influence of core binding factor alpha-1 (CBF-alpha1), which induces expression of OB-specific genes, including alkaline phosphatase, an important enzyme in early osteogenesis, and osteocalcin, a noncollagenous protein deposited within the osteoid. This study investigates the mechanism by which iron inhibits human OB activity, which in vivo may lead to decreased mineralization, osteopenia, and osteoporosis. We demonstrate that iron-provoked inhibition of OB activity is mediated by ferritin and its ferroxidase activity. We confirm this notion by using purified ferritin H-chain and ceruloplasmin, both known to possess ferroxidase activity that inhibited calcification, whereas a site-directed mutant of ferritin H-chain lacking ferroxidase activity failed to provide any inhibition. Furthermore, we are reporting that such suppression is not restricted to inhibition of calcification, but OB-specific genes such as alkaline phosphatase, osteocalcin, and CBF-alpha1 are all downregulated by ferritin in a dose-responsive manner. This study corroborates that iron decreases mineralization and demonstrates that this suppression is provided by iron-induced upregulation of ferritin. In addition, we conclude that inhibition of OB activity, mineralization, and specific gene expression is attributed to the ferroxidase activity of ferritin.

The deposited article is a post-print version (author's manuscript from PMC). This publication hasn't any creative commons license associated. The deposited article version contains attached the supplementary materials within the pdf. Inflammation culminating in fibrosis contributes to progressive kidney disease. Cross-talk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice: a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Using transgenic mice with conditional deletion of FtH in the proximal tubules (FtH(PT-/-)) or myeloid cells (FtH(LysM-/-)), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared with wild-type (FtH(+/+)) controls. However, tubular FtH deletion led to a marked increase in proinflammatory macrophages. Furthermore, injured kidneys from FtH(PT-/-) mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared with kidneys from FtH(+/+) and FtH(LysM-/-) mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscore the critical role of FtH in tubular-macrophage cross-talk during kidney injury. NIH grants: (R01 DK5960, R01 DK5960, P30 DK079337, R01 DK083390); Fundação para a Ciência e Tecnologia grants: (PTDC/SAU-TOX/116627/2010, HMSP-ICT/0022/2010); European Union grant: (ERC-2011-AdG. 294709 DAMAGECONTROL); AHA grant: (11POST7600074). info:eu-repo/semantics/publishedVersion

The prolongation of the QT interval and dispersion could predict ventricular arrhythmias. It is not yet established whether there is a difference between the effects of hemodialysis and hemodiafiltration on QT interval duration and dispersion. Data of thirty patients was investigated while they were receiving hemodiafiltration over a period of 3 months; then the same group of patients was evaluated during treatment with conventional hemodialysis for at least another 3 months. Ionic parameters and surface electrocardiograms (ECG) were analyzed five times during each session, and 2D, M-mode echocardiography and Holter ECGs were performed to acquire additional information. QT interval duration (QTmax) and dispersion (QTd) showed a significant increase during hemodialysis, but not during hemodiafiltration. QTmax was 388.66 ± 31.81 ms at the beginning of hemodialysis and increased to 400.66 ± 39.12 ms even at the 30th minute (p

Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), occurring with hypotension and cardiovascular surgery and inevitably during kidney transplantation. Mortality from AKI is high due to incomplete knowledge of the pathogenesis of IRI and the lack of an effective therapy. Inflammation accompanies IRI and increases the blood level of C-reactive protein (CRP), a biomarker of worsened outcomes in AKI. To test if CRP is causal in AKI we subjected wild-type mice (WT) and human CRP transgenic mice (CRPtg) to bilateral renal IRI (both pedicles clamped for 30 min at 37°C then reperfused for 24 h). Serum human CRP level was increased approximately sixfold after IRI in CRPtg (10.62 ± 1.31 μg/ml at baseline vs. 72.01 ± 9.41 μg/ml at 24 h) but was not elevated by sham surgery wherein kidneys were manipulated but not clamped. Compared with WT, serum creatinine, urine albumin, and histological evidence of kidney damage were increased after IRI in CRPtg mice. RT-PCR analysis of mRNA isolated from whole kidneys of CRPtg and WT subjected to IRI revealed that in CRPtg kidneys 1) upregulation of markers of macrophage classical activation (M1 markers) was blunted, 2) downregulation of markers of macrophage alternative activation (M2 markers) was more robust, and 3) expression of the activating receptor FcγRI was increased. Our finding that CRP exacerbates IRI-induced AKI, perhaps by shifting the balance of macrophage activation and FcγR expression towards a detrimental portfolio, might make CRP a promising therapeutic target for the treatment of AKI.

