Glomerulosclerosis in mice transgenic for growth hormone. Increased mesangial extracellular matrix is correlated with kidney mRNA levels.

Mice transgenic for growth hormone (GH) develop progressive glomerulosclerosis. The compositions of kidney extracellular matrix (ECM) and ECM mRNA were examined. The glomerulosclerotic areas in GH mice contained types I and IV collagen, laminin, and basement membrane heparan sulfate proteoglycan (HSPG), which increased with age. The type IV collagen, laminin B2, and HSPG mRNA levels in GH mice, measured by a solution hybridization RNase protection assay, were increased over normal littermates. These findings suggest that the accumulation of ECM components in the glomeruli of GH mice is regulated at the transcriptional level and that glomerulosclerosis is, in part, due to the excess production of ECM rather than simply a reduction in its turnover. The glomerular lesions in GH mice resemble diabetic nephropathy and may allow further dissection of the molecular basis of certain forms of glomerulosclerosis.

The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction.

We previously reported that one of the main components of the sclerotic material in human glomerular diseases was type IV collagen. In this study we examined the contribution of increased synthesis to this process at the gene expression level. Sufficient material has not been available to study type IV collagen synthesis by normal or sclerotic glomeruli in humans. We took advantage of the availability of nephrectomy specimens from patients with renal carcinoma, and of the observation that approximately 50% of these patients develop varying degrees of glomerulosclerosis. We microdissected glomeruli from 10 patients and analyzed them using in situ reverse transcription coupled with polymerase chain reaction (PCR) analyses (in situ RT-PCR). alpha 2IV collagen mRNA, after reverse transcription into cDNA, was detected in all patients and appeared to be increased in those with glomerulosclerosis (n = 5). A competitive PCR assay was developed to quantitate this change. There was an average 3.7-fold increase in glomerular type IV collagen cDNA in patients with significant sclerosis. This change was not due to an increased number of glomerular cells. Thus, glomerulosclerosis in humans is associated with an elevation of glomerular type IV collagen gene expression, suggesting that increased synthesis of type IV collagen may represent one component of this process.

Isolation, characterization, and propagation in vitro of human glomerular endothelial cells.

Human glomerular endothelial cells have been isolated, cloned, and characterized. They appeared as the first outgrowth from human glomeruli in the presence of platelet-derived growth factor, which was also a requirement for continuous growth. By phase microscopy they appeared as monolayers of polygonal cells. Von Willebrand's factor (VWF) was detected in the cytoplasm of all clones. Their intermediate filaments differed antigenically from that present in human umbilical vein endothelial cells. Like other endothelial cells, they demonstrated high levels of membrane-associated angiotensin-converting enzyme (ACE).

Glomerulosclerosis is transmitted by bone marrow-derived mesangial cell progenitors.

We found that ROP Os/+ (Os/+) mice had diffuse glomerulosclerosis and glomerular hypertrophy and that their mesangial cells (the vascular smooth muscle cells of the glomerulus) displayed an apparent sclerosing phenotype. Since mesangial cells are the major source of scar tissue in glomerulosclerosis, we postulated that the sclerosis phenotype was carried by mesangial cell progenitors and that this phenotype could be derived from the bone marrow (BM). Therefore, we transplanted BM from Os/+ mice into congenic ROP +/+ mice (+/+ mice), which have normal glomeruli. We found that glomeruli of +/+ recipients of Os/+ marrow contained the Os/+ genotype, were hypertrophied, and contained increased extracellular matrix. Clones of recipient glomerular mesangial cells with the donor genotype were found in all +/+ recipients that developed mesangial sclerosis and glomerular hypertrophy, whereas +/+ recipients of +/+ BM had normal glomeruli. Thus, the sclerotic (Os/+) or normal (+/+) genotype and phenotype were present in, and transmitted by, BM-derived progenitors. These data show that glomerular mesangial cell progenitors are derived from the BM and can deliver a disease phenotype to normal glomeruli. Glomerular lesions may therefore be perpetuated or aggravated, rather than resolved, by newly arriving progenitor cells exhibiting a disease phenotype.

Dissociation of glomerular hypertrophy, cell proliferation, and glomerulosclerosis in mouse strains heterozygous for a mutation (Os) which induces a 50% reduction in nephron number.

