Paola Catanuto

Failure to phosphorylate AKT in podocytes from mice with early diabetic nephropathy promotes cell death

Loss of podocytes by apoptosis characterizes the early stages of diabetic nephropathy. To examine its mechanism we studied glomeruli and podocytes isolated from db/db mice with early diabetic nephropathy and albuminuria. Phosphorylation of AKT (protein kinase B, a key survival protein) was found to be lower in the glomeruli of 12 week old db/db compared to db/+ mice. In vitro, insulin phosphorylated AKT solely in podocytes from db/+ mice. Serum deprivation and exposure to tumor necrosis factor-alpha significantly compromised cell viability in podocytes from db/db but not from db/+ mice, and this was associated with a significant decrease in AKT phosphorylation. Inhibition of AKT was necessary to achieve the same degree of cell death in db/+ podocytes. Our study shows that podocyte inability to respond to insulin and susceptibility to cell death may partially account for the decreased podocyte number seen in early diabetic nephropathy.

Activation of the Estrogen Receptor Contributes to the Progression of Pulmonary Lymphangioleiomyomatosis via Matrix Metalloproteinase-Induced Cell Invasiveness

CONTEXT The role of estrogens in the pathogenesis of lymphangioleiomyomatosis (LAM), an aggressive and destructive, eventually fatal lung disease of women, is poorly understood. OBJECTIVE The study was conducted to test the hypothesis that the lung disease in LAM is estrogen mediated and to determine whether estrogens contribute to the invasiveness of LAM. DESIGN In vitro cell culture of spindle-shaped LAM cells (LAMD-SM) were isolated and propagated from affected lungs. Estrogen receptor (ER)-alpha and ERbeta analyses were conducted by RT-PCR. ERalpha and ERbeta, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinases (MMP)-2 had Western blot analysis for protein assessment. Activity assays were performed for MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2. Assessment of MMP-2 promoter function was done via transfection assays. Cell invasion chambers were used to determine and quantitate cell invasiveness. SETTING The study was conducted at an academic medical center. PATIENTS Tissue and cells were obtained from patients as outlined in approved institution review board protocol (97/007). INTERVENTION LAMD-SM cells were treated with a specific MMP-2 antibody or a nonspecific inhibitor, doxycycline. MAIN OUTCOME MEASURES Activity of MMP-2 and invasiveness of LAMD-SM cells were measured. RESULTS LAMD-SM cells express functional ERs (ERalpha and ERbeta), which undergo rapid intracellular turnover in their unbound state. 17beta-estradiol (E(2)) enhances the transcriptional ER activity. E(2)-induced ER activation increases synthesis and activity of MMP-2 through posttranscriptional mechanisms in LAMD-SM. The E(2)/ER-mediated increase of MMP-2 promotes LAMD-SM invasiveness, in assays in vitro, which can be inhibited by specific antibodies against MMP-2 or doxycycline, an inhibitor of MMPs. CONCLUSION The invasion and destruction of lung parenchyma in LAM is, at least partially, an estrogen-MMP-driven process, which has major implications for therapeutic interventions.

Testosterone and 17Β-estradiol have opposite effects on podocyte apoptosis that precedes glomerulosclerosis in female estrogen receptor knockout mice

Podocyte damage and apoptosis are thought to be important if not essential in the development of glomerulosclerosis. Female estrogen receptor knockout mice develop glomerulosclerosis at 9 months of age due to excessive ovarian testosterone production and secretion. Here, we studied the pathogenesis of glomerulosclerosis in this mouse model to determine whether testosterone and/or 17β-estradiol directly affect the function and survival of podocytes. Glomerulosclerosis in these mice was associated with the expression of desmin and the loss of nephrin, markers of podocyte damage and apoptosis. Ovariectomy preserved the function and survival of podocytes by eliminating the source of endogenous testosterone production. In contrast, testosterone supplementation induced podocyte apoptosis in ovariectomized wild-type mice. Importantly, podocytes express functional androgen and estrogen receptors, which, upon stimulation by their respective ligands, have opposing effects. Testosterone induced podocyte apoptosis in vitro by androgen receptor activation, but independent of the TGF-β1 signaling pathway. Pretreatment with 17β-estradiol prevented testosterone-induced podocyte apoptosis, an estrogen receptor-dependent effect mediated by activation of the ERK signaling pathway, and protected podocytes from TGF-β1- or TNF-α-induced apoptosis. Thus, podocytes are target cells for testosterone and 17β-estradiol. These hormones modulate podocyte damage and apoptosis.

