Miki Nagase

Podocyte as the Target for Aldosterone: Roles of Oxidative Stress and Sgk1

Accumulating evidence suggests that mineralocorticoid receptor blockade effectively reduces proteinuria in hypertensive patients. However, the mechanism of the antiproteinuric effect remains elusive. In this study, we investigated the effects of aldosterone on podocyte, a key player of the glomerular filtration barrier. Uninephrectomized rats were continuously infused with aldosterone and fed a high-salt diet. Aldosterone induced proteinuria progressively, associated with blood pressure elevation. Notably, gene expressions of podocyte-associated molecules nephrin and podocin were markedly decreased in aldosterone-infused rats at 2 weeks, with a gradual decrease thereafter. Immunohistochemical studies and electron microscopy confirmed the podocyte damage. Podocyte injury was accompanied by renal reduced nicotinamide-adenine dinucleotide phosphate oxidase activation, increased oxidative stress, and enhanced expression of aldosterone effector kinase Sgk1. Treatment with eplerenone, a selective aldosterone receptor blocker, almost completely prevented podocyte damage and proteinuria, with normalization of elevated reduced nicotinamide-adenine dinucleotide phosphate oxidase activity. In addition, proteinuria, podocyte damage, and Sgk1 upregulation were significantly alleviated by tempol, a membrane-permeable superoxide dismutase, suggesting the pathogenic role of oxidative stress. Although hydralazine treatment almost normalized blood pressure, it failed to improve proteinuria and podocyte damage. In cultured podocytes with consistent expression of mineralocorticoid receptor, aldosterone stimulated membrane translocation of reduced nicotinamide-adenine dinucleotide phosphate oxidase cytosolic components and oxidative stress generation in podocytes. Furthermore, aldosterone enhanced the expression of Sgk1, which was inhibited by mineralocorticoid receptor antagonist and tempol. In conclusion, podocytes are injured at the early stage in aldosterone-infused rats, resulting in the occurrence of proteinuria. Aldosterone can directly modulate podocyte function, possibly through the induction of oxidative stress and Sgk1.

Podocyte Injury Underlies the Glomerulopathy of Dahl Salt-Hypertensive Rats and Is Reversed by Aldosterone Blocker

Recent clinical studies implicate proteinuria as a key prognostic factor for renal and cardiovascular complications in hypertensives. The pathogenesis of proteinuria in hypertension is, however, poorly elucidated. Podocytes constitute the final filtration barrier in the glomerulus, and their dysfunction may play a pivotal role in proteinuria. In the present study, we examined the involvement of podocyte injury in Dahl salt-hypertensive rats, an animal model prone to hypertensive glomerulosclerosis, and explored the effects of inhibition of aldosterone. Four-week–old Dahl salt-resistant and salt-sensitive rats were fed a 0.3% or 8.0% NaCl diet. Some salt-loaded Dahl salt-sensitive rats were treated with a selective aldosterone blocker eplerenone (1.25 mg/g diet) or hydralazine (0.5 mmol/L). After 6 weeks, salt-loaded Dahl salt-sensitive rats developed severe hypertension, proteinuria, and glomerulosclerosis. Immunostaining for nephrin, a constituent of slit diaphragm, was attenuated, whereas expressions of damaged podocyte markers desmin and B7-1 were upregulated in the glomeruli of salt-loaded Dahl salt-sensitive rats. Electron microscopic analysis revealed podocyte foot process effacement. Podocytes were already impaired at as early as 2 weeks of salt loading in Dahl salt-sensitive rats, when proteinuria was modestly increased. Both eplerenone and hydralazine partially reduced systemic blood pressure as measured by indirect and direct methods in salt-loaded Dahl salt-sensitive rats, but only eplerenone dramatically improved podocyte damage and retarded the progression of proteinuria and glomerulosclerosis. Our findings suggest that podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and that inhibition of aldosterone by eplerenone is protective against podocyte damage, proteinuria, and glomerulosclerosis in this hypertensive model.

Enhanced Aldosterone Signaling in the Early Nephropathy of Rats with Metabolic Syndrome: Possible Contribution of Fat-Derived Factors