We reported that the Os mutation in ROP mice induced a 50% reduction in nephron number, glomerular hypertrophy, and severe glomerulosclerosis. We examined two mouse strains with the Os mutation, ROP Os/+ and C57 Os/+ mice, to determine whether the genetic background influenced the development of glomerulosclerosis. Nephron number was decreased by 50% in both ROP Os/+ and C57 Os/+ mice, and a glomerular volume and labeling index were two- to threefold increased in both. Whereas glomerulosclerosis was severe in ROP Os/+ mice, it was absent or minimal in C57 Os/+ mice. ROP Os/+ glomeruli had two- to threefold more type IV collagen, laminin, and tenascin than C57 Os/+ by immunofluorescence microscopy. Glomerular alpha 1IV collagen and tenascin mRNA levels were increased (2.8- and 1.7-fold) in ROP Os/+ and in C57 Os/+ (1.7- and 1.4-fold) mice. Both ROP Os/+ and C57 Os/+ mice had a slight increase (1.5- and 1.7-fold) in 72-kD collagenase mRNA levels. Whereas laminin B1 mRNA levels were twofold higher in ROP +/+ than in C57 +/+ mice, there was no further change in the presence of the Os mutation. Thus, the response to the Os mutation depended on the mouse strain, since severe glomerulosclerosis occurred only in ROP Os/+ mice, even though cell proliferation and glomerular hypertrophy also were present in C57 Os/+ mice.

Transforming growth factor-beta. Murine glomerular receptors and responses of isolated glomerular cells.

Proliferation of resident glomerular cells and the accumulation of mesangial matrix are histologic abnormalities which are observed in the course of many progressive glomerular diseases. We explored the potential regulatory effects of transforming growth factor-beta (TGF-beta) on these processes. We found that cultured mouse glomerular endothelial, mesangial, and epithelial cells as well as isolated intact rat glomeruli possess high-affinity receptors for TGF-beta. We also found that, although TGF-beta consistently inhibited the proliferation of glomerular endothelial and epithelial cells, it acted as a bifunctional regulator of mesangial cell proliferation. TGF-beta significantly increased the production of collagen and fibronectin by glomerular mesangial cells whereas only fibronectin production was augmented in glomerular epithelial cells. The presence of TGF-beta receptors on intact glomeruli and on each glomerular cell type and the demonstrated responsiveness of these cells to TGF-beta combine to suggest that potentially important interactions may occur between resident glomerular cells and TGF-beta in vivo.

IGF-1 decreases collagen degradation in diabetic NOD mesangial cells: implications for diabetic nephropathy.

Nonobese diabetic (NOD) mice develop glomerulosclerosis shortly after the onset of diabetes. We showed that mesangial cells (MCs) from diabetic mice exhibited a stable phenotypic switch, consisting of both increased IGF-1 synthesis and proliferation (Elliot SJ, Striker LJ, Hattori M, Yang CW, He CJ, Peten EP, Striker GE: Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I. Endocrinology 133:1783-1788, 1993). Because the extracellular matrix (ECM) accumulation in diabetic glomerulosclerosis may be partly due to decreased degradation, we examined the effect of excess IGF-1 on collagen turnover and the activity of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in diabetic and nondiabetic NOD-MC. Total collagen degradation was reduced by 58 +/- 18% in diabetic NOD-MCs, which correlated with a constitutive decrease in MMP-2 activity and mRNA levels, and nearly undetectable MMP-9 activity and mRNA. TIMP levels were slightly decreased in diabetic NOD-MC. The addition of recombinant IGF-1 to nondiabetic NOD-MC resulted in a decrease in MMP-2 and TIMP activity. Furthermore, treatment of diabetic NOD-MC with a neutralizing antibody against IGF-1 increased the latent form, and restored the active form, of MMP-2. In conclusion, the excessive production of IGF-1 contributes to the altered ECM turnover in diabetic NOD-MC, largely through a reduction of MMP-2 activity. These data suggest that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis.

Studies on binding and mitogenic effect of insulin and insulin-like growth factor I in glomerular mesangial cells.

