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We found that mice transgenic for native bovine growth hormone (bGH) gene had increased body size and rapidly progressive glomerulosclerosis, whereas mice transgenic for a mutated bGH gene (bGH-m11) had near normal body size and slowly progressive glomerulosclerosis. The aim of this study was to determine whether rapidly and slowly progressive glomerulosclerosis had distinct glomerular extracellular matrix (ECM) and growth factor mRNA levels. ECM and growth factors were quantitated by competitive reverse transcriptase-PCR in microdissected glomeruli from bGH, bGH-m11, and nontransgenic littermate control mice. In rapid progressors (bGH mice) at 2 to 3 months, the levels of mRNA-coding for some glomerular ECM and growth factors were increased (alpha 1(IV) collagen, 7.3-fold; laminin B1, 3.9-fold; tenascin, 8-fold; and tumor growth factor (TGF)-beta 1, 3.4-fold). These levels underwent a further 2.3-fold increase at 6 to 9 months. Platelet-derived growth factor (PDGF)-B mRNA was high at 2 to 3 months (7.4-fold) and 6 to 9 months (9.5-fold) and was associated with an increased [3H]-thymidine-labeling index and glomerular cell number. In slow progressors (bGH-m11 mice), the mRNA levels at 2 to 3 months were approximately one half that of rapid progressors (alpha 1(IV) collagen, 3.4-fold; laminin B1 1.9-fold; tenascin, 3-fold; TGF-beta 1, 2.2-fold). PDGF-B levels were normal. At 6 to 9 months, alpha 1(IV) collagen, TGF-beta 1, and PDGF-B mRNA levels doubled, whereas tenascin and laminin B1 levels remained stable. At 12 to 18 months, the alpha 1(IV) collagen, TGF-beta 1, and tenascin levels increased by nearly another 50%. The labeling index and PDGF levels were not increased at any time. The levels of expression of several glomerular ECM mRNA and growth factors of rapid progressors at 2 to 3 months of age was nearly double that of slow progressors, nearly doubling again by 6 to 9 months. In slow progressors, alpha 1(IV) collagen and TGF-beta 1 mRNA levels continued to increase at a slow rate, but tenascin and laminin mRNA levels were only further increased at 12 to 18 months. Thus, the initial levels of these mRNA and their rate of change correlated with the severity of glomerulosclerosis.

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.

Mice transgenic for bovine growth hormone (bGH) develop progressive mesangial sclerosis resulting in uremia. Mesangial cells from bGH mice were isolated to determine whether the cells maintained a stable phenotypic change in the synthesis and degradation of extracellular matrix, which contribute to the glomerular lesions in vivo. The bGH mesangial cells were 1.2-fold larger than cells from control mice. They had a 1.7-fold increase in doubling time, a 7-fold decrease in labeling index (p < 0.0001), and a 2.4- and 2-fold decrease in c-myc (p < 0.05) and insulin-like growth factor I gene expression, respectively. Collagen synthesis and degradation were studied by PCR, ELISA, and gelatin zymography. bGH mesangial cell alpha 1 collagen IV mRNA levels were increased 2.3-fold (0.47 +/- 0.25 versus 0.20 +/- 0.09 attomoles/500 cells, p < 0.01) whereas secreted collagen IV and collagen IV in the cell lysates were increased 1.4-fold (25.1 +/- 5 versus 17.2 +/- 4 ng/ml/10(5) cells) and 1.8-fold (30.5 +/- 3 versus 16.7 +/- 3 ng/ml/10(5), p < 0.05), respectively. There were no differences in collagen I mRNA levels or in the protein content of either the media or cell lysates. We were not able to detect metalloproteinase 9 (MMP-9) mRNA expression or MMP-9 protein in bGH mesangial cell medium, whereas both mRNA and protein were present in controls. MMP-2 mRNA and enzyme activity in bGH cells were, however, elevated 1.5-fold (p < 0.05) and 2.1-fold (p = 0.05) over controls. Transforming growth factor beta 1 mRNA in bGH cells was 1.6-fold higher than that of controls (p < 0.05). The data suggest that (a) mesangial lesions may result from stable, genetically induced, phenotypic changes in mesangial cells, and (b) alterations of MMP-9 and collagen IV expression by mesangial cells may contribute to an imbalance between extracellular matrix synthesis and degradation and play a critical role in the genesis of glomerulosclerosis.

Streptozotocin-treated C57B1/SJL mice developed glomerular hypertrophy and light microscopic lesions mimicking human diabetic glomerulosclerosis. In contrast, there were no glomerular hypertrophy and lesions in diabetic mice transgenic (TG) for a mutated growth hormone (bGH-G119K) that competes with native endogenous GH and results in dwarfism. We examined the molecular events underlying these findings. The non-transgenic (non-TG) diabetic mouse glomeruli had an increase in mRNA coding for alpha 1IV collagen, laminin B1, TGF-beta 1, 72 kDa collagenase, and TIMP-3. In contrast, glomerular type IV collagen and laminin B1 mRNA levels were normal in diabetic TG dwarf mice. However, the 72 kDa gelatinase, TIMP-3, and TGF-beta 1 mRNAs were elevated in the diabetic dwarfs. Type IV collagen and laminin accumulated in the glomeruli of diabetic non-TG, but not of diabetic dwarf mice, by immunofluorescence microscopy, confirming the mRNA data. GH binding protein mRNA levels were comparable in glomeruli from dwarf and non-TG mice, both diabetic and non-diabetic. We did not detect GH receptor mRNA in glomeruli. These data suggest that diabetic glomerulosclerosis is associated with an increase in type IV collagen and laminin synthesis, and that these changes do not occur in mice transgenic for bGH119K, a functional antagonist of GH. The increase of 72 kDa gelatinase, TIMP-3 and TGF-beta 1 mRNAs, independent of GH, suggested that these changes induced by hyperglycemia were not sufficient for the induction of glomerulosclerosis.

The macrophage mannose receptor, a carbohydrate-binding membrane protein, mediates endocytosis and phagocytosis. This study was undertaken to determine whether mannose receptors were expressed in resting glomerular mesangial and endothelial cells and whether their level was affected by cytokines. Neither mannose receptor mRNA nor proteins were found in resting mesangial or endothelial cells. Mannose receptor mRNA was induced in a dose- and time-dependent manner in mesangial cells by interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) but not by platelet-derived growth factor-B or IL-6. Cell surface receptors were found by fluorescence-activated cell sorter analysis. Binding to stimulated mesangial cells was saturable and inhibited by excess mannose-bovine serum albumin (BSA) but not by galactose-BSA. TNF-alpha and IL-1 alpha also induced apoptosis in mesangial cells. Mannose receptor expression was not restricted to apoptotic stimulated mesangial cells. Neither agonist induced mannose receptor expression or apoptosis in endothelial cells. Because immunoglobulin A, M, and G contain mannose residues, immune aggregates may be removed from the mesangium through cytokine-induced mannose receptors.

An accurate assessment of which patient with glomerular disease will progress to end-stage renal failure would be an important addition to establishing prognosis and evaluating therapeutic strategies. We previously found the development of glomerular scarring in animal models was preceded by an increase in glomerular type IV collagen mRNAs and that the level of scarring predicted the rate of progression. The purpose of this study was to determine whether these findings apply to human glomerular diseases using microdissected glomeruli and assessment of mRNA by competitive PCR. After showing that the levels of type IV collagen mRNAs were elevated in sclerotic glomeruli isolated from nephrectomies, we undertook this preliminary cross-sectional study of type IV collagen subchain mRNAs in renal biopsies in two of the leading causes of glomerulosclerosis, diabetic nephropathy, and membranous glomerulopathy. We found that glomerular type IV collagen mRNA levels were altered in disease-specific ways. The relative levels of the individual alpha-chains of type IV collagen depended on the anatomic site of the glomerular lesions. The alpha 2 type IV/alpha 3 type IV collagen mRNA ratio was high in diabetes mellitus, but not in membranous glomerulopathy. These data, coupled with those obtained from experimental animals, suggest that a dysregulation of basement collagen synthesis underlies progressive glomerular scarring. If these conclusions are verified in prospective studies it will be feasible to assess the risk of developing progressive glomerulosclerosis in the individual patient and to quantitatively assess therapeutic responses in a timely manner.