Metabolic syndrome is an important risk factor for proteinuria and chronic kidney disease independent of diabetes and hypertension; however, the underlying mechanisms have not been elucidated. Aldosterone is implicated in target organ injury of obesity-related disorders. This study investigated the role of aldosterone in the early nephropathy of 17-wk-old SHR/NDmcr-cp, a rat model of metabolic syndrome. Proteinuria was prominent in SHR/NDmcr-cp compared with nonobese SHR, which was accompanied by podocyte injury as evidenced by foot process effacement, induction of desmin and attenuation of nephrin. Serum aldosterone level, renal and glomerular expressions of aldosterone effector kinase Sgk1, and oxidative stress markers all were elevated in SHR/NDmcr-cp. Mineralocorticoid receptors were expressed in glomerular podocytes. Eplerenone, a selective aldosterone blocker, effectively improved podocyte damage, proteinuria, Sgk1, and oxidant stress. An antioxidant tempol also alleviated podocyte impairment and proteinuria, along with inhibition of Sgk1. As for the mechanisms of aldosterone excess, visceral adipocytes that were isolated from SHR/NDmcr-cp secreted substances that stimulate aldosterone production in adrenocortical cells. The aldosterone-releasing activity of adipocytes was not inhibited by candesartan. Adipocytes from nonobese SHR did not show such activity. In conclusion, SHR/NDmcr-cp exhibit enhanced aldosterone signaling, podocyte injury, and proteinuria, which are ameliorated by eplerenone or tempol. The data also suggest that adipocyte-derived factors other than angiotensin II might contribute to the aldosterone excess of this model.

Fluvastatin Ameliorates Podocyte Injury in Proteinuric Rats via Modulation of Excessive Rho Signaling

Statins have been reported to confer renoprotection in several experimental models of renal disease through pleiotropic actions. The roles of statins in glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of fluvastatin on podocyte and tubulointerstitial injury in puromycin aminonucleoside (PAN)-induced nephrosis. PAN induced massive proteinuria and serum creatinine elevation on day 7, which were significantly suppressed by fluvastatin. Immunofluorescence studies of podocyte-associated proteins nephrin and podocin revealed diminished and discontinuous staining patterns in rats with PAN nephrosis, indicating severe podocyte injury. Fluvastatin treatment dramatically mitigated the abnormal staining profiles. Reduction of nephrin expression by PAN and its reversal by fluvastatin were confirmed by quantitative analyses. By electron microscopy, effacement of foot processes was ameliorated in fluvastatin-treated rats. Fluvastatin also mitigated tubulointerstitial damage in PAN nephrosis, with the repression of PAN-induced NF-kappaB and activator protein-1 activation in the kidneys. In addition, expression of activated membrane-bound small GTPase RhoA was markedly increased in the glomeruli of PAN nephrosis, which was inhibited by fluvastatin treatment. In cultured podocytes, fluvastatin suppressed PAN-evoked activation of RhoA and actin cytoskeletal reorganization. Furthermore, fasudil, a specific Rho-kinase inhibitor, successfully ameliorated PAN-induced podocyte damage and proteinuria. In summary, fluvastatin alleviated podocyte and tubulointerstitial injury in PAN nephrosis. The beneficial effects of fluvastatin on podocytes can be attributable to direct modulation of excessive RhoA activity. Our data suggest a therapeutic role for statins in clinical conditions that are relevant to podocyte injury.

Angiogenesis within the developing mouse neural tube is dependent on sonic hedgehog signaling: possible roles of motor neurons

Embryonic morphogenesis of vascular and nervous systems is tightly coordinated, and recent studies revealed that some neurogenetic factors such as Sonic hedgehog (Shh) also exhibit angiogenetic potential. Vascularization within the developing mouse neural tube depends on vessel sprouting from the surrounding vascular plexus. Previous studies implicated possible roles of VEGF/Flk‐1 and Angiopoietin‐1(Ang‐1)/Tie‐2 signaling as candidate molecules functioning in this process. Examining gene expressions of these factors at embryonic day (E) 9.5 and 10.5, we unexpectedly found that both VEGF and Ang‐1 were expressed in the motor neurons in the ventral neural tube. The motor neurons were indeed located in the close vicinity of the infiltrating vessels, suggesting involvement of motor neurons in the sprouting. To substantiate this possibility, we inhibited induction of the motor neurons in the cultured mouse embryos by cyclopamine, a Shh signaling blocker. The vessel sprouting was dramatically impaired by inhibition of Shh signaling, together with nearly complete loss of the motor neurons. Expression of Ang‐1, but not VEGF, within the neural tube was remarkably reduced in the cyclopamine treated embryos. These results suggest that the neural tube angiogenesis is dependent on Shh signaling, and mediated, at least in part, by the Ang‐1 positive motor neurons.

