Synaptopodin Is Dispensable for Normal Podocyte Homeostasis but Is Protective in the Context of Acute Podocyte Injury

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Development of Neuropathic Pain in the Rat Spared Nerve Injury Model Is Not Prevented by a Peripheral Nerve Block

Background: The mechanisms responsible for initiation of persistent neuropathic pain after peripheral nerve injury are unclear. One hypothesis is that injury discharge and early ectopic discharges in injured nerves produce activity-dependent irreversible changes in the central nervous system. The aim of this study was to determine whether blockade of peripheral discharge by blocking nerve conduction before and 1 week after nerve injury could prevent the development and persistence of neuropathic pain-like behavior in the spared nerve injury model.

Methods: Bupivacaine-loaded biodegradable microspheres embedded in fibrin glue were placed in a silicone tube around the sciatic nerve to produce a conduction block. After sensory-motor testing of block efficacy, a spared nerve injury procedure was performed. Development of neuropathic pain behavior was assessed for 4 weeks by withdrawal responses to stimulation (i.e., von Frey filaments, acetone, pinprick, radiant heat) in bupivacaine microspheres-treated animals (n = 12) and in controls (n = 11).

Results: Bupivacaine microspheres treatment produced conduction blockade with a complete lack of sensory responsiveness in the sural territory for 6 to 10 days. Once the block wore off, the degree of hypersensitivity to stimuli was similar in both groups.     

Hemorrhage-Induced Hepatic Injury and Hypoperfusion can be Prevented by Direct Peritoneal Resuscitation

Background  Crystalloid fluid resuscitation after hemorrhagic shock (HS) that restores/maintains central hemodynamics often culminates in multi-system organ failure and death due to persistent/progressive splanchnic hypoperfusion and end-organ damage. Adjunctive direct peritoneal resuscitation (DPR) using peritoneal dialysis solution reverses HS-induced splanchnic hypoperfusion and improves survival. We examined HS-mediated hepatic perfusion (galactose clearance), tissue injury (histopathology), and dysfunction (liver enzymes). Methods  Anesthetized rats were randomly assigned (n = 8/group): (1) sham (no HS); (2) HS (40% mean arterial pressure for 60 min) plus conventional i.v. fluid resuscitation (CR; shed blood + 2 volumes saline); (3) HS + CR + 30 mL intraperitoneal (IP) DPR; or (4) HS + CR + 30 mL IP saline. Hemodynamics and hepatic blood flow were measured for 2 h after CR completion. In duplicate animals, liver and splanchnic tissues were harvested for histopathology (blinded, graded), hepatocellular function (liver enzymes), and tissue edema (wet–dry ratio). Results  Group 2 decreased liver blood flow, caused liver injuries (focal to submassive necrosis, zones 2 and 3) and tissue edema, and elevated liver enzymes (alanine aminotransferase (ALT), 149 ± 28 μg/mL and aspartate aminotransferase (AST), 234 ± 24 μg/mL; p < 0.05) compared to group 1 (73 ± 9 and 119 ± 10 μg/mL, respectively). Minimal/no injuries were observed in group 3; enzymes were normalized (ALT 89 ± 9 μg/mL and AST 150 ± 17 μg/mL), and tissue edema was similar to sham. Conclusions  CR from HS restored and maintained central hemodynamics but did not restore or maintain liver perfusion and was associated with significant hepatocellular injury and dysfunction. DPR added to conventional resuscitation (blood and crystalloid) restored and maintained liver perfusion, prevented hepatocellular injury and edema, and preserved liver function. Presented at the Digestive Disease Week, American Association for the Study of Liver Diseases, Los Angeles, CA, USA, May 2006. No conflicts of interest exist. Grant support: This project was supported by a VA Merit Review grant and by NIH research Grant # 5R01 HL076160-03, funded by the National Heart, Lung, and Blood Institute and the United States Army Medical Resources and Material Command.     

