Restricted nutrition-induced low birth weight, low number of nephrons and glomerular mesangium injury in Japanese quail.

Insufficient nutrition during the perinatal period causes structural alterations in humans and experimental animals, leading to increased vulnerability to diseases in later life. Japanese quail, Coturnix japonica, in which partial (8-10%) egg white was withdrawn (EwW) from eggs before incubation had lower birth weights than controls (CTs). EwW birds also had reduced hatching rates, smaller glomeruli and lower embryo weight. In EwW embryos, the surface condensate area containing mesenchymal cells was larger, suggesting that delayed but active nephrogenesis takes place. In mature EwW quail, the number of glomeruli in the cortical region (mm2) was significantly lower (CT 34.7±1.4, EwW 21.0±1.2); capillary loops showed focal ballooning, and mesangial areas were distinctly expanded. Immunoreactive cell junction proteins, N-cadherin and podocin, and slit diaphragms were clearly seen. With aging, the mesangial area and glomerular size continued to increase and were significantly larger in EwW quail, suggesting compensatory hypertrophy. Furthermore, apoptosis measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis was higher in EwWs than in CTs on embryonic day 15 and postnatal day 4 (D4). Similarly, plasma glucocorticoid (corticosterone) was higher (P<0.01) on D4 in EwW quail. These results suggest that although nephrogenic activity is high in low-nutrition quail during the perinatal period, delayed development and increased apoptosis may result in a lower number of mature nephrons. Damaged or incompletely mature mesangium may trigger glomerular injury, leading in later life to nephrosclerosis. The present study shows that birds serve as a model for 'fetal programming,' which appears to have evolved phylogenetically early.

Early and late outcomes of inflammatory abdominal aortic aneurysms: comparison with the outcomes after open surgical and endovascular aneurysm repair in literature reviews.

AIM: The aim of this study was to analyze the clinical characteristics, operative management and early and late outcomes of Inflammatory abdominal aortic aneurysms (IAAAs) in our vascular service, retrospectively and to compare with the outcomes of open and endovascular aneurysm repair (EVAR) in the patients with IAAAs in literature reviews. METHODS: Between January 1990 and December 2011, this series included twenty-four patients with IAAAs who underwent surgical treatment. The mean length of follow-up was 37.6 months (range 3-108). Kaplan-Meier life tables were used to calculate survival rate. RESULTS: Fifteen patients (60%) were symptomatic including 3 patients with hydronephrosis. Seven patients with severe symptoms underwent emergency operations. Twenty four patients underwent open surgical AAA repair. The 30 - day mortality rates were 0%. All patients had successful post operative courses and followed as outpatients in an elective situation. The ten year cumulative survival rate was 70%. The early and late mortality was the same for open repair compared to non-inflammatory AAA. CONCLUSIONS: IAAA compared with non-IAAA is associated with a higher incidence of preoperative morbidity, however operative mortality rates are lower. EVAR has lower early operative mortality rates, however, there are some problems such as remaining periaortic fibrosis and hydronephrosis in approximately half of IAAA patients after EVAR.

Lung cancer in chronic hypersensitivity pneumonitis.

BACKGROUND: So far, the association of lung cancer with chronic hypersensitivity pneumonitis (CHP) has not been studied. OBJECTIVE: We examined the prevalence and revealed clinical features of lung cancer in CHP. METHODS: We retrospectively reviewed the medical records from 1994 through 2005 and identified 11 patients (15 lesions) with lung cancer among 104 patients with CHP. Their clinical features and histopathological findings were analyzed. RESULTS: Ten men and 1 woman with a median age of 68.9 years were included. All patients had a smoking history. The most prevalent histopathological type of lung cancer was squamous cell carcinoma (53%), and all tumors were located in the peripheral region of the lung. Four patients suffered from lung cancer after the diagnosis of CHP and 1 patient had lung cancer before the diagnosis of CHP. The histological pattern of CHP showed a predominantly usual interstitial pneumonia-like lesion. Tumors were located adjacent to honeycombing in 7 (47%) of 15 lesions, bullae in 4 (27%) lesions, and relatively normal lung in 4 lesions. CONCLUSIONS: Since the prevalence of lung cancer in CHP seems to be high (10.6%) as seen in idiopathic pulmonary fibrosis, physicians should be aware of the possible complication of lung cancer in CHP.

