Distinct developmental reprogramming footprint of macrophages during acute kidney injury across species.

Acute kidney injury (AKI) is a common finding in hospitalized patients, particularly those who are critically ill. The development of AKI is associated with several adverse outcomes including mortality, morbidity, progression to chronic kidney disease, and an increase in healthcare expenditure. Despite the well-established negative impact of AKI and rigorous efforts to better define, identify, and implement targeted therapies, the overall approach to the treatment of AKI continues to principally encompass supportive measures. This enduring challenge is primarily due to the heterogeneous nature of insults that activate many independent and overlapping molecular pathways. Consequently, it is evident that the identification of common mechanisms that mediate the pathogenesis of AKI, independent of etiology and engaged pathophysiological pathways, is of paramount importance and could lead to the identification of novel therapeutic targets. To better distinguish the commonly modulated mechanisms of AKI, we explored the transcriptional characteristics of human kidney biopsies from patients with acute tubular necrosis (ATN), and acute interstitial nephritis (AIN) using a NanoString inflammation panel. Subsequently, we used publicly available single-cell transcriptional resources to better interpret the generated transcriptional findings. Our findings identify robust acute kidney injury (AKI-induced) developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species. These results would expand the current understanding of the pathophysiology of AKI and potentially offer novel targets for additional studies to enhance the translational transition of AKI research.NEW & NOTEWORTHY Our findings identify robust acute kidney injury (AKI)-induced developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species.

Heme Oxygenases in Cardiovascular Health and Disease.

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.

Renal control of disease tolerance to 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.

Counteraction of Myocardial Ferritin Heavy Chain Deficiency by Heme Oxygenase-1.

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.

Expression of lactate dehydrogenase A and B isoforms in the mouse kidney.

Cellular metabolic rates in the kidney are critical for maintaining normal renal function. In a hypoxic milieu, cells rely on glycolysis to meet energy needs, resulting in the generation of pyruvate and NADH. In the absence of oxidative phosphorylation, the continuation of glycolysis is dependent on the regeneration of NAD+ from NADH accompanied by the fermentation of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) isoform A (LDHA), whereas LDH isoform B (LDHB) catalyzes the opposite reaction. LDH is widely used as a potential injury marker as it is released from damaged cells into the urine and serum; however, the precise isoform-specific cellular localization of the enzyme along the nephron has not been characterized. By combining immunohistochemistry results and single-cell RNA-sequencing data on healthy mouse kidneys, we identified that LDHA is primarily expressed in proximal segments, whereas LDHB is expressed in the distal parts of the nephron. In vitro experiments in mouse and human renal proximal tubule cells showed an increase in LDHA following hypoxia with no change in LDHB. Using immunofluorescence, we observed that the overall expression of both LDHA and LDHB proteins decreased following renal ischemia-reperfusion injury as well as in the adenine-diet-induced model of chronic kidney disease. Single-nucleus RNA-sequencing analyses of kidneys following ischemia-reperfusion injury revealed a significant decline in the number of cells expressing detectable levels of Ldha and Ldhb; however, cells that were positive showed increased average expression postinjury, which subsided during the recovery phase. These data provide information on the cell-specific expression of LDHA and LDHB in the normal kidney as well as following acute and chronic kidney disease.NEW & NOTEWORTHY Cellular release of lactate dehydrogenase (LDH) is being used as an injury marker; however, the exact localization of LDH within the nephron remains unclear. We show that LDH isoform A is expressed proximally, whereas isoform B is expressed distally. Both subunit expressions were significantly altered in models of acute kidney injury and chronic kidney disease. Our study provides new insights into basal and postinjury renal lactate metabolism.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

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.

Quantitative 3-dimensional imaging and tissue cytometry reveals lymphatic expansion in acute kidney injury.