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl-xL

Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immune-privileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium.     

尿毒清颗粒对糖尿病大鼠足细胞损伤的保护作用研究

目的：探讨尿毒清颗粒对糖尿病大鼠足细胞损伤的保护作用。方法：将SD大鼠制备成STZ糖尿病模型,实验分3组：正常对照组、糖尿病未干预组、尿毒清颗粒治疗组（2.6g·kg-1·d-1灌胃）,于实验第4周、8周末检测血糖、血肌酐、尿肌酐及尿白蛋白排泄率,光镜、电镜下观察肾组织病理改变并计数足突宽度,免疫组化观察足细胞相关蛋白分子nepherin、podocin的表达,Real time-PCR检测肾皮质nepherin、podocin mRNA表达。结果：尿毒清颗粒可以改善糖尿病大鼠肌酐清除率,降低尿白蛋白排泄,改善肾脏病理,减轻足突融合,维持足细胞相关蛋白分子的分布与表达。结论：初步证实尿毒清颗粒能通过上调足细胞相关蛋白分子水平减轻足细胞损伤,对糖尿病大鼠足细胞损伤具有保护作用。     

Disruption of Myosin 1e Promotes Podocyte Injury

Myosin 1e (Myo1e) is one of two Src homology 3 domain–containing “long-tailed” type I myosins in vertebrates, whose functions in health and disease are incompletely understood. Here, we demonstrate that Myo1e localizes to podocytes in the kidney. We generated Myo1e-knockout mice and found that they exhibit proteinuria, signs of chronic renal injury, and kidney inflammation. At the ultrastructural level, renal tissue from Myo1e-null mice demonstrates changes characteristic of glomerular disease, including a thickened and disorganized glomerular basement membrane and flattened podocyte foot processes. These observations suggest that Myo1e plays an important role in podocyte function and normal glomerular filtration.Myosins are molecular motors that translocate cargo along actin filaments in an ATP-dependent manner. Members of the myosin superfamily, which includes at least 24 myosin classes,¹ contribute to a variety of intracellular functions, including organelle transport, actin reorganization, and cell signaling. Genetic evidence in mice and humans links myosin mutations to hearing and vision defects, neurologic problems, cardiomyopathies, immune system disorders, pigmentation defects, and cancer.²This study focuses on physiologic roles of myosin 1e (myo1e), a class I myosin. Class I myosins consist of a single motor domain, a neck domain that binds one or more calmodulin (or calmodulin-like) light chains, and a cargo-binding tail domain.² Some class I myosins have tails that consist of a single domain, termed tail homology 1 (TH1) domain. This domain is basic in charge and may effect interactions with negatively charged phospholipids. Other class I myosins, including myo1e, have longer tails that in addition to TH1 include a proline-rich TH2 domain and C-terminal Src homology 3 domains. Humans and mice express eight myosin I isoforms (1a through h); functions of three myosin I isoforms (1a, 1c, and 1f) have been previously analyzed using genetic manipulation in mice.³ Mice lacking myo1a, a myosin that is expressed exclusively in intestinal epithelial cells, exhibit defects in the organization of the intestinal brush border.⁴ Myo1c has been implicated in the adaptation by the inner ear sensory hair cells; the importance of myo1c for adaptation has been established, in part, using transgenic mice expressing a mutant version of myo1c that could be selectively inhibited using a modified ADP analog.⁵ Knockout (KO) of myo1f, a long-tailed Myo1 closely related to myo1e, results in defects in neutrophil migration, which are linked to changes in integrin exocytosis and enhanced cell-substrate adhesion.⁶Myo1e is expressed in a wide variety of tissues, including spleen, kidney, small intestine, pancreas, brain, and the immune system.^{6, 7} We previously determined that myo1e tail binds endocytic proteins dynamin and synaptojanin and found that inhibition of myo1e functions in cultured fibroblasts led to defects in endocytosis.⁸ To analyze myo1e functions in vivo, we developed a myo1e-KO mouse and characterized its phenotype. Myo1e was expressed in the glomerular visceral epithelial cells, or podocytes, which play a central role in glomerular filtration. Myo1e-null mice exhibited defects in glomerular filtration and organization, which were similar to glomerular defects observed in inherited renal diseases that have been linked to mutations in podocyte-expressed proteins in humans.^{9, 10} Thus, myo1e seems to be necessary for normal renal filtration and disruption of myo1e functions may contribute to renal disease.     