Subcutaneous and intramuscular adipose tissue stearoyl-coenzyme A desaturase gene expression and fatty acid composition in calf- and yearling-fed Angus steers.

We proposed that stearoyl-CoA desaturase (SCD) activity dictates fatty acid composition of adipose tissue and muscle in beef cattle, regardless of ruminal or hepatic fatty acid hydrogenation or desaturation. Twelve Angus steers were assigned to a calf-fed (CF) group and slaughtered at weaning (8 mo of age; n=4), 12 mo of age (n=4), or 16 mo of age (n=4). Twelve steers were assigned to a yearling-fed (YF) group and slaughtered at 12 mo of age (n=4), 16 mo of age (n=4), and 17.5 mo of age (n=4; 525 kg, market weight). Data were analyzed based on time on the corn-based finishing diet, with terminal age as a covariate, and orthogonal polynomial contrasts were tested on the main effects of treatment group and time on the finishing diet. Fatty acids from duodenal digesta, plasma, liver, LM, and subcutaneous and intramuscular adipose tissue were measured, and SCD gene expression was measured in intramuscular and subcutaneous adipose tissues. In duodenal digesta, palmitic and linoleic acids increased by 100% over the sampling period, α-linolenic acid decreased over the sampling period, and trans-vaccenic acid was greater in YF than in CF steers (all P < 0.01). The proportion of α-linolenic acid decreased over time in all tissues, including liver. The SCD index (ratio of SCD fatty acid products to SCD fatty acid substrates) increased over time in LM and in intramuscular and subcutaneous adipose tissues. The SCD:glyceraldehyde 3-phosphate dehydrogenase mRNA ratio was virtually undetectable at the initial sampling periods in subcutaneous adipose tissue of YF and CF steers, and it increased over time (P < 0.01). The SCD index and SCD:glyceraldehyde 3-phosphate dehydrogenase ratio were greater in intramuscular adipose tissue of CF steers than in that of YF steers. The SCD index did not change over time in liver and decreased over time in duodenal digesta. We conclude that, unlike essential fatty acids, the SFA and MUFA composition of adipose tissue is regulated by adipose tissue fatty acid desaturation, with little contribution from hepatic or duodenal fatty acids.

A notable case report of May-Hegglin anomaly with immune complex-related nephropathy: a genetic and histological analysis.

May-Hegglin anomaly (MHA) is a rare autosomal dominant disease characterized by macrothrombocytopenia and leukocyte inclusions with microfilaments in the ribosomes. Mutations in the MYH9 gene, encoding non-muscle myosin heavy chain IIA (NMMHC-IIA) have been identified in patients with MHA and other MYH9-related diseases. Two young males (an older and younger brother) presented with macrothrombocytopenia and leukocyte inclusion bodies. Electron microscopy (EM) revealed parallel filaments in leukocyte inclusion bodies characteristic of MHA. Immunofluorescence microscopy (IF) showed NMMHC-IIA antibodies in 1 - 2 leukocyte inclusion bodies. These findings were consistent with MHA and they were identified to express the MYH9 mutation, D1424H. The older brother underwent a renal biopsy because of persistent proteinuria. Histology revealed mesangial proliferative glomerulonephritis with granular deposits of IgG and C1q. EM showed that the dense deposits were located in subendothelial cells, mesangial cells and Bowman's capsule. Immunocytochemistry revealed that NMMHC-IIA antibodies were localized in podocyte and endothelial cells in the glomerulus. Moreover, the expression of nephrin and podocin, slit diagram protein, was normal. An inflammatory mechanism may occur separately from MYH9-related disease. This report presents a case of MHA with immune complex-related nephropathy.

Independent histological risk factors for lymph node metastasis of superficial esophageal squamous cell carcinoma; implication of claudin-5 immunohistochemistry for expanding the indications of endoscopic resection.