Background Renal replacement therapy may have a favorable effect on diastolic left ventricular function, but it is not clear whether hemodiafiltration is superior to hemodialysis in this field. Nitric oxide (NO) and asymmetric dimethylarginine (ADMA) may play a role in the changes of intracardiac hemodynamics, but it is not clear whether the different renal replacement methods have disparate influence on the metabolism of these materials. Methods Thirty patients on renal replacement therapy were investigated. First, data was analyzed while patients received hemodiafiltration over a period of three months. Then, the same patients were evaluated during treatment with hemodialysis for at least another three months. Echocardiography was performed before and after renal replacement therapy. Results No significant difference was found in the volume removals between hemodialysis and hemodiafiltration. The left atrial diameter and transmitral flow velocities (E/A) decreased significantly only during hemodiafiltration. A positive correlation was observed between the left atrial diameter and E/Ea representing the left ventricular pressure load during hemodiafiltration. Significant correlations between NO and A and E/A were observed only in the case of hemodiafiltration. Conclusion Hemodiafiltration has a beneficial effect on echocardiographic markers representing left ventricular diastolic function. This could be attributed to the differences between the dynamics of volume removal and its distribution among liquid compartments.

Acute kidney injury (AKI) is a common, heterogeneous, and detrimental clinical condition that has significant attributable morbidity and mortality. Despite major advances in understanding the epidemiology, pathogenesis, and outcomes of AKI, preventive measures remain inadequate and therapeutic approaches (except for renal replacement therapy) have largely proven futile so far. Critical to the process of designing rational therapies is translational research, which involves the transition between the basic research discoveries and everyday clinical applications to prevent, diagnose, and treat human diseases. Progress in innovative approaches has been hampered due in part to the reliance on functional markers (serum creatinine and blood urea nitrogen) that are neither sensitive nor specific to diagnose AKI. This limitation has created a great deal of interest and intense investigation to identify a “troponin‐like marker” that would facilitate recognition of AKI and allow for timely implementation of the precise therapeutic agent. The other major obstacle in this field is the diverse and complex nature of AKI that involves multiple independent and overlapping pathways, making it difficult to cure AKI with a single approach. In this review, we will summarize the advances, ongoing studies, and future perspectives in the field of translational research of AKI. Clin Trans Sci 2012; Volume 5: 93–101

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-β1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-β1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.

Iron is essential for almost all living organisms. It participates in a wide variety of fundamental metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, when present in excess, iron poses a threat to cells and tissues. The toxicity of iron is largely based on its ability to catalyze the generation of free radicals, which attack and damage cellular membranes, protein, and DNA. Under physiological circumstances this threat is reduced because meticulously regulated mechanisms have evolved to move iron across biological membranes and to ensure that its distribution in multicellular organisms is carefully orchestrated. These mechanisms are responsible to keep free iron concentrations at the lowest sufficient level under healthy conditions. High tissue iron concentrations have been associated with the development and progression of several pathological conditions, including certain cancers, liver and cardiovascular diseases, diabetes, hormonal derangements, skeletal abnormalities, several neurodegenerative disorders, and immune system dysfunctions. In this chapter, we discuss oxidative stress related to iron metabolism and provide an overview of several diseases that are linked to iron overload and toxicity.

peritubular fibroblasts are a major fraction of interstitial cells in the kidney that produce erythropoietin. In an issue of the American Journal of Physiology-Renal Physiology , Ponnusamy and colleagues ([14][1]) have explored the mechanism linking renal tubular epithelial cell death and injury to