Obesity-Induced Endothelial Dysfunction Is Prevented by Deficiency of P-Selectin Glycoprotein Ligand-1

Endothelial dysfunction precedes atherosclerosis and represents an important link between obesity and cardiovascular events. Strategies designed to prevent endothelial dysfunction may therefore reduce the cardiovascular complications triggered by obesity. We tested the hypothesis that deficiency of P-selectin glycoprotein ligand-1 (Psgl-1) would improve the endothelial dysfunction associated with obesity. Psgl-1-deficient (Psgl-1^−/−) and wild-type (Psgl-1^+/+) mice were fed standard chow or a high-fat, high-sucrose diet (diet-induced obesity [DIO]) for 10 weeks. DIO increased mesenteric perivascular adipose tissue (mPVAT) macrophage content and vascular oxidative stress in Psgl-1^+/+ mice but not in Psgl-1^−/− mice. Pressure myography using mesenteric arteries demonstrated that relaxation responses to acetylcholine were significantly impaired in DIO Psgl-1^+/+ mice, whereas DIO Psgl-1^−/− mice were protected from endothelial dysfunction with similar relaxation responses to Psgl-1^+/+ or Psgl-1^−/− mice fed standard chow. The superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) partially recovered impaired endothelial function induced by DIO. A neutralizing Psgl-1 antibody was also effective in preventing endothelial dysfunction and reducing mPVAT macrophage content induced by DIO. These results indicate that obesity in mice leads to PVAT inflammation and endothelial dysfunction that is prevented by Psgl-1 deficiency. Psgl-1 inhibition may be a useful treatment strategy for targeting vascular disease associated with obesity.Obesity is epidemic in the United States and is associated with increased risk for cardiovascular complications (1). Excess visceral adipose tissue may be the primary driver of the vascular risk associated with obesity because visceral fat is strongly associated with a chronic, low-grade inflammatory state characterized by increased macrophage activity in adipose tissue (2,3). The mechanism(s) by which obesity promotes vascular disease is unclear, but endothelial dysfunction has been demonstrated even in children with obesity (4,5). Endothelial dysfunction due to impairment of nitric oxide (NO) activity represents an early stage of many cardiovascular diseases (6).P-selectin glycoprotein ligand-1 (Psgl-1) is the primary leukocyte ligand for P-selectin (P-sel) and an important ligand for E-selectin (E-sel) (7). Psgl-1 deficiency has been shown to reduce leukocyte-endothelial interactions in obesity and to reduce macrophage accumulation in gonadal fat pads (8). The purpose of this study was to determine the effect of Psgl-1 deficiency on endothelial dysfunction associated with obesity.     

Protamine-induced Cardiotoxicity Is Prevented by Anti-TNF-α Antibodies and Heparin

Background : We investigated the role of tumor necrosis factor [alpha] (TNF-[alpha]) in protamine-induced cardiotoxicity and the possibility of preventing or decreasing this effect by anti TNF-[alpha] antibodies and heparin.