Inhibition of C-jun N-terminal kinase improves insulin sensitivity but worsens albuminuria in experimental diabetes.

C-jun N-terminal kinase (JNK) regulates both the development of insulin resistance and inflammation. Podocytes of the widely used db/db mouse model of diabetic nephropathy lose their ability to respond to insulin as albuminuria develops, in comparison to control db/+ mice. Here we tested whether JNK inhibition or its gene deletion would prevent albuminuria in experimental diabetes. Phosphorylated/total JNK was significantly increased in vivo in glomeruli of db/db compared to db/+ mice. Treatment of podocytes isolated from these two strains of mice with tumor necrosis factor-alpha caused greater phosphorylation of JNK in those obtained from diabetic animals. When db/db mice were treated with a cell-permeable TAT-JNK inhibitor peptide, their insulin sensitivity and glycemia significantly improved compared to controls. We induced diabetes in JNK1 knockout mice with streptozotocin and found that they had significantly better insulin sensitivity compared to diabetic wild-type or JNK2 knockout mice. Albuminuria was, however, worse in all mice treated with the JNK inhibitor and in diabetic JNK2 knockout mice compared to controls. Nephrin expression was also reduced in JNK inhibitor-treated mice compared to controls. A similar degree of mesangial expansion was found in all diabetic mice. Our study shows that targeting JNK to improve systemic insulin sensitivity does not necessarily prevent diabetic nephropathy.

17 Β-estradiol and tamoxifen upregulate estrogen receptor Β expression and control podocyte signaling pathways in a model of type 2 diabetes

Diabetic nephropathy remains one of the most important causes of end-stage renal disease. This is particularly true for women from racial/ethnic minorities. Although administration of 17beta-estradiol to diabetic animals has been shown to reduce extracellular matrix deposition in glomeruli and mesangial cells, effects on podocytes are lacking. Given that podocyte injury has been implicated as a factor leading to the progression of proteinuria and diabetic nephropathy, we treated db/db mice, a model of type 2 diabetic glomerulosclerosis, with 17beta-estradiol or tamoxifen to determine whether these treatments reduce podocyte injury and decrease glomerulosclerosis. We found that albumin excretion, glomerular volume, and extracellular matrix accumulation were decreased in these mice compared to placebo treatment. Podocytes isolated from all treatment groups were immortalized and these cell lines were found to express the podocyte markers WT-1, nephrin, and the TRPC6 cation channel. Tamoxifen and 17beta-estradiol treatment decreased podocyte transforming growth factor-beta mRNA expression but increased that of the estrogen receptor subtype beta protein. 17beta-estradiol, but not tamoxifen, treatment decreased extracellular-regulated kinase phosphorylation. These data, combined with improved albumin excretion, reduced glomerular size, and decreased matrix accumulation, suggest that both 17beta-estradiol and tamoxifen may protect podocytes against injury and therefore ameliorate diabetic nephropathy.

Therapeutic benefits of young, but not old, adipose-derived mesenchymal stem cells in a chronic mouse model of bleomycin-induced pulmonary fibrosis.

The observation that pulmonary inflammatory lesions and bleomycin (BLM)-induced pulmonary fibrosis spontaneously resolve in young mice, whereas remaining irreversible in aged mice suggests that impairment of pulmonary regeneration and repair is associated with aging. Because mesenchymal stem cells (MSCs) may promote repair after injury, we postulated that differences in MSCs from aged mice may underlie postinjury fibrosis in aging. The potential for young-donor MSCs to inhibit BLM-induced pulmonary fibrosis in aged male mice (>22 months) has not been studied. Adipose-derived MSCs (ASCs) from young (4 months) and old (22 months) male mice were infused 1 day after intratracheal BLM administration. At 21-day sacrifice, aged BLM mice demonstrated lung fibrosis by Ashcroft score, collagen content, and α(v)-integrin messenger RNA (mRNA) expression. Lung tissue from aged BLM mice receiving young ASCs exhibited decreased fibrosis, matrix metalloproteinase (MMP)-2 activity, oxidative stress, and markers of apoptosis vs BLM controls. Lung mRNA expression of tumor necrosis factor-alpha was also decreased in aged BLM mice receiving young-donor ASCs vs BLM controls. In contrast, old-donor ASC treatment in aged BLM mice did not reduce fibrosis and related markers. On examination of the cells, young-donor ASCs had decreased mRNA expression of MMP-2, insulin-like growth factor (IGF) receptor, and protein kinase B (AKT) activation compared with old-donor ASCs. These results show that the BLM-induced pulmonary fibrosis in aged mice could be blocked by young-donor ASCs and that the mechanisms involve changes in collagen turnover and markers of inflammation.