Endoscopic resection is curative for superficial esophageal squamous cell carcinoma (ESCC) limited to the lamina propria. Endoscopic resection is not recommended for superficial ESCC invading muscularis mucosa or submucosa, however, because of the high frequency of lymph node metastasis (LNM) in such patients. Methods to more accurately predict LNM by analysis of endoscopically resected specimens are needed. Patients with superficial ESCC who underwent surgery without prior chemoradiotherapy (n= 110) were retrospectively examined to determine whether LNM correlated with immunohistochemical parameters and conventional histological parameters, including depth of invasion and vascular permeation. Cancer cell expression of claudins-1, 5, and 7, E-cadherin, beta-catenin, and matrix metalloproteinase 7 was evaluated. Univariate analysis revealed that LNM correlated with claudin-5 expression, but not any other immunohistochemical parameter examined. Multivariate analysis revealed three independent risk factors for LNM: aberrant claudin-5 expression in cancer cells (odds ratio; OR [95% confidence interval]= 4.61[1.44-14.77]), depth of submucosal invasion greater than 200 microm (3.55 [1.02-13.17]), and positive lymphatic permeation (3.34 [1.22-9.15]). LNM was found in one of 29 (3.4%) patients with none of these three risk factors, and in 32 of 81 (39.5%) patients with one or more of these risk factors. In superficial ESCC, routine analysis of claudin-5 expression in cancer cells together with depth of invasion and lymphatic permeation may be useful for predicting LNM and thereby reducing the number of patients undergoing additional surgery after successful endoscopic resection.

Endocytoscopic observation for esophageal squamous cell carcinoma: can biopsy histology be omitted?

We examined whether endocytoscopic observation of esophageal squamous cell carcinoma can replace the histologic examination of biopsy specimens. In a basic investigation, we examined 57 iodine-unstained areas in the resected specimens of the esophagus from 28 individuals. The endocytoscopic findings were graded from 0 to 3 in tandem with observations of the iodine staining. For endocytoscopic observation, we sprayed 1% methylene blue or toluidine blue as a vital dye on the surface of the esophageal mucosa, allowing 15-20 s for sufficient staining. One endoscopist observed the target lesions and decided their endocytoscopic type classification. Histological diagnoses were made by two pathologists who were unaware of the endoscopic findings. We then compared the endocytoscopic diagnosis and conventional histological diagnosis. In an in vivo investigation, we examined 71 lesions of esophageal squamous cell carcinoma. Two endoscopists diagnosed the type classification in consultation with a pathologist with regard to 'nuclear density,''nuclear abnormality,' and 'whether biopsy histology could have been omitted on the basis of endocytoscopic findings.' For the in vivo observation, we utilized XEC120U (higher magnification type [x1100]), XEC300F (lower magnification type [x450]), and XGIF-Q260EC1 (lower magnification type [x450]) instruments. In the basic investigation, among the 11 areas classified as Type 1, 10 (91%) were category 1 by the Vienna classification. Among the 39 lesions classified as Type 3, 36 (92%) were category 4 or 5. The sensitivity of endocytoscopy for malignant lesions (Vienna classification categories 4 and 5) was 94.7%, if Type 3 was considered malignant. The specificity was 84.2% according to the same criteria. In the in vivo observation, two endoscopists diagnosed more than 90% of esophageal squamous cell carcinomas as neoplasms using each type of endocytoscope. With regard to nuclear density, the pathologist considered it to be increased in 98% of cases with the XEC120U, in 94% with the XEC300F, and in 93% with the XGIF-Q260EC1. With regard to nuclear abnormality, the positivity rate was 90% with the XEC120U, 78% with the XEC300F, and 80% with the XGIF-Q260EC1. As to whether or not biopsy histology examination was considered necessary, the pathologist made a 'Yes' judgment for 84% of cases observed with the XEC120U, 66% with the XEC300F, and 67% with the XGIF-Q260EC1. Cancerous lesions diagnosed as Type 3 by both endoscopists using the XEC120U accounted for 46 (90.2%) of the 51 cases. Among these 46 cases, biopsy histology was considered unnecessary by the pathologist in 43 (93.5%). We believe that endocytoscopic observation has the potential to reduce the extent of histologic examination of biopsy specimens in cases corresponding to Types 1 and 3 of our classification.

Cellular regulation of bovine intramuscular adipose tissue development and composition.