Methods : Isolated rat hearts were perfused for 60 min with Krebs-Henseleit solution (KH). The control group was perfused with KH alone, the KH > protamine > KH group was treated from the 20th to the 40th minute with protamine, and the KH + anti-TNF > protamine + anti-TNF > KH + anti-TNF group was treated the same as the KH > protamine > KH group but with anti-TNF-[alpha] antibodies added throughout perfusion. The KH + heparin > protamine + heparin > KH + heparin group was treated the same as the KH > protamine > KH group but with heparin added to KH throughout perfusion. The KH > protamine > KH + heparin was perfused the same as the KH> protamine > KH group but with heparin added to KH for the last 20 min. Left ventricular (LV) function and coronary flow were measured every 10 min. TNF-[alpha] was measured in the coronary sinus effluent. Left ventricular TNF messenger RNA was determined in the control and KH > protamine > KH groups at baseline and after the 40-min perfusion.

Results : Protamine caused a significant decrease of peak systolic pressure and dP/dt (to 25% of baseline). Significant amounts of TNF-[alpha] in the effluent in the KH > protamine > KH group (102.3 +/- 15.5 pg/min) and TNF messenger RNA expression in left ventricular samples were detected. TNF-[alpha] was below detectable concentrations in the control, KH + anti-TNF > protamine + anti-TNF > KH + anti-TNF, and KH + heparin > protamine + heparin > KH + heparin groups. TNF-[alpha] concentrations correlated with depression of LV peak systolic pressure (r = 0.984;P = 0.01) and first derivate of the increase of LV pressure (r = 0.976;P = 0.001). Heparin improved LV recovery and decreased protamine-induced TNF-[alpha] release (KH > protamine > KH + heparin group).     

Unraveling the Role of Podocyte Turnover in Glomerular Aging and Injury

Podocyte loss is a major determinant of progressive CKD. Although recent studies showed that a subset of parietal epithelial cells can serve as podocyte progenitors, the role of podocyte turnover and regeneration in repair, aging, and nephron loss remains unclear. Here, we combined genetic fate mapping with highly efficient podocyte isolation protocols to precisely quantify podocyte turnover and regeneration. We demonstrate that parietal epithelial cells can give rise to fully differentiated visceral epithelial cells indistinguishable from resident podocytes and that limited podocyte renewal occurs in a diphtheria toxin model of acute podocyte ablation. In contrast, the compensatory programs initiated in response to nephron loss evoke glomerular hypertrophy, but not de novo podocyte generation. In addition, no turnover of podocytes could be detected in aging mice under physiologic conditions. In the absence of podocyte replacement, characteristic features of aging mouse kidneys included progressive accumulation of oxidized proteins, deposits of protein aggregates, loss of podocytes, and glomerulosclerosis. In summary, quantitative investigation of podocyte regeneration in vivo provides novel insights into the mechanism and capacity of podocyte turnover and regeneration in mice. Our data reveal that podocyte generation is mainly confined to glomerular development and may occur after acute glomerular injury, but it fails to regenerate podocytes in aging kidneys or in response to nephron loss.Chronic loss of kidney function is of significant public health importance not only because of high prevalence but also because it is a major independent risk factor for cardiovascular morbidity and mortality.¹ Aged kidneys display an increased susceptibility for progressive diseases, suggesting that overlapping molecular programs contribute to organ aging and disease progression.² Recent data indicate that a decrease in the number of glomerular podocytes is an important predictor for kidney aging, and podocytes are considered the weak link in the progression of CKD.³ Animal models have shown that podocyte depletion of up to 20% can be tolerated before a scarring response takes place,⁴ but residual podocytes are unable to undergo cell division.^5–7 Although it is widely believed that glomeruli have only a limited capacity to resolve lesions, cases of potential podocyte regeneration and disease reversal have been described.^8–10 These recent observations have instigated hope of finding ways to stimulate kidney regeneration. So far, two potential podocyte progenitor niches, bone marrow cells and parietal epithelial cells, have been characterized.^11–14 Bone marrow transplantation has an ameliorating effect in several animal models of glomerular disease,^15–18 but the underlying mechanisms remain poorly understood. In humans, Y-chromosome–positive podocytes have been detected in kidneys transplanted from females to males.¹² Located directly adjacent to podocytes, parietal epithelial cells express the stem cell markers CD24 and CD133 in human tissue and are capable of self-renewal, as well as differentiation into several cell types, including podocytes.^11,14,19In this study, a novel flow cytometry–based quantitative method was devised to assess the regenerative capacity of podocytes during diphtheria toxin (DT)–mediated acute podocyte loss, models of chronic nephron loss, and kidney aging.     