Subtype specific estrogen receptor action protects against changes in MMP-2 activation in mouse retinal pigmented epithelial cells

Eyes with age-related macular degeneration (AMD) demonstrate accumulation of specific deposits and extracellular matrix (ECM) molecules under the retinal pigment epithelium (RPE). AMD is about two times more prevalent in aging postmenopausal women. Therefore we studied whether 17beta-estradiol (E(2)) modulates the expression and activity of the trimolecular complex (MMP-2, TIMP-2 and MMP-14), molecules which are of major importance for ECM turnover in RPE. We used cell lines isolated from estrogen receptor knockout mice (ERKO) to determine which ER (estrogen receptor) subtype was important for ECM regulation in RPE cells. We found that mouse RPE sheets had higher baseline MMP-2 activity in the presence of ERbeta. This correlated with higher MMP-2 activity in RPE cell lines isolated from ERKOalpha mice. Exposure to E(2) increased MMP-2 activity in mouse RPE cell lines. In addition E(2) increased transcriptional activation of the MMP-2 promoter through a functional Sp1 site which required the presence of ERbeta, but not ERalpha. E(2) also maintained levels of pro MMP-2, and MMP-14 and TIMP-2 activity after oxidant injury. Since the direct effects of E(2) on MMP-2 transcriptional activation and the regulation of the trimolecular complex after oxidant-induced injury requires ERbeta, this receptor subtype may have a role as a potential therapeutic target to prevent changes in activation of MMP-2.

Estrogens and Progression of Diabetic Kidney Damage

It is generally accepted that estrogens affect and modulate the development and progression of chronic kidney diseases (CKD) not related to diabetes. Clinical studies have indeed demonstrated that the severity and rate of progression of renal damage tends to be greater among men, compared with women. Experimental studies also support the notion that female sex is protective and male sex permissive, for the development of CKD in non-diabetics, through the opposing actions of estrogens and testosterone. However, when we consider diabetes-induced kidney damage, in the setting of either type 1 or type 2 diabetes, the contribution of gender to the progression of renal disease is somewhat uncertain. Previous studies on the effects of estrogens in the pathogenesis of progressive kidney damage have primarily focused on mesangial cells. More recently, data on the effects of estrogens on podocytes, the cell type whose role may include initiation of progressive diabetic renal disease, became available. The aim of this review will be to summarize the main clinical and experimental data on the effects of estrogens on the progression of diabetes-induced kidney injury. In particular, we will highlight the possible biological effects of estrogens on podocytes, especially considering those critical for the pathogenesis of diabetic kidney damage.

Estrogen receptor β protects against in vivo injury in RPE cells

Epidemiological data suggest that estrogen deficiency in postmenopausal women may contribute to the severity of AMD. We discovered that 17beta-estradiol (E2) was a crucial regulator of the severity of extracellular matrix turnover (ECM) dysregulation both in vivo and in vitro. We also found in vitro that the presence of estrogen receptor (ER)beta regulates MMP-2 activity. Therefore in an attempt to delineate the role of the ER subtypes, female estrogen receptor knockout (ERKO) mice were fed a high-fat diet, and the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, Bruchs membrane changes, and choriocapillaris endothelial morphology. We found that changes in the trimolecular complex of pro-MMP-2, MMP-14 and TIMP-2 correlated with increased Bruchs membrane thickening or sub-retinal deposit formation (basal laminar deposits) in ERKObeta mice. In addition RPE isolated from ERKObeta mice had an increase in expression of total collagen and a decrease in MMP-2 activity. Finally we found that ERK an intermediate signaling molecule in the MMP pathway was activated in RPE isolated from ERKObeta mice. These data suggest that mice which lack ERbeta are more susceptible to in vivo injury associated with environmental light and high fat diet.

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 18-month-old (estrogen receptor knockout) ERKOalpha and ERKObeta mice and their C57Bl/6 wildtype littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER)alpha and ERbeta protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology.