Glomerular aging in females is a multi-stage reversible process mediated by phenotypic changes in progenitors.

The glomeruli of postmenopausal C57BL6 mice, and age-matched males, show progressive hypertrophy and glomerulosclerosis. We asked whether this was a multistage process, was due to alterations in glomerular progenitors, and was reversible in female mice. Using cross bone marrow transplants (BMT) between young and old females, we found that BMT delivered a phenotype that was donor age-specific. The fact that lesions in young recipients were more severe if the donors were in late rather than early menopause suggested that new progenitor phenotypes had appeared. Postmenopausal recipients of BMT from young donors had reduced glomerular hypertrophy and sclerosis, implying that the aging lesions in females were reversible and that progenitors, rather than the local environment, determined the glomerular profile. The altered phenotype included increased extracellular matrix synthesis and decreased matrix metalloproteinase-2 levels as well as cell hypertrophy. The mechanism of the cellular hypertrophy was due to uncoupling of hypertrophy from proliferation, resulting from elevated p27 levels. Thus, glomerular hypertrophy and sclerosis in aging females is a multistage process, is reversible, and may be determined by the phenotype of bone marrow-derived progenitor cells.

The glomerulosclerosis of aging in females: contribution of the proinflammatory mesangial cell phenotype to macrophage infiltration.

Age-associated renal changes may be an important cause of renal failure. We recently found that aged female B6 mice developed progressive glomerular lesions. This was associated with macrophage infiltration, a frequent finding in glomerulosclerosis. We used these mice as a model for studying the mechanisms of glomerular aging. We compared the gene expression profile of intact glomeruli from late postmenopausal (28-month-old) mice to that of intact glomeruli from premenopausal (5-month-old) mice. We found that inflammation-related genes, especially those expressed by activated macrophages, were up-regulated in the glomeruli of 28-month-old mice, a result correlating with the histological observation of glomerular macrophage infiltration. The mechanism for macrophage recruitment could have been stable phenotypic changes in mesangial cells because we found that mesangial cells isolated from 28-month-old mice expressed higher levels of RANTES and VCAM-1 than cells from 5-month-old mice. The elevated serum tumor necrosis factor (TNF)-alpha levels present in aged mice may contribute to increased RANTES and VCAM-1 expression in mesangial cells. Furthermore, cells from 28-month-old mice were more sensitive to TNF-alpha-induced RANTES and VCAM-1 up-regulation. The effect of TNF-alpha on RANTES expression was mediated by TNF receptor 1. Interestingly, mesangial cells isolated from 28-month-old mice had increased nuclear factor-kappaB transcriptional activity. Inhibition of nuclear factor-kappaB activity decreased baseline as well as TNF-alpha-induced RANTES and VCAM-1 expression in mesangial cells isolated from 28-month-old mice. Thus, phenotypic changes in mesangial cells may predispose them to inflammatory stimuli, such as TNF-alpha, which would contribute to glomerular macrophage infiltration and inflammatory lesions in aging.

Development of albuminuria and glomerular lesions in normoglycemic B6 recipients of db/db mice bone marrow: the role of mesangial cell progenitors.

The pathologic hallmarks of diabetic nephropathy are excess mesangial extracellular matrix (ECM) and mesangial cell proliferation. We previously showed that mesangial cell phenotypic changes play an important role in the pathogenesis of diabetic nephropathy. We concluded that phenotypic changes were present in bone marrow (BM)-derived mesangial cell progenitors, as transplantation of BM from db/db mice, a model of type 2 diabetic nephropathy, transferred the db genotype and a nephropathy phenotype to naive B6 mice recipients. The recipients did not develop diabetes; however, they did develop albuminuria and glomerular lesions mirroring those in the donors (i.e., glomerular hypertrophy, increased ECM, and increased cell number with cell proliferation). We found that matrix metalloproteinase 2 (MMP-2) facilitated invasion of the mesangial cells into ECM and proliferation in vitro. Thus, increased MMP-2 activity in db/db mesangial cell progenitors may partially explain increased mesangial cell repopulation and proliferation in B6 recipients of db/db BM. In summary, BM-derived mesangial cell progenitors may play a crucial role in the development and progression of ECM accumulation and mesangial cell proliferation in this model of diabetic nephropathy in type 2 diabetes.

Effects of ventricular unloading on apoptosis and atrophy of cardiac myocytes.

BACKGROUND: Ventricular unloading decreases cardiac ventricular mass. This loss of ventricular mass can be due to either atrophy (a reversible process) or apoptosis (an irreversible process) of the cardiac myocytes. We investigated the effect of ventricular unloading on atrophy and apoptosis of cardiac myocytes, using working and nonworking transplant heart models in rats. MATERIALS AND METHODS: ACI rats underwent heterotopic heart transplantation with two different techniques to create working and nonworking cardiac grafts. Cardiac grafts were harvested at different time points after transplantation. TUNEL, caspase-3 assay, and electron microscopy were used to assess the degree of apoptosis while cellular atrophy was estimated by calculation of the cytoplasmic index (CI = mean sectional cytoplasmic area/nucleus). RESULTS: Ventricular mass reduction was more pronounced in nonworking than in working hearts (P < 0.05). Apoptotic index and caspase-3 activities increased in both groups, peaking at 3 days after transplantation, but were not significantly different between the two models. The cytoplasmic index was significantly lower in nonworking than in working grafts (P < 0.05). CONCLUSIONS: These data suggest that cellular atrophy is the primary mechanism that accounts for myocardial weight reduction following ventricular unloading. The inference is that ventricular unloading by ventricular assist devices may not cause permanent loss of cardiac myocytes, thus allowing for functional recovery.

Glucose induces clonal selection and reversible dinucleotide repeat expansion in mesangial cells isolated from glomerulosclerosis-prone mice.

Clonal selection has been proposed as a pathogenetic mechanism in various chronic diseases, such as scleroderma, hypertension, pulmonary fibrosis, interstitial fibrosis of the kidney, atherosclerosis, and uterine leiomyomatosis. We previously found that mesangial cells from ROP mice prone to develop glomerulosclerosis changed their phenotype in response to high glucose concentrations. Here, we investigate whether clonal selection might contribute to this phenotype change. We found that in ROP mice at least two distinct mesangial cell clones exist. They are characterized by a different length of the d(CA) repeat in the MMP-9 promoter and exhibit a significantly different gene expression profile. Exposure of ROP mesangial cells to 25 mmol/l glucose for 35 days induces both clonal selection and reversible dinucleotide repeat expansion. None of these findings were present in mesangial cells isolated from C57BL/6 mice, which are not sclerosis-prone. We conclude that mesangial cell michrochimerism may be a marker for the susceptibility to glomerulosclerosis, that dinucleotide repeat expansion may be a novel mechanism for glucose-induced changes in gene expression, and that clonal selection may partially explain the change in mesangial cell phenotype in diabetes.

Response to sex hormones differs in atherosclerosis-susceptible and -resistant mice.

Genetic factors that determine the degree of susceptibility to atherosclerosis may also influence the effects of estrogens and progestins in arterial vessel disease. We examined and compared estrogen receptor (ER) and progesterone receptor (PR) expression and the effects of 17beta-estradiol (E2) and progesterone (P) on collagen synthesis and matrix metalloproteinase (MMP) activities in the aortic arch and in cultured aortic smooth muscle cells (ASMC) of atherosclerosis-susceptible (C57Bl6/J, B6) or -resistant (C3H/HeJ, C3H) mice. ERalpha, ERbeta, and PR levels were higher in the aorta and ASMC of atherosclerosis-susceptible B6 mice. In transfection studies using an estrogen response element-driven reporter plasmid, E2 elicited a >2-fold increase in luciferase activity in ASMC of B6 (B6-ASMC), which demonstrated the transcriptional activity of ER in atherosclerosis-susceptible cells. Importantly, the response of endogenous target genes to E2 and P was different in B6-ASMC and C3H-ASMC. E2 decreased collagen synthesis but had no effect on MMP activities in B6-ASMC. P decreased MMP-2 and MMP-9 activity in B6-ASMC. In contrast, E2 increased MMP-2 and decreased MMP-9 activity but had no effect on collagen synthesis in C3H-ASMC. P had no effect on collagen synthesis and MMP activity in C3H-ASMC. These differences in response to sex hormones may have important implications for women who receive hormone replacement therapy.

The molecular basis of age-related kidney disease.

Renal disease affects 11% of people in the United States over the age of 65, not including those with diabetes or hypertension. Although glomerular disease is the most common underlying etiology of age-related renal dysfunction, the cause of glomerular disease and whether it is the only contributor to renal failure are not known. Our studies in female mice show that renal disease in the postmenopausal period is associated with progressive glomerular enlargement and scarring, as well as abnormal renal function. To study the underlying causes of aging-related glomerular disease, we isolated and characterized glomerular smooth muscle (mesangial) cells from female mice of various ages. We found that the cells from older mice exhibit a variety of phenotypic changes, including increased concentrations of p27, a protein that serves to inhibit progression from the G1 to the S phase of the cell cycle. Because the bone marrow (BM) contains mesangial cell progenitors that can transfer the donor glomerular phenotype (normal or diseased) to recipients, we exchanged BM between postmenopausal and premenopausal mice and found that aging-related glomerular enlargement and scarring are transferred to young recipient glomeruli. In addition, BM from normal, young donors led to the regression of aging-related glomerular disease in postmenopausal recipients; namely, both glomerular enlargement and scarring were reduced. Thus, aging-related glomerular disease is an entity distinct from all other causes of renal disease, is characterized by phenotypic changes in mesangial cell progenitors, and is reversible when the phenotype of the progenitors is returned to normal.

Estrogen deficiency accelerates progression of glomerulosclerosis in susceptible mice.

Estrogen deficiency may contribute to the development and progression of glomerulosclerosis in postmenopausal women. The responsiveness to estrogens could be controlled by genetic traits related to those that determine the susceptibility to glomerular scarring. This study was undertaken to determine whether the intensity of the sclerotic response was modified by the estrogen status in sclerosis-prone ROP Os/+ mice. Ovariectomized ROP Os/+ mice developed more severe renal dysfunction and glomerulosclerosis than intact, ie, estrogen sufficient age-matched female mice. Ovariectomized ROP Os/+ exhibited increased accumulation of extracellular matrix, predominantly of laminin, and a marked distortion of the glomerular architecture. We found an increase in macrophage infiltration in the mesangium of ovariectomized ROP Os/+. Estrogen deficiency decreased glomerular estrogen receptor expression in ROP Os/+ mice, which we had previously found to be low in the parental ROP strain. Thus, although physiological estrogen levels in young ROP Os/+ mice could not prevent the development of glomerulosclerosis, estrogen deficiency accelerated the progression of glomerular scarring in this mouse strain. This suggests that estrogen replacement will slow but not prevent the progression of glomerulosclerosis. It underscores the importance of the genetic composition of individuals that determines the susceptibility to diseases as well as the response to treatment.

Resistance to glomerulosclerosis in B6 mice disappears after menopause.

The frequency of chronic renal failure increases with age, especially in women after menopause. Glomerulosclerosis is a common cause of chronic renal failure in aging. We reported that pre-menopausal female C57BL6 (B6) mice are resistant to glomerulosclerosis, irrespective of the type of injury. However, we now show that B6 mice develop progressive glomerulosclerosis after menopause. Glomerular lesions, first recognized in 18-month-old mice, consisted of hypertrophy, vascular pole sclerosis, and mesangial cell proliferation. Diffuse but moderate mesangial sclerosis and more marked hypertrophy were present at 22 months. At 28 to 30 months the glomerulosclerosis was diffuse and increased levels of type I and type IV collagen and transforming growth factor-beta 1 mRNA were present. Urine albumin excretion was significantly increased in 30-month-old mice. Mesangial cells isolated from 28-month-old mice retained their sclerotic phenotype in vitro. Comparison of the effects of uninephrectomy (Nx) in 20-month-old and 2.5-month-old mice revealed a 1.7-fold increase in urine albumin excretion, accelerated glomerulosclerosis, and renal function insufficiency in 20-month-old Nx mice, but not in 2.5-month-old Nx mice. Glycemic levels, glucose, insulin tolerance, and blood pressure were normal at all ages. Thus, B6 mice model the increased frequency of chronic renal failure in postmenopausal women and provide a model for studying the mechanism(s) of glomerulosclerosis in aging women.

Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium.

PURPOSE: Age-related macular degeneration (ARMD) is characterized by progressive thickening and accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE). ECM dysregulation probably contributes to the pathologic course of ARMD. By activating estrogen receptors (ERs), estrogens regulate the expression of genes relevant in the turnover of ECM, among them matrix metalloproteinase (MMP)-2. Estrogen deficiency may predispose to dysregulated synthesis and degradation of ECM, leading to accumulation of collagens and other proteins between the RPE and its basement membrane. The purposes in the current study were to confirm the expression of ERs in human RPE, to elucidate whether these ERs are functional, and to test whether 17beta-estradiol (E(2)) regulates expression of ERs and MMP-2. METHODS: Expression of ERs was examined in freshly isolated human RPE monolayer and in cultured human RPE cells, by using total RNA for RT-PCR and protein extracts for Western blot analysis. Supernatants were collected from freshly isolated human RPE and from cultured human RPE to assess MMP-2 activity by zymography and protein expression by Western blot. The transcriptional activity of ERs was studied in transfection experiments with an estrogen-responsive reporter construct. All these studies were preformed in the presence or absence of E(2) (10(-11) and 10(-7) M). RESULTS: Human RPE isolated from female and male individuals expressed both ER subtypes alpha and beta at the mRNA and protein levels. Treatment of cultured RPE cells with 10(-10) M E(2) increased expression of mRNA and protein of both receptor subtypes. E(2) (10(-10) M) also increased MMP-2 activity (approximately 2.2-fold) and protein expression (approximately 2.5-fold). In contrast, there was no change in ER levels and MMP-2 activity at higher E(2) concentrations (10(-8) M), compared with baseline. Preincubation of cells with 10(-7) M pyrrolidinedithiocarbamate (PDTC), an inhibitor of nuclear factor (NF)-kappaB, abolished the increase in MMP-2 activity and protein expression induced by E(2) at 10(-10) M. CONCLUSIONS: Both ER subtypes are expressed in RPE and regulated in a dose-dependent fashion by E(2). Estrogens similarly regulate MMP-2. This estrogen-induced effect is, at least in part, mediated through NF-kappaB. These data support the hypothesis that estrogens may exert biological function in RPE through ERs and that estrogen deficiency or excess may cause dysregulation of molecules that influence the turnover of ECM in Bruch's membrane associated with ARMD.

Restoration of glomerular haemodynamics and renal injury independent of arterial hypertension in rats with subtotal renal ablation.

To study whether prevention of renal injury using the anti-inflammatory drugs pentosan polysulphate (PPS) and mycophenolate mofetil (MMF) is associated with improvement of glomerular haemodynamics, PPS and MMF were compared with losartan. The awake systolic blood pressure (SBP), proteinuria (Uprot) and micropuncture studies were performed 30 days after five-sixths nephrectomy in untreated rats and in rats treated with PPS (100 mg/kg per day), MMF (30 mg/kg per day) or losartan (30 mg/kg per day). In the rats receiving no treatment, there was a rise in SBP (to 180-200 mmHg) and in Uprot, which were prevented by losartan. In the PPS and MMF groups, the SBP was elevated but the Uprot did not increase. In the untreated rats the total glomerular filtration rate (GFR) decreased (-80%) and the single-nephron GFR (37-42%), plasma flow (67-127%) and glomerular pressure (10-15 mmHg) increased. These changes were prevented by PPS and MMF to the same extent as by losartan: the rise in single-nephron GFR and plasma flow were reduced by 50% and the glomerular pressure was normal. In rats receiving losartan, this was due to the fall in arterial pressure, whereas in PPS- and MMF-treated rats it was due to a rise in afferent resistance, indicating autoregulatory capacity. Total GFR was similar, despite the lower single-nephron GFR in treated groups, suggesting a larger proportion of functioning nephrons. Losartan, PPS and MMF significantly reduced glomerular sclerosis and tubular dilation and atrophy in association with a reduction in the lymphocyte and macrophage infiltrate. These results suggest an interaction between the haemodynamic and inflammatory changes that perpetuate each other during progression of renal injury. Renal protection provided by anti-inflammatory drugs is partially mediated by the prevention of glomerular haemodynamic alterations.

Association of a decreased number of d(CA) repeats in the matrix metalloproteinase-9 promoter with glomerulosclerosis susceptibility in mice.

The genetic background plays an important role in the development of progressive glomerulosclerosis. However, no marker is available for the reliable prediction of genetic susceptibility to glomerulosclerosis. Because matrix metalloproteinase-9 (MMP-9) levels are decreased in models of glomerulosclerosis and MMP-9 promoter polymorphism has been observed among patients with diabetic nephropathy, MMP-9 could be one such marker. The object of this study was to determine whether MMP-9 promoter polymorphism was associated with altered MMP-9 expression in mesangial cells (MC) from two mouse strains, i.e., ROP (glomerulosclerosis prone) and B6SJL (glomerulosclerosis resistant). ROP MC expressed 12-fold less MMP-9 mRNA. The MMP-9 promoter in ROP MC contained fewer d(CA) repeats, which was associated with lower MMP-9 expression and activity. Phorbol-12-myristate-13-acetate (3 to 60 ng/ml) increased MMP-9 expression in both MC types (3- to 4.5-fold), but the level in ROP MC never reached that in B6SLJ MC. Although reciprocal transfection of ROP and B6SJL MMP-9 promoter constructs into B6SJL and ROP cells revealed that the promoters were functional in both cell types, the B6SJL promoter was less responsive to phorbol-12-myristate-13-acetate stimulation when transfected into ROP MC, suggesting a role for other factors. In conclusion, the MMP-9 promoter exhibits a decreased number of d(CA) repeats in the sclerosis-prone strain. Because fewer d(CA) repeats associated with decreased MMP-9 expression in MC, it might be a genetic marker for glomerulosclerosis.

Estrogen-related abnormalities in glomerulosclerosis-prone mice: reduced mesangial cell estrogen receptor expression and prosclerotic response to estrogens.

The development and progression of glomerulosclerosis (GS) is determined by the genetic background. The incidence of end-stage renal disease is increased in postmenopausal women, suggesting that estrogen deficiency may play a role in the accumulation of extracellular matrix by mesangial cells (MCs), which are primarily responsible for the synthesis and degradation of this matrix. Using mouse models that are prone or resistant to the development of GS, we compared the expression of estrogen receptor (ER)-alpha and ER-beta subtypes in GS-prone and GS-resistant glomeruli and isolated MCs, and examined the effects of estrogens on ER, collagen, and matrix metalloproteinase (MMP) expression in MCs. Glomeruli and MCs from GS-prone mice had decreased expression of ER-alpha and ER-beta subtypes and ER transcriptional activity was also decreased in their MCs. Importantly, although 17 beta-estradiol treatment resulted in decreased collagen accumulation and increased MMP-9 expression and activity in MCs from GS-resistant mice, there was, paradoxically, no effect on collagen accumulation and decreased MMP-9 expression and activity in MCs from GS-prone mice. Thus, GS susceptibility is associated with diminished ER expression in MCs. The renal protective effects of estrogens, including decreased collagen accumulation and increased MMP-9 expression, seem to be blunted in GS-prone MCs.

Upregulation of type I collagen by TGF-beta in mesangial cells is blocked by PPARgamma activation.

We found that peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA was reduced by 77% in glomeruli of diabetic mice. Because mesangial cells play an important role in diabetic nephropathy, we examined regulation of type I collagen expression by PPARgamma and transforming growth factor-beta(1) (TGF-beta(1)) in mouse mesangial cells in the presence of 6 and 25 mM glucose. Mesangial cells contained functionally active PPARgamma. Exposure to 25 mM glucose resulted in reduced PPARgamma expression and transcriptional activity, accompanied by increased type I collagen expression. Restoration of PPARgamma activity to normal levels in cells cultured in 25 mM glucose, by transfection with a PPARgamma expression construct and treatment with the PPARgamma agonist troglitazone, returned type I collagen levels toward normal values. Activation of PPARgamma by troglitazone also decreased type I collagen mRNA and blocked TGF-beta(1)-mediated upregulation of type I collagen mRNA and protein. Moreover, PPARgamma activation suppressed basal and activated TGF-beta(1) responses in mesangial cells. This action was blocked by transfection of cells with a dominant-negative PPARgamma construct. In summary, PPARgamma suppresses the increased type I collagen mRNA and protein expression mediated by TGF-beta(1) in mesangial cells.