The lymphatic system plays an integral role in physiology and has recently been identified as a key player in disease progression. Tissue injury stimulates lymphatic expansion, or lymphangiogenesis (LA), though its precise role in disease processes remains unclear. LA is associated with inflammation, which is a key component of acute kidney injury (AKI), for which there are no approved therapies. While LA research has gained traction in the last decade, there exists a significant lack of understanding of this process in the kidney. Though innovative studies have elucidated markers and models with which to study LA, the field is still evolving with ways to visualize lymphatics in vivo. Prospero-related homeobox-1 (Prox-1) is the master regulator of LA and determines lymphatic cell fate through its action on vascular endothelial growth factor receptor expression. Here, we investigate the consequences of AKI on the abundance and distribution of lymphatic endothelial cells using Prox1-tdTomato reporter mice (ProxTom) coupled with large-scale three-dimensional quantitative imaging and tissue cytometry (3DTC). Using these technologies, we describe the spatial dynamics of lymphatic vasculature in quiescence and post-AKI. We also describe the use of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) as a marker of lymphatic vessels using 3DTC in the absence of the ProxTom reporter mice as an alternative approach. The use of 3DTC for lymphatic research presents a new avenue with which to study the origin and distribution of renal lymphatic vessels. These findings will enhance our understanding of renal lymphatic function during injury and could inform the development of novel therapeutics for intervention in AKI.

Heme Burden and Ensuing Mechanisms That Protect the Kidney: Insights from Bench and Bedside.

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.

Soluble Fas affects erythropoiesis in vitro and acts as a potential predictor of erythropoiesis-stimulating agent therapy in patients with chronic kidney disease.

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.

Potential Role of H-Ferritin in Mitigating Valvular Mineralization.

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). 3H-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-1ß (interleukin-1ß) 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.

Dynamic signature of lymphangiogenesis during acute kidney injury and chronic kidney disease.

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.

Heme Oxygenase 1 as a Therapeutic Target in Acute Kidney Injury.

A common clinical condition, acute kidney injury (AKI) significantly influences morbidity and mortality, particularly in critically ill patients. The pathophysiology of AKI is complex and involves multiple pathways, including inflammation, autophagy, cell-cycle progression, and oxidative stress. Recent evidence suggests that a single insult to the kidney significantly enhances the propensity to develop chronic kidney disease. Therefore, the generation of effective therapies against AKI is timely. In this context, the cytoprotective effects of heme oxygenase 1 (HO-1) in animal models of AKI are well documented. HO-1 modulates oxidative stress, autophagy, and inflammation and regulates the progression of cell cycle via direct and indirect mechanisms. These beneficial effects of HO-1 induction during AKI are mediated in part by the by-products of the HO reaction (iron, carbon monoxide, and bile pigments). This review highlights recent advances in the molecular mechanisms of HO-1-mediated cytoprotection and discusses the translational potential of HO-1 induction in AKI.

Pharmacological induction of ferritin prevents osteoblastic transformation of smooth muscle cells.

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 ß-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.

Proximal tubule-targeted heme oxygenase-1 in cisplatin-induced acute kidney injury.

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.

Macrophage and epithelial cell H-ferritin expression regulates renal inflammation.

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.

Hemodiafiltration beneficially affects QT interval duration and dispersion compared to hemodialysis.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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 < 0.05). QTd was found to be 31.33 ± 10.08 ms before the commencement of hemodialysis with the largest prolongation being seen at the 240th minute (51.33 ± 14.56 ms, p < 0.05). The occurrence of ventricular premature beats was significantly higher during hemodialysis (p = 0.018). The left atrial diameter significantly decreased at the end of hemodiafiltration (at the beginning 45.1 ± 5.25 mm, at the end 40.77 ± 5.76 mm; p < 0.05). CONCLUSION: Our results suggest a beneficial effect of hemodiafiltration on the studied electrocardiographic parameters compared to hemodialysis. The larger decrease in the left atrial diameter suggests a more efficient intracardiac volume-decreasing potential of hemodiafiltration.

C-reactive protein exacerbates renal ischemia-reperfusion injury.

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.

Mitochondria-targeted heme oxygenase-1 decreases oxidative stress in renal epithelial cells.

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.

Endothelial nitric oxide synthase inhibits the development of autoimmune-mediated vasculitis in mice.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Hemodialysis and hemodiafiltration differently modulate left ventricular diastolic function.

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.