Grande, Joseph P.

Neointimal hyperplasia contributes to failure of hemodialysis arteriovenous fistulas (AVFs). Increased expression of matrix metalloproteinase (MMP)-9 occurs in AVFs, and MMP-9 is implicated in neointimal hyperplasia and vascular injury. Recent studies demonstrate that MMP-9, by degrading N-cadherin, leads to increased expression of β-catenin and β-catenin-dependent proliferation of smooth muscle cells. The present study examined this pathway in the venous limb of a murine AVF model. Western analyses demonstrate that, in this model, there is diminished expression of N-cadherin accompanied by increased expression of β-catenin, c-Myc, and proliferating cell nuclear antigen (PCNA). By immunohistochemistry, β-catenin and c-Myc localized to proliferating smooth muscle cells in the venous limb of the AVF. Increased expression of β-catenin was accompanied by augmented expression of phosphorylated (p)-glycogen synthase kinase (GSK)-3β, GSK-3β, and integrin-linked kinase. The administration of doxycycline suppressed MMP-9 expression but did not reduce venous histological injury in the AVF, or increase AVF patency assessed 6 wk after its creation. Doxycycline did not influence expression of β-catenin, c-Myc, GSK-3β, or integrin-linked kinase. Thus, in this vascular injury model, the upregulation of β-catenin cannot be readily attributed to MMP-9 upregulation; increased β-catenin expression may reflect either the upregulation of p-GSK-3β, GSK-3β, or integrin-linked kinase. This study provides the first exploration of β-catenin in an AVF, demonstrating substantial upregulation of this mitogenic signaling molecule and uncovering possible mechanisms that may account for such upregulation.

Abstract Tissue injury triggers reparative processes that often involve endothelial progenitor cells (EPCs) recruitment. We hypothesized that atherosclerotic renal artery stenosis (ARAS) activates homing signals that would be detectable in both the kidney and EPCs, and attenuated on renal repair using selective cell-based therapy. Pigs were treated with intrarenal autologous EPC after 6 weeks of ARAS. Four weeks later, expression of homing-related signals in EPC and kidney, single kidney function, microvascular (MV) density, and morphology were compared with untreated ARAS and normal control pigs (n = 7 each). Compared with normal EPC, EPC from ARAS pigs showed increased stromal cell-derived factor (SDF)-1, angiopoietin-1, Tie-2, and c-kit expression, but downregulation of erythropoietin (EPO) and its receptor. The ARAS kidney released the c-kit-ligand stem cell factor, uric acid, and EPO, and upregulated integrin β2, suggesting activation of corresponding homing signaling. However, angiopoietin-1 and SDF-1/CXCR4 were not elevated. Administration of EPC into the stenotic kidney restored angiogenic activity, improved MV density, renal hemodynamics and function, decreased fibrosis and oxidative stress, and attenuated endogenous injury signals. The ARAS kidney releases specific homing signals corresponding to cognate receptors expressed by EPC. EPC show plasticity for organ-specific recruitment strategies, which are upregulated in early atherosclerosis. EPC are renoprotective as they attenuated renal dysfunction and damage in chronic ARAS, and consequently decreased the injury signals. Importantly, manipulation of homing signals may potentially allow therapeutic opportunities to increase endogenous EPC recruitment.

Atherosclerotic renal artery stenosis reduces blood flow and perfusion pressures to the poststenotic kidney producing renovascular hypertension and threatening glomerular filtration rate. Little is known regarding regional tissue oxygenation in human renovascular disease that develops slowly. We compared stenotic and contralateral kidneys regarding volume, tissue perfusion, blood flow measured by multidetector computed tomography, and blood oxygen level–dependent magnetic resonance values in the cortex and medulla in 14 patients with unilateral stenosis (mean: 71% by quantitative computed tomography) and in 14 essential hypertensive patients during 150 mEq/d of sodium intake and renin-angiotensin blockade. Stenotic kidney volume was reduced compared with the contralateral kidney (118.6±9.9 versus 155.4±13.7 mL; P <0.01), as was total blood flow (269.7±42.2 versus 383.7±49; P =0.02), mainly because of reduced cortical volume. Tissue perfusion was similar but lower than essential hypertension (1.5 versus 1.2 mL/min per milliliter; P <0.05). Blood oxygen level–dependent MR at 3 T confirmed elevated R2* values (a measure of deoxyhemoglobin) in deep medullary regions in all 3 sets of kidneys (38.9±0.7 versus cortex 17.8±0.36 s −1 ; P <0.0001). Despite reduced blood flow, R2* values did not differ between atherosclerotic and essential hypertensive kidneys, although furosemide-suppressible fall in medullary R2* was reduced in stenotic kidneys (5.7±1.8 versus 9.4±1.9 s −1 ; P <0.05). Renal venous oxygen levels from the stenotic kidney were higher than those from essential hypertensives (65.1±2.2 versus 58.1±1.2; P =0.006). These data indicate that, although stenosis reduced blood flow and volume, cortical and medullary oxygenation was preserved under these conditions.

To investigate the role of HLA-DQ2 in the pathogenesis of associated immune disorders, we generated transgenic mice that expressed HLA-DQ2 in the absence of endogenous murine class II molecules (AE 0 DQ2). These AE 0 DQ2 mice with a mixed genetic background spontaneously developed skin lesions on their ears, whereas control AE 0 DQ6 genotype control mice (also with a mixed genetic background) did not. The skin lesions were characterized by deep subepidermal blistering with hydropic degeneration and lymphoid infiltration in the subepidermal area as determined by histopathology. Immunofluorescence analysis revealed thick band-like granular deposition of IgG, IgM, and a thin band of IgA deposition along the basement membrane. AE 0 DQ2 mice also developed significant and progressive hematuria and proteinuria as compared with the AE 0 DQ6 mice (p < 0.05). Histopathology showed immune complex deposits in the glomeruli of AE 0 DQ2 mice. Immunofluorescence analysis showed progressive mesangial and capillary wall deposition of IgA, IgM, IgG and C1q in the kidney. With electron microscopy, the deposits showed a ‘fingerprint’ substructure; and tubuloreticular structures were identified within endothelial cells. Conversely, these changes were not observed in AE 0 DQ6 mice. Serum anti-double stranded (ds)DNA IgM and IgG levels were also significantly elevated among AE 0 DQ2 mice compared with AE 0 DQ6 mice (p < 0.001). In conclusion, AE 0 DQ2 mice spontaneously develop an autoimmune lupus-like syndrome and are useful model for this disease. It remains to be determined whether genetic admixture played a role in the development of this systemic lupus erythematosus-like syndrome in HLA-DQ2 transgenic mice. Lupus (2010) 19, 815—829.

Abstract To gain insights into 1,25-dihydroxyvitamin D3 receptor (VDR) function during fetal bone development, we examined fetal rat tissues from gestational days 13–21 for the presence and distribution of VDR using immuno-histochemistry. Prior to ossification, VDR epitopes were observed in the mesenchyme condensing to form skeletal tissues, on day 13 in the developing vertebral column and limbs, and on day 17 of gestation in developing calvaria. Immunostaining for VDR was seen in proliferating and hypertrophic chondrocytes and in osteoblasts of limb buds and the vertebral column by day 17 of gestation. In calvaria, VDR epitopes were observed in osteoblasts by gestational day 19. VDR immunostaining was also evident in the skin of fetal limbs at all gestational ages examined. We show for the first time that the VDR appears very early in the developing fetal rat skeleton, suggesting that the VDR, in concert with its ligand, 1,25-dihydroxyvitamin D3, may play a role in the differentiation of mesenchymal precursors into bone tissue.

Acute ischemic insults to the kidney are recognized complications of human sickle cell disease (SCD). The present study analyzed in a transgenic SCD murine model the early renal response to acute ischemia. Renal hemodynamics were profoundly impaired following ischemia in sickle mice compared with wild-type mice: glomerular filtration rate, along with renal plasma flow and blood flow rates, were markedly reduced, while renal vascular resistances were increased more than threefold in sickle mice following ischemia. In addition to these changes in renal hemodynamics, there were profound disturbances in renal signaling processes: phosphorylation of members of the MAPK and Akt signaling proteins occurred in the kidney in wild-type mice after ischemia, whereas such phosphorylation did not occur in the kidney in sickle mice after ischemia. ATP content in the postischemic kidney in sickle mice was less than half that observed in wild-type mice. Examination of the expression of candidate genes uncovered changes that may predispose to increased sensitivity of the kidney in sickle mice to ischemia: increased expression of inducible nitric oxide synthase and decreased expression of endothelial nitric oxide synthase, and increased expression of TNF-α. Inducibility of anti-inflammatory, cytoprotective genes, such as heme oxygenase-1 and IL-10, was not impaired in sickle mice after ischemia. We conclude that the kidney in SCD is remarkably vulnerable to acute ischemic insults. We speculate that such sensitivity of the kidney to ischemia in SCD may underlie the occurrence of acute kidney injury in patients with SCD and may set the stage for the emergence of chronic kidney disease in SCD.

Abstract ETS variant 1 (ETV1), also known as ETS-related protein 81, is overexpressed in prostate tumors, but whether and how this transcription factor affects tumorigenesis has remained elusive. Here, we show that ETV1 is primarily overexpressed in the most aggressive human prostate tumors. Transgenic ETV1 mice developed prostatic intraepithelial neoplasia as well as hyperplasia/neoplasia in seminal vesicles. Moreover, ETV1 cooperated with the androgen receptor (AR) to bind to the prostate-specific antigen enhancer and stimulate gene transcription. Consistent with its ability to physically interact with AR, ETV1 rendered an ETV1 binding site–driven reporter androgen inducible, and, on the other hand, ETV1 superinduced transcription from an AR binding site on androgen stimulation. In conclusion, our study substantiates that ETV1 overexpression is an underlying cause in the development of prostate and possibly also seminal vesicle cancer. Its interaction with and activation of AR provides a molecular mechanism on how ETV1 exerts its deleterious function. Thus, inhibiting ETV1 or blocking its interaction with AR may represent novel strategies in prostate cancer therapy. [Cancer Res 2009;69(20):8102–10]