Distinct Roles for Basal and Induced COX-2 in Podocyte Injury

Transgenic mice that overexpress cyclooxygenase-2 (COX-2) selectively in podocytes are more susceptible to glomerular injury by adriamycin and puromycin (PAN). To investigate the potential roles of COX-2 metabolites, we studied mice with selective deletion of prostanoid receptors and generated conditionally immortalized podocyte lines from mice with either COX-2 deletion or overexpression. Podocytes that overexpressed COX-2 were virtually indistinguishable from wild-type podocytes but were significantly more sensitive to PAN-induced injury, produced more prostaglandin E₂ and thromboxane B₂, and had greater expression of prostaglandin E₂ receptor subtype 4 (EP₄) and thromboxane receptor (TP). Treatment of COX-2-overexpressing podocytes with a TP antagonist reduced apoptosis, but treatment with an EP₄ antagonist did not. In contrast, podocytes from COX-2-knockout mice exhibited increased apoptosis, markedly decreased cell adhesion, and prominent stress fibers. In vivo, selective deletion of podocyte EP₄ did not alter the increased sensitivity to adriamycin-induced injury observed in mice overexpressing podocyte COX-2. In contrast, genetic deletion of TP in these mice prevented adriamycin-induced injury, with attenuated albuminuria and foot process effacement. These results suggest that basal COX-2 may be important for podocyte survival, but overexpression of podocyte COX-2 increases susceptibility to podocyte injury, which is mediated, in part, by activation of the thromboxane receptor.Podocytes play a crucial role in regulation of glomerular function, and podocyte injury is an essential feature of progressive glomerular diseases. Although our understanding of podocyte biology has dramatically increased in recent years, mechanisms underlying functional and structural podocyte disturbances in renal diseases are still incompletely understood.¹ Recent studies indicate that local podocyte damage can spread to induce injury in otherwise healthy podocytes and further affect both glomerular endothelial and mesangial cells, implying that even limited podocyte injury might initiate a vicious cycle of progressive glomerular damage.²Mice with cyclooxygenase-2 (COX-2) gene deletion exhibit impaired glomerulogenesis and renal cortical development.³ However, increased expression of COX-2 in podocytes has been reported in various experimental models of progressive glomerular injury^4,5 and in cultured podocytes stimulated by mechanical stress.⁶ Furthermore, in models of renal ablation, diabetic nephropathy, and salt-sensitive hypertension, inhibition of COX-2 activity by selective COX-2 inhibitors significantly decreases proteinuria and progressive renal injury.^7–11 To determine if increased podocyte COX-2 expression plays a pathogenic role in glomerular injury, we recently generated COX-2 transgenic mice driven by a nephrin promoter, successfully inducing selective upregulation of COX-2 expression in podocytes. Although glomerular structure and function were normal at baseline in these transgenic mice, administration of either adriamycin or puromycin (PAN) led to significantly increased albuminuria compared with wild-type mice and induced further upregulation of COX-2 and downregulation of the slit diaphragm molecule nephrin. These studies suggested that increased podocyte COX-2 in response to injury may predispose the podocyte to further injury.^12,13 In cultured podocytes, mechanical stress induced COX-2 and expression of the prostaglandin E₂ (PGE₂) receptor subtype 4 (EP₄), and PGE₂ stimulation of stretched podocytes resulted in a loss of actin stress fiber organization.⁶ These results suggest that enhanced prostanoid signaling may be a facilitating event for morphologic changes and may directly influence podocyte function under pathophysiological conditions that promote synthesis of COX-2 metabolites. To investigate potential roles of COX-2 metabolites in podocyte function, we generated conditionally immortalized podocyte lines with either deficiency or overexpression of COX-2. In further in vitro and in vivo studies, we identified a role for COX-2-derived prostanoids as potential mediators of podocyte injury.     