It is well documented that grain feeding stimulates adipogenesis in beef cattle, whereas pasture feeding depresses the development of adipose tissues, including intramuscular (i.m.) adipose tissue. Additionally, production practices that depress adipocyte differentiation also limit the synthesis of MUFA. Marbling scores and MUFA increase in parallel suggesting that stearoyl-coenzyme A desaturase (SCD) gene expression is closely associated with and necessary for marbling adipocyte differentiation. Similarly, marbling scores and fatty acid indices of SCD activity are depressed in response to dietary vitamin A restriction. In bovine preadipocytes, vitamins A and D both decrease glycerol-3-phosphate dehydrogenase (GPDH) activity, an index of adipocyte differentiation, whereas incubation of bovine preadipocytes with l-ascorbic acid-2-phosphate increases GPDH activity. Exposing bovine preadipocytes to zinc also stimulates adipogenesis, putatively by inhibiting nitric oxide (NO) production. However, incubation of bovine preadipocytes with arginine, a biological precursor of NO, strongly promotes differentiation in concert with increased SCD expression. This suggests that the effect of either arginine or zinc on adipogenesis is independent of NO synthesis in bovine preadipocytes. Enhanced expression of SCD is associated with a greater accumulation of MUFA both in bovine preadipocyte cultures and during development in growing steers. In bovine preadipocytes, trans-10, cis-12 CLA strongly depresses adipocyte differentiation and SCD gene expression, thereby reducing MUFA concentrations. The bovine preadipocyte culture studies suggest that any production practice that elevates vitamins A or D or trans-10, cis-12 CLA in bovine adipose tissue will reduce i.m. adipose tissue development. Conversely, supplementation with vitamin C or zinc may promote the development of i.m. adipose tissue.

Reliability of endoscopic esophageal mucosectomy using TxHood, a multipurpose treatment hood.

Endoscopic mucosectomy, comprising both endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD), is a minimally invasive treatment for patients with early esophageal carcinoma. The use of ESD is appropriate for mucosal lesions of any size. However, ESD techniques are relatively difficult and can lead to serious complications such as perforation and massive bleeding, which have been reported more frequently after ESD than after EMR. This study describes a novel technique for ESD using a newly designed multipurpose treatment hood (TxHood) as well as basic experiments to ensure its safety. The TxHood includes various therapeutic tools such as an electric needleknife, a snare wire, and an injection needle, and the lines can be selected freely before insertion of an enodoscope covered by a TxHood. The main techniques for ESD are endoscopic submucosal saline injections on demand through a working channel of the endoscope or TxHood and a cut or swing cut with a needleknife attached to the TxHood. Moreover, the target area can be grasped with a grasping forceps through a working channel of the endoscope to obtain effective countertraction. In these experiments, an electric needleknife set parallel to the shaft of the endoscope offered safety and ease of handling for the dissecting procedures. Altogether, 16 resections of mucosa with an average size of 3.5 x 2.5 cm (range, 2 x 2 to 7 x 4 cm) were performed. The average time required for each targeted endoscopic resection area was about 15 min. No perforations or instances of uncontrollable bleeding occurred. In conclusion, this basic study demonstrates that the new ESD technique with the TxHood provides a useful treatment for early esophageal carcinoma and may be applicable for all mucosal or submucosal tumors in the gastrointestinal tract.

Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis.

Phosphorylation of tyrosine residue (Y1204) of rat nephrin by Fyn kinase allows Nck adaptor protein binding to nephrin motifs, which include the phosphorylated tyrosine. This phosphorylation-dependent switch induces actin polymerization in a cell culture system. Here, we generated an antibody recognizing phosphorylated nephrin at the Nck binding sites pY1204 and pY1228 to determine the phosphorylation status of nephrin using a rat model of puromycin aminonucleoside-induced nephrosis. Changes in globular actin (G-actin) and filamentous actin (F-actin) contents in isolated glomeruli were measured by western blot. Before experimental nephrosis, both Y1204 and Y1228 were phosphorylated, and most of the actin was filamentous. Before the onset of overt proteinuria, however, phosphorylation of both Y1204 and Y1228 rapidly decreased and became almost undetectable. During this period, the amount of F-actin in glomeruli began to decrease, whereas G-actin increased. Phosphorylation of nephrin at Y1228 in glomeruli of patients with minimal change nephrosis was significantly decreased compared with that in normal glomeruli. Our study suggests that tyrosine phosphorylation of nephrin by regulating F-actin formation may be important for the maintenance of normal podocyte morphology and function.

Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes.

Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat nephrin by the Fyn kinase markedly increased its interaction with a regulatory subunit of PI3K. Stable transfection of rat nephrin in the podocytes with podocin led to nephrin tyrosine phosphorylation, PI3K-dependent phosphorylation of Akt, increased Rac1 activity, and an altered actin cytoskeleton with decreased stress fibers and increased lamellipodia. These changes were reversed with an inhibitor of PI3K and not seen when the nephrin-mutant Y1152F replaced wild-type nephrin. Rac1 and Akt1 contributed to lamellipodia formation and decreased stress fibers, respectively. Finally, in the rat model of puromycin aminonucleoside nephrosis, nephrin tyrosine phosphorylation, nephrin-PI3K association, and glomerular Akt phosphorylation were all decreased. Our results suggest that PI3K is involved in nephrin-mediated actin reorganization in podocytes. Disturbed nephrin-PI3K interactions may contribute to abnormal podocyte morphology and proteinuria.

Ephrin-B1 localizes at the slit diaphragm of the glomerular podocyte.

Ephs and ephrins are a family of membrane-bound proteins that function as receptor-ligand pairs. Members of the Eph-ephrin-B family have recently been reported to regulate the paracellular permeability of epithelial cells. In this study, we analyzed the expression and the function of ephrin-B1 in glomeruli. Using immunofluorescence (IF), we found that ephrin-B1 was expressed along the glomerular capillary loop. Immunoelectron microscopy revealed that ephrin-B1 expression was restricted at the slit diaphragm. Dual labeled IF showed ephrin-B1 colocalized with the slit diaphragm proteins nephrin and CD2-associated protein. Ephrin-B1 colocalized with nephrin at the late capillary loop stage of kidney development. Additionally, injection of rats with a nephritogenic anti-nephrin antibody (ANA) reduced ephrin-B1 expression. When podocytes were cultured in vitro, they extruded processes that co-stained for ephrin-B1 and for CD2-associated protein. When these podocytes were treated in culture with small interfering RNA for ephrin-B1, CD2-associated protein was reduced in the processes, with a remaining faint perinuclear staining. We suggest that ephrin-B1 has a role in maintaining barrier function at the slit diaphragm.

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression.

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Lipogenesis and stearoyl-CoA desaturase gene expression and enzyme activity in adipose tissue of short- and long-fed Angus and Wagyu steers fed corn- or hay-based diets.

Angus and Wagyu steers consuming high-roughage diets exhibit large differences in adipose tissue fatty acid composition, but there are no differences in terminal measures of stearoyl-CoA desaturase (SCD) activity or gene expression. Also, adipose tissue lipids of cattle fed corn-based diets have greater MUFA:SFA ratios than cattle fed hay-based diets. We hypothesized that any changes in SCD gene expression and activity would precede similar changes in adipose tissue lipogenesis between short- and long-fed endpoints. Furthermore, changes in SCD activity and gene expression between production endpoints would differ between corn- and hay-fed steers and between Wagyu and Angus steers. Angus (n = 8) and Wagyu (n = 8) steers were fed a corn-based diet for 8 mo (short-fed; 16 mo of age) or 16 mo (long-fed; 24 mo of age), whereas another group of Angus (n = 8) and Wagyu (n = 8) steers was fed a hay-based diet for 12 mo (short-fed; 20 mo of age) or 20 mo (long-fed; 28 mo of age) to match the end point BW of the corn-fed steers. Acetate incorporation into lipids in vitro was greater (P < 0.01) in corn-fed steers than in hay-fed steers and tended (P = 0.06) to be greater in Wagyu than in Angus s.c. adipose tissue because the rate in Wagyu was twice that of Angus adipose tissue in the corn-fed, short-fed steers. There were diet x end point interactions for lipogenesis in i.m. and s.c. adipose tissues (both P < 0.01) because lipogenesis was 60 to 90% lower in the long-fed cattle than in short-fed cattle fed the corn-based diet. The greatest SCD enzyme activity in Angus s.c. adipose tissue was observed at 24 mo of age (corn-based diet), but activity in Wagyu adipose tissue was greatest at 28 mo of age (hay-based diet; breed x diet x end point interaction, P = 0.08). For short- vs. long-fed endpoints in Angus, s.c. adipose tissue SCD activity was less (hay diet) or the same (corn diet). Conversely, SCD gene expression was greatest in long-fed Wagyu steers fed the hay- or corn-based diets (breed x end point interaction; P < 0.01). Contrary to our hypotheses, SCD activity increased over time, whereas lipogenesis from acetate decreased. However, the developmental pattern of SCD gene expression and activity differed markedly between hay-fed Angus and Wagyu adipose tissues, which may explain the differences in the MUFA:SFA ratios observed in adipose tissues from these cattle.