Abstract The effect of chronic (CCR) and intermittent (ICR) caloric restriction on serum insulin-like growth factor (IGF)-I levels and mammary tumor (MT) development was investigated. Ten-week-old MMTV-TGF-α female mice were assigned to ad libitum–fed (AL; AIN-93M diet), ICR [3-week 50% caloric restriction using AIN-93M-mod diet, 2× protein, fat, vitamins, and minerals followed by 3 weeks of daily 100% AL consumption of AIN-93M (∼75% of AL for each 6-week cycle)], and CCR (calorie and nutrient intake matched for each 6-week ICR cycle) groups. Half of the mice from each group were sacrificed at 79 (end of restriction) or 82 (end of refeeding) weeks of age. Serum was obtained at euthanasia and in cycles 1, 3, 5, 8, and 11. MT incidence was 71.0%, 35.4%, and 9.1% for AL, CCR, and ICR mice. ICR-Restricted mice had significantly lower terminal serum IGF-I and IGF-I/IGF binding protein-3 (IGFBP-3) ratio than CCR, ICR-Refed, and AL mice. There were no differences in terminal IGFBP-3. Final body, internal, and mammary fat pad weights correlated positively with IGF-I and negatively with IGFBP-3. Few changes were found for protein expression of IGF-IRα and IGFBP-3 in mammary tissue and MTs. During the study, IGF-I levels of ICR-Restricted mice were reduced, whereas refeeding allowed partial recovery. For all groups, elevated IGF-I levels preceded MT detection, although not all values were significant versus mice without MTs. However, the specific role of IGF-I in the protective effect of calorie restriction remains to be determined. These results confirm that ICR prevents MT development to a greater extent than CCR.

Unilateral renal artery stenosis (RAS) leads to atrophy of the stenotic kidney and compensatory enlargement of the contralateral kidney. Although the two-kidney, one-clip (2K1C) model has been extensively used to model human RAS, the cellular responses in the stenotic and contralateral kidneys, particularly in the murine model, have received relatively little attention. We studied mice 2, 5, and 11 wk after unilateral RAS. These mice became hypertensive within 1 wk. The contralateral kidney increased in size within 2 wk after surgery. This enlargement was associated with a transient increase in expression of phospho-extracellular signal-regulated kinase (p-ERK), the proliferation markers proliferating cell nuclear antigen and Ki-67, the cell cycle inhibitors p21 and p27, and transforming growth factor-β, with return to baseline levels by 11 wk. The size of the stenotic kidney was unchanged at 2 wk but progressively decreased between 5 and 11 wk. Unlike the contralateral kidney, which showed minimal histopathological alterations, the stenotic kidney developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Surprisingly, the stenotic kidney showed a proliferative response, which involved largely tubular epithelial cells. The atrophic kidney had little evidence of apoptosis, despite persistent upregulation of p53; expression of cell cycle regulatory proteins in the stenotic kidney was persistently increased through 11 wk. These studies indicate that in the 2K1C model, the stenotic kidney and contralateral, enlarged kidney exhibit a distinct temporal expression of proteins involved in cell growth, cell survival, apoptosis, inflammation, and fibrosis. Notably, an unexpected proliferative response occurs in the stenotic kidney that undergoes atrophy.

Abstract Vitamin D and vitamin D metabolites such as 25-hydroxyvitamin D and 1α,25-dihydroxyvitamin D [1α,25(OH)2D3] circulate in the serum of fish. The receptor for 1α,25(OH)2D3 (VDR) has previously been cloned from fish intestine, and ligand binding assays have shown the presence of the VDR in the gills, intestine, and liver of fish. Using immunohistochemical methods with specific antibodies against the VDR, we now report that the VDR is widely expressed in tissues of the adult male and female zebrafish, Danio rerio, specifically in epithelial cells of gills, tubular cells of the kidney, and absorptive cells in the intestine. Additionally, the VDR is expressed in the skin, the olfactory organ, the retina, brain, and spinal cord. Sertoli cells of the testis, oocytes, acinar cells of the pancreas, hepatocytes, and bile duct epithelial cells express substantial amounts of the receptor. Osteoblast-like cells and chondrocytes also express VDR. Preimmune serum and antiserum preadsorbed with Danio VDR protein fails to detect VDR in the same tissues. The VDR is also present in the developing eye, brain, and otic vesicle of 48- and 96-h postfertilization zebrafish embryos. Parenteral administration of 1α,25(OH)2D3 increases concentrations of VDR in intestinal epithelial cells but not in epithelial cells of the gills. Lithocholic acid, however, does not alter concentrations of VDR after parenteral administration. The data suggest that VDR is widely distributed in tissues of the zebrafish, D. rerio, and is likely to play important roles in epithelial transport, bone, and endocrine function. Furthermore, concentrations of the receptor seem to be regulated by its ligand, 1α,25-dihydroxyvitamin D but not by lithocholic acid. Zebrafish may serve as a useful model in which to assess the function of the VDR in diverse tissues.