Autocrine activation of the IGF-I signaling pathway in mesangial cells isolated from diabetic NOD mice.

Mesangial cells isolated from NOD mice after the onset of diabetes have undergone a stable phenotypic change. This phenotype is characterized by increased expression of IGF-I and downregulation of collagen degradation, which is associated with decreased MMP-2 activity. Here, we investigated the IGF-I signaling pathway in mesangial cells isolated from NOD mice before (nondiabetic NOD mice [ND-NOD]) and after (diabetic NOD mice [D-NOD]) the onset of diabetes. We found that the IGF-I signaling pathway in D-NOD cells was activated by autocrine IGF-I. They had phosphorylation of the IGF-I receptor beta-subunit, phosphorylation of insulin receptor substrate (IRS)-1, and association of the p85 subunit (phosphatidylinositol 3-kinase [PI3K]) with the IGF-I receptor and IRS-1 in D-NOD cells in the basal state. This was also associated with increased phosphorylation of ERK2 in D-NOD mesangial cells. Inhibiting autocrine IGF-I from binding to its receptor using an IGF-I-neutralizing antibody or inhibiting IGF-I signaling pathways using a specific PI3K inhibitor or a specific mitogen-activated protein kinase/extracellular response kinase kinase inhibitor decreased phosphorylated ERKs in D-NOD cells. Importantly, this was associated with increased MMP-2 activity. The addition of exogenous IGF-I to ND-NOD activated signal transduction. Therefore, we conclude that the IGF-I signaling pathway is intact in both D-NOD and ND-NOD cells. However, the phenotypic change in D-NOD cells is associated with constitutive activation of the IGF-I signaling pathways, which may participate in the development and progression of diabetic glomerulosclerosis.

Reversibility of glucose-induced changes in mesangial cell extracellular matrix depends on the genetic background.

Adequate glycemic control protects most patients with diabetes from nephropathy, but a substantial fraction of patients develop progressive disease despite lowering glycemia. We isolated mesangial cells (MC) from the glomeruli of mouse strains that model these two outcomes in patients with diabetes, namely those that have the propensity (ROP) or resistance (B6) to develop progressive diabetic nephropathy. We determined the nature and reversibility of changes in selected extracellular matrix-related molecules after chronic exposure to elevated glucose concentration. MC were exposed to 25 mmol/l glucose for 5 weeks followed by 6 mmol/l glucose and 19 mmol/l mannitol for an additional 5 weeks. Matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta(1) (TGF-beta(1)) levels increased in B6 MC exposed to 25 mmol/l glucose but returned to baseline levels when the glucose concentration was reduced to 6 mmol/l. MMP-2 and TGF-beta(1) were higher in ROP MC at baseline and increased in response to 25 mmol/l glucose, but remained elevated when glucose concentration was reduced. Type I collagen expression and accumulation increased in a reversible manner in B6 MC exposed to 25 mmol/l glucose. However, type I collagen expression was higher in ROP MC at baseline and remained unaffected by changes in glucose concentration. Thus, 25 mmol/l glucose induced reversible changes in MMP-2, TGF-beta(1), and type I collagen in MC of sclerosis-resistant mice but not in MC from sclerosis-prone mice. Therefore, progressive diabetic nephropathy may be secondary to stable alterations in the phenotype of MC as a result of the interplay between the genetic background and elevated glucose concentrations.

Pentosan polysulfate prevents glomerular hypertension and structural injury despite persisting hypertension in 5/6 nephrectomy rats.

Five/six nephrectomy induces systemic and glomerular hypertension, glomerulosclerosis, proteinuria, and tubulointerstitial fibrosis. Polysulfate pentosan (PPS) decreases mesangial proliferation and extracellular matrix accumulation. The aim of this study was to determine whether PPS prevents glomerular hemodynamic changes and renal damage. Micropuncture studies were performed in three groups of eight male Wistar rats. Two groups included rats with 5/6 nephrectomy-one of which was treated with PPS in drinking water (100 mg/kg body wt) and the second of which received normal drinking water-and the third group consisted of normal rats that served as controls. Five/six nephrectomy produced systemic hypertension, a 50% reduction in GFR, and a 67% increase in single-nephron GFR due to elevated glomerular pressure and single-nephron plasma flow as well as proteinuria. Hypertension persisted in PPS-treated animals. Despite a similar reduction in GFR, PPS prevented the rise in single-nephron GFR, glomerular capillary hydrostatic pressure, and proteinuria. By morphometry, glomerular volume was increased by 46% and mesangial area by 94%. Fractional glomerular capillary area decreased by 24%. PPS prevented these changes. Tubular dilatation, epithelial cell atrophy, and increased interstitial area were largely prevented by PPS, as was the interstitial inflammatory infiltrate. These results suggest that the renal protection conferred by PPS was mediated both by prevention of glomerular hypertension as well as suppression of the inflammatory response. It was postulated that this was partly due to the preservation of a greater fraction of functional nephrons.

Expression and regulation of estrogen receptors in mesangial cells: influence on matrix metalloproteinase-9.

Diabetic glomerulosclerosis is characterized by the accumulation of extracellular matrix (ECM) in the mesangium. Estrogens seem to retard whereas estrogen deficiency seems to accelerate progressive glomerulosclerosis. Thus, mesangial cells (MC) may be a target for estrogens. Estrogen action is mediated via estrogen receptor (ER) subtypes ERalpha and ERbeta. Both ER subtypes were expressed in human and mouse MC. Using an estrogen-responsive reporter construct in transfection assays, it also was demonstrated that the nuclear ER were transcriptionally active. In the presence of 17beta-estradiol (E2; 10(-10) to 10(-8) M), there was a progressive increase in the mRNA levels of both ERalpha (approximately 1.8-fold and approximately 2.7-fold after 24 and 72 h, respectively) and ERbeta (approximately 1.3-fold and approximately 2.2-fold after 24 and 72 h, respectively). ERalpha protein levels increased approximately 2.5-fold after 24 h (10(-10) M, E2) and up to approximately 5.4-fold after 72 h (10(-9) M, E2). ERbeta protein levels increased approximately 2.1-fold in the presence of E(2) (10(-9) M) after 24 h. Thus, estrogens positively regulate the expression of the ER subtypes, thereby maintaining or increasing MC responsiveness to estrogens. Because diabetic glomerulosclerosis may be due partly to a decrease in ECM degradation, the effects of estrogens on matrix metalloproteinases (MMP) were studied. It was found that E2 (10(-10) to 10(-8) M) increased both MMP-9 mRNA and MMP-9 activity in MC. This may be an important mechanism by which estrogens influence ECM turnover and protect against progression of diabetic glomerulosclerosis.

Growth hormone increases inducible nitric oxide synthase expression in mesangial cells.

Mice transgenic for bovine growth hormone (GH) develop progressive glomerulosclerosis. However, the proximal signaling events that lead to increased matrix deposition in this pathologic condition are still unclear. Components of the L-arginine metabolic pathway, especially inducible nitric oxide (NO) synthase (iNOS), ornithine aminotransferase (OAT), and ornithine decarboxylase (ODC), have been associated with glomerular scarring. In this study, mesangial cells were treated with GH, and the expression of iNOS, ODC, and OAT was determined using reverse transcription-PCR. In addition, nitrite accumulation in the conditioned media of mesangial cell cultures was measured in the presence or absence of GH. The findings revealed that GH increased iNOS transcript levels in a dose-dependent manner, with the highest levels being attained at GH concentrations of 20 to 50 ng/ml. The GH-induced increase in iNOS transcript levels was accompanied by a significant increase in nitrite concentrations in conditioned media, which was blocked by the addition of L-N(G)-monomethylarginine. The effect of GH (50 ng/ml) in eliciting nitrite production was as potent as that of bacterial lipopolysaccharide (10 microg/ml). The expression of OAT and ODC, in contrast, was not altered at any of the GH concentrations tested. GH receptor mRNA was also expressed by mesangial cells, independently of the GH concentration present in the cell culture medium. These data indicate that GH may interact with its receptor to regulate the L-arginine/NO pathway in mesangial cells, by directly modulating iNOS expression and NO production, without altering the arginase/OAT/ODC pathway.