Expression of allograft inflammatory factor-1 in kidneys: A novel molecular component of podocyte.

Our comprehensive gene expression profiles of the kidneys in an anti-glomerular basement membrane (GBM) nephritis model using DNA arrays revealed that allograft inflammatory factor-1 (AIF-1) was one of the highly expressed genes. Here, we explored the pathological significance of AIF-1 expression in the kidneys. The expression pattern of AIF-1 mRNA and protein in the kidneys of normal and diseased rats, such as anti-GBM nephritis and puromycin aminonucleoside nephrosis, was investigated by in situ hybridization, immunohistochemistry, and immunoelectron microscopy. Furthermore, the expression of AIF-1 in human kidneys and urinary sediments was examined. AIF-1 was expressed at both mRNA and protein levels in podocytes of normal and diseased rats, and in infiltrating cells in anti-GBM nephritis kidneys. The expression of AIF-1 in podocytes was constitutive; positive in podocytes of both normal and diseased rats. In humans, AIF-1 was expressed in podocytes and infiltrating inflammatory cells, similarly. Moreover, it was detected in urinary podocytes from patients with immunoglobulin A nephropathy. These data document for the first time that AIF-1, a constitutively expressed protein in rat and human podocytes, is a novel molecular component of podocytes, and that the upregulation of AIF-1 in an anti-GBM nephritis model may mainly be a consequence of its expression in infiltrating cells.

Transcription factor Ets-1 is essential for mesangial matrix remodeling.

Most advanced glomerular diseases are characterized by abnormal extracellular matrix (ECM) accumulation in the glomeruli, and matrix metalloproteinases (MMPs) play a pivotal role in ECM remodeling in various glomerular diseases. The proto-oncogene, ets-1, is a transcription factor regulating the expression of various matrix proteinases, including MMP-1, MMP-3, and MMP-9. The goal of the present study was to characterize the role of Ets-1 in the progression of glomerular diseases. Overexpression of Ets-1 in cultured mesangial cells prevented transforming growth factor (TGF)-beta-induced inhibition of DNA-binding activity and TGF-beta-induced type I collagen production. In addition, exogenous Ets-1 abolished TGF-beta-induced collagen gel contraction. The in vivo transfection of the ets-1 gene into nephritic kidney resulted in the increases in glomerular MMP-1, MMP-3, and MMP-9 mRNA, decreases in mesangial ECM deposition, and attenuation of fibronectin extradomain A (EDA) and type I collagen expression. In contrast, knockdown of Ets-1 in glomeruli resulted in severe ECM deposition in diseased glomeruli. In conclusion, Ets-1 promotes degradation of ECM proteins and is critical for integral glomerular reorganization.

Blockade of VEGF accelerates proteinuria, via decrease in nephrin expression in rat crescentic glomerulonephritis.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that maintains the glomerular and peritubular capillary (PTC) network in the kidney. The soluble form of the VEGF receptor-1 (soluble fms-like tyrosine kinase 1 (sFlt-1)) is known to regulate VEGF activity by binding VEGF in the circulation. We hypothesized that VEGF may be beneficial for maintaining glomerular filtration barrier and vascular network in rats with progressive glomerulonephritis (GN). For blockade of VEGF activity in vivo, rats were transfected twice with plasmid DNA encoding the murine sFlt-1 gene into femoral muscle 3 days before and 2 weeks after the induction of antiglomerular basement membrane antibody-induced GN. Inhibition of VEGF with sFlt-1 resulted in massive urinary protein excretion, concomitantly with downregulated expression of nephrin in nephritic rats. Further, blockade of VEGF induced mild proteinuria in normal rats. Administration of sFlt-1 affected neither the infiltration of macrophages nor crescentic formation. In contrast, treatment of sFlt-1 accelerated the progression of glomerulosclerosis and interstitial fibrosis accompanied with renal dysfunction and PTC loss at day 56. VEGF may play a role in maintaining the podocyte function as well as renal vasculature, thereby protecting glomeruli and interstitium from progressive renal insults.

Microarray analysis of a reversible model and an irreversible model of anti-Thy-1 nephritis.

A single intravenous injection of anti-Thy-1 monoclonal antibody (mAb) 1-22-3 is known to cause reversible mesangial proliferative glomerulonephritis. However, mAb 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis and tubulointerstitial change with an irreversible course. To identify genes that play an important role in the irreversible progression of renal injury, we used microarray technology to identify differences in gene expression between these models. Rats were intravenously injected with mAb 1-22-3 1 week after unilateral nephrectomy (irreversible model) or a sham operation (reversible model), and rats were killed on days 4, 7, 14, 42, and 56 after the injection. complementary DNA probes prepared from kidney messenger RNAs were hybridized with oligonucleotide microarrays containing 4854 rat genes. The microarray identified 189 differentially expressed genes, having at least a two-fold difference in expression level between the two models, and they were classified into five clusters. One of the clusters consisted of genes whose expression was markedly upregulated in the irreversible model. This cluster included the genes encoding osteopontin, kidney injury molecule-1, and thymosin beta10. Increased expression of thymosin beta10 was localized mainly in macrophages in the fibrotic interstitium, and upregulation of thymosin beta10 expression was also observed in a unilateral ureteral obstruction model. The microarray analysis yielded information on the molecular mechanisms responsible for the difference in disease progression between the reversible and irreversible model of anti-Thy-1 nephritis. Thymosin beta10 may play an important role in the progression of kidney disease.

Exploring RNA interference as a therapeutic strategy for renal disease.

The short synthetic interfering RNA duplexes (siRNAs) can selectively suppress gene expression in somatic mammalian cells without nonselective toxic effects of double-stranded RNA (dsRNA). However, a selective in vivo delivery of siRNA transfer has not been reported in kidney. Here, we investigated whether injection of synthetic siRNAs via renal artery followed by electroporation could be effective and therapeutic in silencing specific gene in glomerulus. We investigated the effect of siRNA in rat cultured mesangial cells (MCs) and showed that siRNA sequence-specific suppression of transgene expression was over a 1000-fold more potent than that by antisense oligodeoxynucleotide (ASODN). Transfection of siRNA targeting luciferase into rat kidneys significantly inhibited expression of a cotransfected luciferase expression vector in vivo. The delivery of siRNA targeting enhanced green fluorescent protein (EGFP) in the transgenic 'green' rat reduced endogenous EGFP expression, mainly in glomerular MCs. Furthermore, RNAi targeting against TGF-beta1 significantly suppressed TGF-beta1 mRNA and protein expression, thereby ameliorated the progression of matrix expansion in experimental glomerulonephritis. In addition, vector-based RNAi also inhibited TGF-beta1 expression in vitro and in vivo. In conclusion, siRNA-directed TGF-beta1 silencing may be of therapeutic value in the prevention and treatment of fibrotic diseases.

Superior renoprotective effects of combination therapy with ACE and AGE inhibition in the diabetic spontaneously hypertensive rat.

AIMS/HYPOTHESIS: Diabetic renal disease has been postulated to progress as a result of an interaction between metabolic and haemodynamic pathways. Our aim was to assess the functional, structural, molecular and cellular aspects of renal disease in an experimental model of diabetes with associated hypertension. METHOD: Streptozotocin-induced diabetic spontaneously hypertensive rats were randomised to no treatment, the ACE inhibitor, perindopril (2 mg/l), the AGE formation inhibitor, aminoguanidine (1 g/l) and a combination of both agents and were followed for 32 weeks. RESULTS: Diabetes was associated with a considerable increase in albumin excretion rate. Both aminoguanidine and perindopril retarded the increase in albuminuria, which was completely abrogated by combination therapy. Glomerulosclerosis and tubulointerstitial damage was reduced by both monotherapies with further renoprotection afforded by combination therapy in both cases. Combination therapy was also associated with a superior restoration in diabetes-induced nephrin protein depletion compared to either monotherapy. TGFbeta1 expression as assessed by in situ hybridisation was increased in the diabetic rats and reduced by perindopril and aminoguanidine. CONCLUSION/INTERPRETATION: These findings indicate that in the context of diabetes-related renal injury, blocking both the renin-angiotensin and advanced glycation pathways offers superior renoprotection and could be considered as a therapeutic strategy in the prevention and retardation of progressive-diabetic renal injury.