Heme-mediated oxidative modification of low-density lipoprotein (LDL) plays a crucial role in early atherogenesis. It has been shown that hydrogen sulfide (H2S) produced by vascular smooth muscle cells is present in plasma at a concentration of about 50 mu mol/L. H2S is a strong reductant which can react with reactive oxygen species like superoxide anion and hydrogen peroxide. The current study investigated the effect of H2S on hemin-mediated oxidation of LDL and oxidized LDL (oxLDL)-induced endothelial reactions. H2S dose dependently delayed the accumulation of lipid peroxidation products-conjugated dienes, lipid hydroperoxides (LOOH), and thiobarbituric acid reactive substances-during hemin-mediated oxidation. Moreover, H2S decreased the LOOH content of both oxidized LDL and lipid extracts derived from soft atherosclerotic plaque, which was accompanied by reduced cytotoxicity. OxLDL-mediated induction of the oxidative stress responsive gene, heme oxygenase-1, was also abolished by H2S. Finally we have shown that H2S can directly protect endothelium against hydrogen peroxide and oxLDL-mediated endothelial cytotoxicity. These results demonstrate novel functions of H2S in preventing hemin-mediated oxidative modification of LDL, and consequent deleterious effects, suggesting a possible antiatherogenic action of H2S. (C) 2008 Elsevier Inc. All rights reserved.

Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease. Human aortic smooth muscle cells (HSMCs) undergo mineralization in response to elevated levels of inorganic phosphate (Pi) in an active and well-regulated process. This process involves increased activity of alkaline phosphatase and increased expression of core binding factor alpha-1, a bone-specific transcription factor, with the subsequent induction of osteocalcin. Mounting evidence suggests an essential role for the heme oxygenase 1 (HO-1)/ferritin system to maintain homeostasis of vascular function. We examined whether induction of HO-1 and ferritin alters mineralization of HSMCs provoked by high Pi. Upregulation of the HO-1/ferritin system inhibited HSMC calcification and osteoblastic differentiation. Of the products of the system, only ferritin and, to a lesser extent, biliverdin were responsible for the inhibition. Ferritin heavy chain and ceruloplasmin, which both possess ferroxidase activity, inhibited calcification; a site-directed mutant of ferritin heavy chain, which lacked ferroxidase activity, failed to inhibit calcification. In addition, osteoblastic transformation of HSMCs provoked by elevated Pi (assessed by upregulation of core binding factor alpha-1, osteocalcin, and alkaline phosphatase activity) was diminished by ferritin/ferroxidase activity. We conclude that induction of the HO-1/ferritin system prevents Pi-mediated calcification and osteoblastic differentiation of human smooth muscle cells mainly via the ferroxidase activity of ferritin.

Vascular access dysfunction causes morbidity in hemodialysis patients. This study examined the generation and pathobiological significance of superoxide anion in a rat femoral arteriovenous fistula (AVF). One week after AVF creation, there was increased production of superoxide anion accompanied by decreased total superoxide dismutase (SOD) and Cu/Zn SOD activities and induction of the redox-sensitive gene heme oxygenase-1. Immunohistochemical studies of nitrotyrosine formation demonstrated that peroxynitrite, a product of superoxide anion and nitric oxide, was present in increased amounts in endothelial and smooth muscle cells in the AVF. Because uncoupled NOS isoforms generate superoxide anion, and NOS coupling requires tetrahydrobiopterin (BH(4)) as a cofactor, we assessed NOS uncoupling by determining the ratio of BH(4) to dihydrobiopterin (BH(2)); the BH(4)-to-BH(2) ratio was markedly attenuated in the AVF. Because Src is a vasculopathic signaling species upstream and downstream of superoxide anion, such expression was evaluated; expression of Src and phosphorylated Src was both markedly increased in the AVF. Expression of NADPH oxidase (NOX) 1, NOX2, NOX4, cyclooxygenase (COX) 1, COX2, p47(phox), and p67(phox) was all unchanged, as assessed by Western analyses, thereby suggesting that these proteins may not be involved in increased production of superoxide anion. Finally, administration of tempol, a superoxide anion scavenger, decreased neointima formation in the juxta-anastomotic venous segment and improved AVF blood flow. We conclude that the AVF exhibits increased superoxide anion generation that may reflect the combined effects of decreased scavenging by SOD and increased generation by uncoupled NOS, and that enhanced superoxide anion production promotes juxta-anastomotic stenosis and impairs AVF function.