Glucocorticoid-Induced Osteoporosis in Growing Mice Is Not Prevented by Simultaneous Intermittent PTH Treatment

Glucocorticoids (GCs) are widely used in medicine for treatment of chronic diseases. Especially in children, prolonged treatment causes growth retardation and early onset of osteoporosis. Human parathyroid hormone (PTH) has an anabolic effect on bone when administrated intermittently. The aim of the present study was to examine whether a combined therapy of dexamethasone (DEX) and PTH could prevent the detrimental effects of GC on cortical and trabecular bone in the femur and vertebrae of growing mice. Three-week-old female FVB mice were treated with control, DEX, PTH, or a combination of DEX and PTH by daily subcutaneous injections. After 4 weeks, animals were killed and the femur and L5 vertebra were isolated. Cortical and trabecular bone parameters and relative calcium density were measured by high-resolution X-ray micro-computed tomography (micro-CT). In the femur, PTH can reverse the effects of DEX on bone volume to control. However, it cannot reverse the undermineralization, which most likely is a strong contributor to bone fragility. In the vertebra, PTH improves bone volume to some extent but does not restore the values to normal. It augments the negative effect of DEX on mineralization. We conclude that the detrimental effects of DEX in the growing skeleton cannot be prevented by simultaneous PTH treatment.     

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype

Parietal epithelial cells have been identified as potential progenitor cells in glomerular regeneration, but the molecular mechanisms underlying this process are not fully defined. Here, we established an immortalized polyclonal human parietal epithelial cell (hPEC) line from naive human Bowman’s capsule cells isolated by mechanical microdissection. These hPECs expressed high levels of PEC-specific proteins and microRNA-193a (miR-193a), a suppressor of podocyte differentiation through downregulation of Wilms’ tumor 1 in mice. We then investigated the function of miR-193a in the establishment of podocyte and PEC identity and determined whether inhibition of miR-193a influences the behavior of PECs in glomerular disease. After stable knockdown of miR-193a, hPECs adopted a podocyte-like morphology and marker expression, with decreased expression levels of PEC markers. In mice, inhibition of miR-193a by complementary locked nucleic acids resulted in an upregulation of the podocyte proteins synaptopodin and Wilms’ tumor 1. Conversely, overexpression of miR-193a in vivo resulted in the upregulation of PEC markers and the loss of podocyte markers in isolated glomeruli. Inhibition of miR-193a in a mouse model of nephrotoxic nephritis resulted in reduced crescent formation and decreased proteinuria. Together, these results show the establishment of a human PEC line and suggest that miR-193a functions as a master switch, such that glomerular epithelial cells with high levels of miR-193a adopt a PEC phenotype and cells with low levels of miR-193a adopt a podocyte phenotype. miR-193a–mediated maintenance of PECs in an undifferentiated reactive state might be a prerequisite for PEC proliferation and migration in crescent formation.     

ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage

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Sensitization to Minor Antigens Is a Significant Barrier in Bone Marrow Transplantation and Is Prevented by CD154:CD40 Blockade

Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation rejection. The mechanism of sensitization to minor histocompatibility antigens (Mi‐HAg) has not been thoroughly explored. We used a mouse model of allosensitization to Mi‐HAg to study the Mi‐HAg sensitization barrier in bone marrow transplantation (BMT). AKR mice were sensitized with MHC congenic Mi‐HAg disparate B10.BR skin grafts. Adaptive humoral (B‐cells) and cellular (T cells) responses to Mi‐HAg are elicited. In subsequent BMT, only 20% of sensitized mice engrafted, while 100% of unsensitized mice did. In vivo cytotoxicity assays showed that Mi‐HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes significantly more rapidly than naïve AKR mice but less rapidly than MHC‐sensitized recipients. Sera from Mi‐HAg sensitized mice also reacted with cells from other mouse strains, suggesting that Mi‐HAg peptides were broadly shared between mouse strains. The production of anti‐donor‐Mi‐HAg antibodies was totally prevented in mice treated with anti‐CD154 during skin grafting, suggesting a critical role for the CD154:CD40 pathway in B‐cell reactivity to Mi‐HAg. Moreover, anti‐CD154 treatment promoted BM engraftment to 100% in recipients previously sensitized to donor Mi‐HAg. Taken together, Mi‐HAg sensitization poses a significant barrier in BMT and can be overcome with CD154:CD40 costimulatory blockade.     

Mechanisms of Podocyte Injury in Diabetes: Role of Cytochrome P450 and NADPH Oxidases

OBJECTIVE

We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.

RESULTS

Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.

CONCLUSIONS

Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.Diabetic nephropathy in humans is characterized by increased urinary albumin excretion (microalbuminuria), which often progresses to proteinuria, one of the most important prognostic risk factors for kidney disease progression (1). Glomerular visceral epithelial cells, or podocytes, play a critical role in maintaining the structure and function of the glomerular filtration barrier. Careful morphometric analyses of renal biopsy in subjects with type 1 and type 2 diabetes (2–4) demonstrate that the density of podocytes is reduced not only in individuals with diabetic nephropathy, but also in patients with short duration of diabetes before the onset of microalbuminuria (4,5). Studies in experimental models of type 1 and type 2 diabetes have also documented that podocyte depletion represents one of the earliest cellular lesions affecting the diabetic kidney (6,7). Among various morphologic characteristics, the decreased number of podocytes in glomeruli is the strongest predictor of progression of diabetic nephropathy, where fewer cells predict more rapid progression (3,4). Although these observations identify podocyte depletion as one of the earliest cellular features of diabetic kidney disease, the mechanisms that underlie the loss of podocytes in diabetic nephropathy remain poorly understood.High glucose induces apoptosis (8), and there is evidence that podocyte apoptosis contributes to reduced podocyte number (9). High glucose, transforming growth factor-β (TGF-β), and angiotensin II (ANGII) induce apoptosis of cultured podocytes (9–12). ANGII appears to induce apoptosis in cultured rat glomerular epithelial cells at least partially via TGF-β because its apoptotic effect is attenuated by an anti–TGF-β antibody (12). There is also evidence that reactive oxygen species (ROS) contribute to podocyte apoptosis and depletion in cells exposed to high glucose and in experimental diabetic nephropathy (7). However, the sources of ROS and the kinetics of their generation have not been well characterized. We and others (13–15) have recently identified NADPH oxidases as major sources of ROS in kidney cortex and glomeruli of rats with type 1 diabetes. Six homologs of the cytochrome subunit of the phagocyte NADPH oxidase (Nox2/gp91^phox) have been cloned (16). At least three different Nox isoforms are expressed in the kidney cortex: Nox1, Nox2, and Nox4 (16). Cytochromes P450 (CYP450s) are significant sources of ROS in many tissues (17,18). CYP450 metabolizes arachidonic acid into hydroxyeicosatetraenoic acids (20-HETEs) and EETs (epoxyeicosatrienoic acids). 20-HETE, the ω-hydroxylation product of arachidonic acid, is one of the major CYP eicosanoids produced in the kidney cortex (19–21). The predominant CYP450 in the kidney cortex that synthesizes 20-HETE is cytochrome P450 of the 4A family (CYP4A) (19–21). 20-HETE has multiple and opposing functions depending on the site of production and target cells/tissues (19,22–24).In this study, we demonstrate that high glucose induces ROS production and apoptosis in cultured mouse podocytes through the upregulation of CYP4A with increased production of 20-HETE and upregulation of NADPH oxidases. Inhibition of 20-HETE production prevented podocyte apoptosis in vitro and decreased oxidative stress, podocyte apoptosis, and proteinuria in an in vivo model of type 1 diabetes.     

Hematopoietic MicroRNA-126 Protects against Renal Ischemia/Reperfusion Injury by Promoting Vascular Integrity

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow–derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin⁻/Sca-1⁺/cKit⁺ hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin⁻/Sca-1⁺/cKit⁺ cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin⁻/Sca-1⁺/cKit⁺ cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell–derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.Ischemia/reperfusion injury (IRI) is a central event in such clinical conditions as AKI and in organ transplantation, and it is strongly associated with delayed graft function and long-term graft survival.^1–3 Emerging evidence indicates that the renal microvascular endothelium of the outer medullary peritubular network is the primary site of injury in the pathogenesis of ischemia-induced renal dysfunction.⁴ Following ischemia, perfusion of the peritubular capillary network is rapidly impaired as a consequence of endothelial cell (EC) swelling,⁵ impaired vasorelaxation,⁶ and increased leukocyte adhesion.⁷ In addition, microvascular destabilization initiated by the loss of EC–EC interaction⁸ and EC–pericyte interactions can lead to significant reductions in peritubular capillary density due to microvascular rarefaction.^8,9 The resulting loss in renal perfusion can further exacerbate medullary ischemia and drive the development of interstitial fibrosis by stimulation of profibrogenic factors, such as TGF-β.¹⁰ As a consequence, integrity of the peritubular capillary network is a key determinant for the preservation of renal function. Indeed, clinical biopsy studies have shown an association between loss of tubular structure and function on the one hand and capillary rarefaction on the other.^11,12Because of their limited replicative capacity, renal ECs are thought to be insufficiently capable to completely repair the injured endothelium of the peritubular capillary plexus after IRI.^13,14 Therefore, current therapeutic strategies to prevent microvascular loss have focused on the prevention of pericyte perturbation to reduce capillary rarefaction.^15–18 However, once rarefaction has occurred, these strategies may fail to induce sufficient revascularization required to reverse renal dysfunction.¹⁹ In search of ways to augment neovascularization in the injured kidney, many laboratories have investigated the biology and therapeutic use of circulating vascular progenitor cells originating from the bone marrow (BM) compartment.²⁰ These progenitor cells were shown to incorporate into the injured microvasculature in experimental models for GN,²¹ ischemic nephropathy,^22,23 and interstitial fibrosis.²⁴ This phenomenon has been particularly observed when extensive or repetitive endothelial injury occurs, for example in kidney transplantation.²⁵ Microvascular incorporation of BM-derived progenitor cells has been linked to preservation of the vasculature because they may serve as an alternative cellular source to facilitate re-endothelialization.²⁶ In addition, the CXCR4⁺ fraction of progenitor cells is mobilized to the ischemic kidney by local secretion of the chemokine stromal cell–derived factor-1 (SDF-1)^23,27 and has been shown to exert renoprotective effects in a paracrine fashion.Phosphoinositide 3-kinase (PI3K)/AKT signaling in progenitor cells plays a critical role in mobilization of progenitors from the BM via the SDF-1/CXCR4 axis²⁸ and their subsequent differentiation toward vascular cells.²⁹ MicroRNA-126 (miR-126) is a key regulator of PI3K/AKT signaling by direct targeting of the negative regulator PI3K regulatory subunit 2 (PI3KR2/p85-β) and targets genes that play key roles in angiogenesis and inflammation.^29–31 In addition, miR-126 was shown to coregulate the expansion and mobilization of hematopoietic stem cells and progenitor cells.^32,33 We hypothesized that miR-126 overexpression in the hematopoietic compartment of mice can enhance the vasoprotective potential of these progenitors and that this will translate to decreased renal injury.