Gene expression changes of sonic hedgehog signaling cascade in a mouse embryonic model of fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) is a congenital anomaly attributable to prenatal maternal excessive intake of ethanol. The authors made a mammalian model of FAS by culturing mouse embryos with high ethanol for embryonic day 7.8 to 9.5 in the whole embryo culture system. The embryos exposed to high ethanol were smaller and less advanced in development than were the embryos in the control group and showed craniofacial abnormalities, such as a fusion defect of the neural tube. The expression patterns of CRABP-I and AP-2 as markers of the neural crest cells were mostly unchanged in the in situ hybridization. However, the density and area of the expression were decreased, possibly because of the death of the neural crest cells. The expression patterns of the Sonic hedgehog signaling cascade genes (Shh, Ptc-1 and Gli-1) were mostly unchanged in the in situ hybridization, but the quantitative expressions of Ptc-1 and Gli-1 were increased in real-time reverse transcriptase-polymerase chain reaction analyses, quite contrary to the findings of a previous study using chick embryos. These findings suggest Shh signaling also is involved in the pathogenesis of FAS in mammalian embryo, but in a mode different from that in the chick embryo.

Hedgehog signaling: a biophysical or biomechanical modulator in embryonic development?

Abstract:u2002 Although embryonic development is inevitably affected by biophysical or biomechanical processes, it has yet to be elucidated to what extent molecular mechanisms of development are modulated by such physical factors. The hedgehog family, including Sonic hedgehog (Shh), is the most well‐known morphogens involved in the developmental pattern formation of various organs, such as the nervous system, face, limbs, and skin appendages. There are several unique features in hedgehog signaling including long‐range diffusion or positive and negative feedback loops, suggesting the possible modification of hedgehog signaling by biophysical or biomechanical factors. Especially, the period of embryonic day 8–10 is characterized by various biomechanically regulated processes in mouse development, such as axial rotation and vasculoangiogenesis. We executed a series of experiments using a mouse whole embryo culture system to investigate the biomechanical roles of hedgehog signaling during this period. In this review, we examine various examples in which biophysical and biomechanical aspects of hedgehog signaling in development are revealed, including our own data using the mouse whole embryo culture system.

Defects in aortic fusion and craniofacial vasculature in the holoprosencephalic mouse embryo under inhibition of sonic hedgehog signaling.

Sonic hedgehog (Shh) is a well-known morphogen indispensable in facial and nervous development, and recently it has also garnered much attention as a potent angiogenic factor. We previously created an animal model of holoprosencephaly by administration of cyclopamine, a specific inhibitor of hedgehog signaling, to the mouse embryos cultured in vitro, and found several types of angiogenic defects. In this study, we focused on other angiogenic phenotypes in the same model. When cyclopamine was added for embryonic day (E) 8.0-9.5, a pair of immature dorsal aortae, which normally fuse to form the single aorta by E9.5, remained to be separated. Expressions of vascular endothelial growth factor and bone morphogenetic protein 4, putative mediators of aortic fusion, were also reduced around the aorta by blockade of Shh signaling. When cyclopamine was added for E8.5-10.5, vessels on the surface of craniofacial region (possibly external cardinal veins) were extended and malformed. These results suggest that Shh signaling is essential for some aspects of embryonic angiogenesis, and that pathophysiology of holoprosencephaly may involve, at least in part, the Shh-dependent angiogenesis.

Neurospheres From Human Adipose Tissue Transplanted Into Cultured Mouse Embryos can Contribute to Craniofacial Morphogenesis : A Preliminary Report

Adipose-derived stromal cells (ASCs) are one of the most promising stem cell populations that differentiate into the mesodermal as well as neural lineages in vitro. In this study, we examined the neural differentiating potential of human ASCs by a neurosphere culture method. Neurospheres derived from human ASCs expressed Nestin and Musashi-1 genes, which are marker genes for neural stem cells. When these cells were labeled with green fluorescent protein gene transfection by Sendai virus vector and transplanted into the head region of mouse embryos using a whole embryo culture system, these cells were incorporated into the craniofacial development. Some transplanted cells appeared to migrate along the second branchial arches, implicating some similarity to the cranial neural crest cells. Although preliminary, our results support an idea that ASC-derived neurospheres have properties of neural progenitors in vitro and in vivo.

Changes in angiogenic gene expression in a case of expanded capillary malformation: does an expanded capillary malformation grow?

We examined a 59-year-old woman with a capillary malformation (CM) in her thigh, which was serially excised. Interestingly, the remnant CM after the first excision was enlarged at the time of the second excision. To investigate whether this phenomenon was caused by mere passive expansion or regrowth, the CM specimens of the first operation (nonexpanded) and the second operation (expanded) were examined. Expressions of angiogenic genes Tie2 and Angiopoietin-1 were up-regulated within the expanded CM compared with the nonexpanded lesion, suggesting angiogenesis in the expanded CM. Expression pattern of the endothelial marker von Willebrand factor and the capillary densities were unchanged after the excision, suggesting that angiogenesis seen in the expanded CM resulted in reorganization of vascular networks. We consider that our data support a hypothesis that the expanded lesion in this case was caused by regrowth, not a passive expansion, of the CM.