Blockade of mineralocorticoid receptor has been shown to improve the clinical outcomes of proteinuric kidney diseases. However, little is known about the regulation of mineralocorticoid receptor-dependent transcriptional activity in renal disease. Here we identify a new role for Rac1, a member of the Rho family GTPases, as a potent activator of mineralocorticoid receptor signal transduction both in vitro and in vivo. Transient transfection assays in HEK 293 cells revealed that constitutively active Rac1 (CA-Rac1) enhanced mineralocorticoid receptor-dependent reporter activity, which was accompanied by increased nuclear translocation of mineralocorticoid receptor. CA-Rac1 facilitated mineralocorticoid receptor nuclear accumulation also in podocytes via p21-activated kinase phosphorylation. In mice lacking Rho GDP-dissociation inhibitor-alpha (Arhgdia(-/-) mice), renal abnormalities, including heavy albuminuria and podocyte damage, were associated with increased Rac1 (but not RhoA) and mineralocorticoid receptor signaling in the kidney, without alteration in systemic aldosterone status. Pharmacological intervention with a Rac-specific small-molecule inhibitor diminished mineralocorticoid receptor overactivity and renal damage in this model. Furthermore, albuminuria and histological changes in Arhgdia(-/-) mice were suppressed by mineralocorticoid receptor blockade, confirming the pathological role of Rac1-mineralocorticoid receptor interaction. Our results provide evidence that signaling cross-talk between Rac1 and mineralocorticoid receptor modulates mineralocorticoid receptor activity and identify Rac1 as a therapeutic target for chronic kidney disease.

Abstract-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.

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.

Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP-dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.

Abstract-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.

made equal contributions to this study. We thank Prof. M. Tohvama for a critical review of the manuscriot and Dr. Y. Kihou for helpful suggestions relating to the structure of the novel protein. We thank A. Okabe, K. &to, and A. Hirata for their technical assistance for electroencephalograms and imaging analysis. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.

Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/ insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys 32 -Ile 33 bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.Some proteases have been suggested to be related to neural cell dynamics in such processes as cell death, migration, cellto-cell adhesion and de-adhesion, process elongation, pathfinding, and axonal rearrangement (1-5). These phenomena have been investigated by supplying known proteases involved in blood coagulation, fibrinolysis, or digestion to neural cell cultures. However, the observations that the proteases are mainly localized in and released from non-neural cells do not support all of such neural effects (5-7). Thus, we postulated that neurons themselves may produce and release their own proteases to participate in the neural cell dynamics described above. Neuropsin (NP)1 was cloned from the mouse brain and was shown to be localized in mouse hippocampal pyramidal neurons (8). These results and the observation that its mRNA showed marked activity-dependent changes caused by plasticity-inducible stimuli are suggestive of some neural effects in limbic plasticity (8, 9). However, it is still not known whether NP protein has enzyme activity as suggested by the deduced amino acid sequence (8). We postulated that the enzyme activity might be a molecular basis for the physiological responses induced by various stimuli. Therefore, in the present study, we examined whether recombinant NP (r-NP) and brain NP had proteolytic activity against synthetic and natural substrates. EXPERIMENTAL PROCEDURESMaterials-Mono S, Sepharose 2B, CNBr-activated Sepharose 4B and CL-6B, Superdex-75HR, Superose 12, Resource S, and Protein G-Sepharose were from Amersham Pharmacia Biotech. Silver staining kits were from Bio-Rad. Diisopropyl fluorophosphate (DFP), benzamidine, bestatin, soybean trypsin inhibitor, human plasma thrombin (EC 3.4.4.13), and TNM-FH insect cel...

Lamellar granules (LG) of the epidermis appear as discrete round or oblong shaped granules in classical transmission electron micrographs, but a recent cryo-transmission electron microscopy study has claimed that LG are in fact branched tubular structures. LG contain various cargoes including lipids, hydrolytic enzymes, and several other proteins. It is not known whether there are any differences in the timing of expression among them and whether they are sorted into the granules individually or collectively. In order to address these questions, we studied the expression of glucosylceramides (GlcCer), cathepsin D (CatD), corneodesmosin (Cdsn), kallikrein (KLK)7, and KLK8 in normal human epidermis using confocal laser scanning microscopy and immunoelectron microscopy. The results were consistent with the model that LG are parts of a branched tubular structure. In this structure, all the components were shown to be distributed as separate aggregates. In the trans-Golgi network (TGN), bulbous protrusions containing GlcCer, Cdsn, KLK7 and KLK8, and small CatD-positive vesicles were observed. The molecules were shown to be delivered to the apical region of granular keratinocytes. This study provides strong evidence for the sequential synthesis and independent trafficking of various LG cargoes, including for the first time CatD and KLK8, from TGN.