The mesangial cells, as part of their smooth muscle cell function, are actively involved in regulating glomerular hemodynamics. Their overlying endothelium is fenestrated; therefore, these cells are directly exposed to plasma substances, including hormones such as insulin and insulin-like growth factor I (IGF-I). These peptides may contribute to the mesangial sclerosis and cellular hyperplasia that characterize diabetic glomerulopathy. We report herein the characterization of the receptors and the mitogenic effects of IGF-I and insulin on mouse glomerular mesangial cells in culture. The IGF-I receptor was characterized on intact cells. The Kd of the IGF-I receptor was 1.47 X 10(-9) M, and the estimated number of sites was 64,000 receptors/cell. The binding was time, temperature, and pH dependent, and the receptor showed down-regulation after exposure to serum. The expression of the receptor did not change on cells at different densities. The specific binding for insulin was too low to allow characterization of the insulin receptor on intact cells. However, it was possible to identify the insulin receptor in a wheat germ agglutinin-purified preparation of solubilized mesangial cells. This receptor showed the characteristic features of the insulin receptor, including pH dependence of binding and a curvilinear Scatchard plot. The mitogenic effects of insulin and IGF-I on mesangial cells were measured by the incorporation of [3H]thymidine into DNA. IGF-I was more potent than insulin. The half-maximal response to IGF-I stimulation occurred at 1.3 X 10(-10) M, and a similar increase with insulin was observed at concentrations in the range of 10(-7) M, suggesting that this insulin action was mediated through the IGF-I receptor. These data show that the mouse microvascular smooth muscle cells of the glomerulus express a cell surface receptor for IGF-I in vitro and that this peptide is a potent mitogen for these mesangial cells. It may, therefore, play a role in glomerular proliferative lesions. The insulin receptor is present in small numbers and does not mediate mitogenesis in mesangial cells.

Co-expression of bovine growth hormone (GH) and human GH antagonist genes in transgenic mice.

Bovine growth hormone (bGH) transgenic (Tg) mice have been shown to possess enhanced growth phenotypes and exhibit severe glomerulosclerosis. One amino acid substitution in GH, i.e. G119R in bGH or G120R in human (h) GH, results in GH antagonists (GHAs). GHA-Tg mice exhibit dwarf phenotypes and normal kidneys. In order to investigate the possibility of GHAs as pharmaceutical agents for the treatment of human diseases with excessive GH levels, we cross bred mice that express bGH with those that express hGHA. Double positive Tg mice were identified that express both genes although at different levels. Kidney histological studies revealed that the double positive Tg mice with high GHA/GH expression ratios possessed normal or near normal kidneys, whereas those with low GHA/GH ratios exhibited glomerulosclerosis similar to GH-Tg mice. Thus, co-expression of GH and GHA genes in vivo results in animal phenotypes and kidney histopathologies which are a reflection of the relative expression levels of each gene.

Protection against diabetes-induced nephropathy in growth hormone receptor/binding protein gene-disrupted mice.

To further investigate the role of GH in diabetic nephropathy, experimental diabetes was induced with streptozotocin (STZ) in mice in which the GH receptor/binding protein gene was disrupted. Body weight, blood glucose, and renal histology and morphometry were studied 10 weeks after diabetes induction in wild-type (+/+) mice and in mice heterozygous (+/-) and homozygous (-/-) for the disruption. Equivalent levels of hyperglycemia developed in all diabetic groups. Normal weight gain was absent in +/+ and +/- diabetic groups, and -/- diabetics lost weight during the study. Diabetic +/+ and +/- groups both showed evidence of glomerulosclerosis, increases in glomerular volume, and increases in the ratio of mesangial area to total glomerular area, whereas diabetic -/- mice showed none of these pathological changes. These results extend our previous findings of protection against diabetes-associated kidney damage in transgenic mice expressing a GH antagonist. Taken together, the results argue for an important role of GH in the development of diabetes induced end-organ damage.

Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I.

Experimental evidence has suggested that insulin-like growth factor-I (IGF-I) may contribute to diabetic complications. Previously, we and others have shown that normal glomerular mesangial cells have receptors for, synthesize, and exhibit a mitogenic response to IGF-I. We investigated the IGF-I response in cells derived from a genetic model of diabetes, the nonobese diabetic (NOD) mouse. Mesangial cell lines were derived from diabetic (D-NOD) and nondiabetic adult mice. D-NOD cells released more IGF-I into the supernatant and had a decreased binding of IGF-I to surface receptors. Analysis according to Scatchard revealed a decreased number of receptor sites on D-NOD cells, although the structure of the IGF-I receptor visualized by cross-linking was identical for both cell types. Preincubation of D-NOD cells with an antibody to IGF-I resulted in an increase in the number of receptor sites. This suggested that autocrine IGF-I was responsible for the decrease in D-NOD receptor number and that diabetes had resulted in a stable phenotypic change.

Effects of streptozotocin treatment in growth hormone (GH) and GH antagonist transgenic mice.

To investigate GH's role in diabetic end organ damage, experimental diabetes was induced with streptozotocin (STZ) in bovine GH (bGH) or bGH antagonist transgenic mice and in their nontransgenic (NTG) litter mates. Body growth, blood glucose, serum insulin-like growth factor-I levels, liver GH receptor (GHR) binding, and kidney histology of these animals were evaluated. After administration of multiple low doses of STZ, 90% of the mice developed hyperglycemia. The diabetic animals, especially those expressing GH and GH antagonist transgenes, demonstrated retarded body growth and reduced insulin-like growth factor-I levels when compared with their nondiabetic litter mates. Kidney histology revealed severe glomerulosclerosis in diabetic and nondiabetic bGH transgenic mice. Diabetic NTG mice exhibited moderate kidney lesions. Diabetic bGH antagonist transgenic mice possessed normal glomeruli indistinguishable from those seen in nondiabetic NTG mice. GHR-binding assays revealed that liver GHR-binding sites were significantly reduced in diabetic NTG mice and transgenic dwarf mice when compared with their nondiabetic controls. Conversely, liver GHR-binding ability was significantly increased in bGH transgenic mice as compared with their NTG littermates and remained high during diabetes. It is concluded that transgenic mice that express a GH antagonist are protected from diabetes and or GH-induced nephropathy.

Matrix accumulation in mesangial cells exposed to cyclosporine A requires a permissive genetic background.

BACKGROUND: Chronic nephrotoxicity is an important adverse effect of cyclosporine A (CsA) therapy. Tubulo-interstitial lesions and arteriolopathy are common histologic findings. Glomerular lesions are also described, but they are of variable severity. The aim of our study is to determine whether CsA has a direct effect on mesangial cells and whether the cellular response depends on the genetic background. METHODS: We studied mesangial cells isolated from mice susceptible (ROP/Le-+Es1(b)+Es1(a), ROP) and resistant to glomerulosclerosis (B6SJLF1, C57). We previously showed that sclerosis-prone and sclerosis-resistant phenotypes are maintained in vitro. We examined whether CsA exposure directly affected extracellular matrix turnover in mesangial cells and whether the response is determined by the genetic background. Extracellular matrix synthesis and degradation were studied by proline incorporation, ELISA, reverse transcription-polymerase chain reaction, zymography, and reverse zymography. We chose a CsA dose that induced neither cytotoxicity nor apoptosis (1 microg/ml). RESULTS: At the dose of 1 microg/ml total collagen accumulation was increased in ROP but not in C57 cells. Matrix metalloproteinase (MMP)-2 activity and mRNA levels were selectively decreased in ROP cells. CsA exposure did not affect tissue inhibitors of MMP (TIMP)-1 and -2 activity or TGF-beta1 mRNA expression and protein synthesis in either cell line. CONCLUSION: CsA increases total collagen accumulation in mesangial cells from sclerosis-prone mice by decreasing MMP-2 activity, but does not affect cells from sclerosis-resistant mice. Thus, CsA directly affects mesangial cells, but only those with a permissive genetic background for glomerulosclerosis.

Pentosan polysulfate treatment reduces cyclosporine-induced nephropathy in salt-depleted rats.

BACKGROUND: Long-term cyclosporine (CsA) treatment leads to a decreased glomerular filtration rate, hyalinosis of afferent arterioles, and striped cortical tubulo-interstitial fibrosis. We showed previously that pentosan polysulfate (SP54) prevented the development of microvascular and interstitial lesions in mouse models of progressive glomerulosclerosis. In this study, we examined the effect of pentosan polysulfate on the development of CsA nephropathy. METHODS: Pair-fed Sprague-Dawley rats were fed a low-sodium (0.03%) diet and received CsA (15 mg/kg, subcutaneously, in olive oil)/5% glucose, pentosan polysulfate (10 mg/kg, subcutaneously in 5% glucose) plus CsA, olive oil/pentosan polysulfate, or olive oil/5% glucose for 30 days. Creatinine clearance (CrCl) was determined at three time points. Afferent arteriolar lesions, glomerular volume, and tubulo-interstitial lesions were quantitated. RNA was extracted from cortex. RESULTS: Severe lesions were found in the CsA group. A reduction in the number of affected arterioles (32%) and the degree of chronic tubulo-interstitial lesions (44%) was found in pentosan polysulfate/CsA-treated rats. A 20% decrease in glomerular volume was found in CsA rats, but not in pentosan polysulfate/CsA-treated rats. Pentosan polysulfate treatment did not prevent the CsA-induced decrease in CrCl (approximately 30%) at 4 weeks. CsA did not affect cortical endothelial or neuronal nitric-oxide synthase or mRNA levels, but there was small increase in neuronal nitric-oxide synthase mRNA levels in the pentosan polysulfate/CsA-treated group. CONCLUSIONS: Pentosan polysulfate reduced structural renal lesions in CsA-treated, salt-depleted Sprague-Dawley rats.

The contribution of glomerular mesangial cells to progressive glomerulosclerosis.

These data from in vitro studies, partially confirmed on intact glomeruli, suggest that glomerular cells are not only responsive to a number of growth-regulatory peptides but they are also important sources of several of these agents. The studies on transgenic mice confirm the potential role of the insulin-like peptides. This model also provides clear evidence that genetically directed modifications of cell behavior are important in the pathogenesis of glomerulosclerosis. The ability of glomerular cells to undergo proliferation and participate in hypertrophy of the glomerulus may be a critical determinant in the development of progressive glomerulosclerosis. That this ability is controlled by genetic differences between species and individuals seems well-established in both animal studies and clinical experience.

Administration of AGEs in vivo induces genes implicated in diabetic glomerulosclerosis.

Administration of advanced glycosylation end products (AGEs), prepared on mouse albumin, to normal young adult mice, resulted in an increase in mean glomerular volume and up-regulation of laminin B1 and alpha 1 type IV collagen mRNAs measured by competitive PCR in single microdissected glomeruli. Both glomerular hypertrophy and overexpression of genes coding for extracellular matrix were abrogated when aminoguanidine, an inhibitor of AGE cross-linking, was added to the AGEs injections, suggesting that the glomerular response to AGEs was specific. The effects of AGEs administration in vivo are comparable to those occurring in the early stage of diabetic nephropathy, suggesting a participation of AGEs in these events.

Studies by competitive PCR of glomerulosclerosis in growth hormone transgenic mice.

We have shown that the glomerulosclerotic lesions of mice transgenic for bovine growth hormone (bGH mice) consisted of a change in the phenotype of glomerular collagens and an elevation of the mRNAs for these collagens in whole kidney. The purpose of this study was to determine whether these phenotypic and quantitative changes were present in the glomeruli. We used the increased sensitivity afforded by reverse transcription followed by the polymerase chain reaction (RT-PCR) to detect type I collagen mRNA and a quantitative PCR assay to quantitate type IV collagen mRNA in microdissected glomeruli. There was a six- to eightfold increase in alpha 1IV collagen mRNA in the glomeruli of bGh mice. alpha 1(I) collagen mRNA was present in glomeruli of bGH mice, which is consistent with our previous findings that the sclerotic mesangium contained type I collagen peptides by immunofluorescence microscopy. Normal glomeruli did not contain detectable amounts of alpha 1I collagen mRNA. In summary, we found a phenotypic change in glomeruli of mice transgenic for bGH consisting of increased type IV collagen mRNA levels and the appearance of type I collagen mRNA. Thus, the development of glomerulosclerosis appeared to be at least partially regulated at a pretranslational level.

Glomerulosclerosis in mice transgenic for native or mutated bovine growth hormone gene.

Mice transgenic for bovine growth hormone (bGH) develop an increase in body weight and glomerular lesions characterized by a disproportionate increment in glomerular volume and progressive mesangial sclerosis. The relationship between glomerular size and body growth in bGH mice was further investigated by examining mice transgenic for a mutated GH gene (bGH-m11) which failed to enhance body growth. The glomeruli in bGH-m11 mice exhibited an increase in size and glomerulosclerosis comparable to those found in bGH mice. The levels of alpha 1 type IV collagen mRNA, as measured by the competitive polymerase chain reaction in isolated microdissected glomeruli, were markedly elevated in mice transgenic for both bGH and bGH-m11 genes. These data suggest that body growth on one hand, and glomerular hypertrophy and sclerosis on the other hand, are mediated by different portions of GH or different second messenger signaling systems.

Collagen and collagenase mRNAs in normal and sclerotic glomeruli: predictors of progression and response to therapy.

Progressive glomerulosclerosis is associated with decreasing kidney function, eventuating in end-stage renal failure. There are multiple components of the extracellular matrix, and the exact composition in various renal diseases is not known. Thus, we examined some of the major components of the extracellular matrix (ECM) in murine and human glomerular diseases. We studied matrix synthesis and degradation at the level of gene expression and ECM composition in the intact glomerulus. To determine whether the composition of sclerosis was similar among diseases, we examined a normal mouse strain and compared it with strains which spontaneously developed glomerulosclerosis. The baseline levels of matrix components varied between different mouse strains, and this level correlated with their propensity to develop glomerulosclerosis. In addition, when glomerulosclerosis was induced, the baseline ECM mRNA level predicted the subsequent outcome. We studied mice transgenic for bovine growth hormone, since they develop progressive glomerulosclerosis. Treatment with heparin substantially decreased the lesions without changes in type IV collagen mRNAs. However, there was an up-regulation of both the mRNA and enzyme activity for the 92 kD matrix metalloproteinase. In contrast, when these mice were treated with either angiotensin converting enzyme inhibitors or angiotensin II (Ang II) receptor antagonists, the glomerulosclerosis was accentuated histologically and the ECM synthetic and degradative mRNAs were elevated. These data suggest that the mRNA levels reflect response to therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Glomerulosclerosis, arteriosclerosis, and vascular graft stenosis: treatment with oral heparinoids.

At present there is no known treatment for established glomerulosclerosis or atherosclerosis. Since the principal lesion in glomerulosclerosis involves mesangial cells, a vascular smooth muscle cell, we searched for new therapeutic approaches affecting vascular smooth muscle function, especially with respect to modifying the turnover of extracellular matrix. We used mice transgenic for bovine growth hormone (bGH), since these mice develop end-stage renal disease due to progressive glomerulosclerosis. We previously showed that the subcutaneous injection of a non-anticoagulant heparin reduced glomerulosclerosis in bGH mice. Since injectable drugs are not a practical means of controlling glomerulosclerosis in humans, we assessed oral heparin-like compounds and found that oral pentosan polysulfate (PPS) reduced glomerulosclerosis in bGH mice at non-toxic doses. Because the positive therapeutic response in the bGH model could have been principally hormone-mediated, we examined other models of non-immune mediated glomerulosclerosis, including ROP Os/+ non-diabetic and diabetic mice. We found that an oral PPS (Elmiron), which is approved for other indications in humans, reduced sclerosis in all of these forms of chronic, progressive glomerulosclerosis. Based on the similarity of the cellular events in glomerulosclerosis and arteriosclerosis, we assessed the effect(s) of PPS in congenital (Watanabe rabbits) and induced (New Zealand White lipid-fed rabbits) models of arteriosclerosis. The extent and severity of the lesions was significantly reduced in both models by PPS treatment. Finally, we asked whether the proliferative and sclerotic lesion, which is the cause of vascular graft stenosis, might also respond to PPS treatment. To do this we cultured cells from the materials removed from stenotic arteriovenous grafts in hemodialysis patients. We found that PPS inhibits the proliferation and matrix production in a dose-dependent manner.