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.

The role that advanced glycosylation end-products (AGEs) may play in the development of diabetic nephropathy is still not completely understood. In order to elucidate the nature of their effect, the consequences of exogenously administered AGEs on extracellular matrix gene expression were examined in mice by competitive PCR. Normal adult mice receiving repeated injections of AGEs had an increase in the expression of genes coding for type IV collagen and laminin in the glomeruli. The increase was accompanied by up-regulation of TGF-beta 1 but not PDGF-B expression. The expression of smooth muscle and beta-actin did not change, showing that the increase in gene expression was specific for genes implicated in the early stages of diabetic nephropathy. The co-administration of aminoguanidine, a drug that inhibits AGEs cross-links, prevented the up-regulation of gene expression in AGEs-injected mice. Thus, AGEs can induce extracellular matrix genes in the absence of hyperglycaemia.

Background: Progressive glomerulosclerosis associated with decreasing kidney function, resulting in end-stage renal failure, is the major finding in diabetic nephropathy. There are multiple components of the extracellular matrix, and neither the exact composition nor the factors responsible for its increase in diabetic nephropathy is known., Methods: We examined matrix synthesis and degradation at the level of gene expression and ECM composition, in the glomeruli of diabetic mice transgenic for a mutated bGH and their littermate controls, and in renal biopsies of patients with diabetic nephropathy., Results: We found that streptozotocin-treated C57BL/SJL mice developed histological lesions closely resembling human diabetic glomerulosclerosis, whereas there were no glomerular lesions in diabetic mice transgenic for a growth hormone analogue which competes with native GH and results in dwarfism. We found that diabetic glomerulosclerosis in mice was associated with an increase in type IV collagen and laminin synthesis, and that these changes are selectively inhibited in dwarf mice transgenic for the mutant GH analogue. Preliminary examination of glomeruli isolated from renal biopsies revealed a homogeneous elevation in the alpha 2/alpha 3IV ratio among diabetics., Conclusions: These data suggested that diabetic nephropathy is characterized by modifications in ECM gene regulation in both mouse models and in diabetics, and that GH may promote the susceptibility to this complication. Finally, the data from the Diabetes Control and Complications Trial is updated, emphasizing the importance of close glucose control in the amelioration of an elevated albumin excretion rate as a continuous function of the level of control.

Antibodies to glutamic acid decarboxylase (GAD), a pancreatic islet beta-cell antigen, are present in > 80% of newly diagnosed insulin-dependent diabetes mellitus (IDDM), and are found in nonobese diabetic (NOD) mice, a murine model of spontaneous IDDM. To determine whether GAD is a target antigen in the kidney damage of NOD mice, we studied GAD mRNAs (GAD65 and GAD67) by RT-PCR in mesangial cells, isolated glomeruli, and kidney cortex and medulla in NOD and SJL/C57BL mice. GAD mRNAs were detected in the cortex of both diabetic and nondiabetic NOD and SJL/C57BL mice and GAD antigen was present in proximal and distal tubules by immunofluorescence microscopy. Neither GAD antigen nor mRNA were present in mesangial cells or glomeruli of diabetic or nondiabetic mice. Thus, the expression of GAD in renal tubules raises the possibility that GAD antigens may play a role in diabetic tubulointerstitial disease, whereas the absence of these antigens in glomeruli suggests that GAD-triggered autoimmunity is not directly involved in the glomerular lesions.

Changes in the composition of the mesangial extracellular matrix (ECM) and cell turnover are present in glomerular disease. To determine if ECM changes play a role in perpetuating mesangial cell dysfunction, we examined a line of mouse mesangial cells cultured on films or gels of several ECM components and also on methyl cellulose, an inert substrate that prevents attachment. Cells on films of fibronectin or type IV or I collagen had persistently high growth rates and high levels of alpha 1-I and alpha 1-IV collagen mRNAs. In contrast, on gels of type IV or I collagen or matrigel, the growth rate was low. The alpha 1-IV collagen mRNA levels were low on type IV collagen gel or matrigel, whereas the alpha 1-I collagen mRNA levels remained high. In contrast, the alpha 1-I collagen mRNA levels were low on type I collagen gel, and the alpha 1-IV collagen mRNA levels were high. Cells on methyl cellulose formed floating aggregates, did not proliferate, and had a 5- to 10-fold decrease in both alpha 1-I and alpha 1-IV collagen mRNA levels. These phenotypic changes were largely reversible. Finally, when matrigel was layered over cells on fibronectin films, alpha 1-IV collagen mRNA levels decreased, but alpha 1-I collagen mRNA levels and proliferation remained high. Thus proliferation and alpha 1-I and alpha 1-IV collagen mRNA levels in mesangial cells were independently regulated and depended on attachment and the nature of the adjacent matrix.

Oligosyndactyly mice (ROP Os/+) are a radiation-induced mutant strain with reduced glomerular number and increased glomerular size. We found that they develop glomerulosclerosis. At 3 mo, ROP Os/+ mice had diffuse mesangial expansion by light microscopy, whereas their +/+ littermates did not. Electron microscopic morphometry revealed a twofold increase in mesangial areas but no changes in the thickness of glomerular basal laminae. Mean glomerular volume was increased 1.8-fold. Cell number and thymidine labeling index were increased 1.3- and 2.4-fold, respectively. The amount of glomerular type IV collagen and tenascin but not laminin was increased by immunofluorescence microscopy. mRNA levels in microdissected glomeruli were measured by competetive reverse transcription-polymerase chain reaction and corrected for cell number. alpha 1-Chain type IV collagen and tenascin mRNAs were increased 3.2-fold and 1.8-fold, whereas laminin B1 mRNA levels were not. The levels of 72-kDa collagenase mRNA were increased 1.6-fold. Transforming growth factor-beta 1 mRNA levels were elevated 1.8-fold, but platelet-derived growth factor-B mRNA levels remained normal. This is the first analysis of glomerular molecular and cellular changes in a model of congenital nephron reduction.

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.

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)

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.

Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin-dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription-competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes.

Mice transgenic for bovine growth hormone (bGH) develop progressive diffuse glomerulosclerosis. Because murine mesangial cells in vitro were found to express the genes for 72-kd gelatinase and the metalloproteinase inhibitor TIMP-1, the expression of these genes in vivo in isolated whole glomeruli from bGH mice and normal control littermates was examined. Intact glomeruli were isolated by microdissection and subjected to reverse transcription. TIMP-1 cDNA was not detected by standard polymerase chain reaction (PCR) in glomeruli from bGH or control mice. In contrast, cDNA for 72-kd gelatinase was detected by standard PCR in both bGH and control mice, and the level was subsequently measured by quantitative competitive PCR. The gelatinase cDNA level was 14.7 +/- 2.8 x 10(-4) attomoles/glomerulus in 2- to 3-month-old control mice and was unchanged in 6-month-old controls. The bGH mice had 3.5-fold and 4.5-fold higher cDNA levels at 2 to 3 months and 6 months of age, respectively. Finally, zymography of glomerular extracts revealed increased levels of 72-kd and 96- to 100-kd gelatinase activity in bGH glomeruli in comparison to that in controls. In summary, whereas the genes for both TIMP-1 and 72-kd gelatinase are expressed in vitro in cultured mesangial cells, only the gelatinase gene appeared to be expressed in vivo in intact glomeruli. In addition, there was an up-regulation in the glomerular expression of the 72-kd gelatinase in bGH mice, a murine model of glomerulosclerosis.

High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur in diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosis of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were administered daily to healthy rats alone or in combination with the AGE inhibitor aminoguanidine. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electron microscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), significant periodic acid/Schiff reagent-positive deposits, basement membrane widening, and mesangial extracellular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with significant loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and functional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. The effects are AGE-specific, as they are ameliorated by a pharmacological AGE inhibitor, aminoguanidine.

Background: Angiotensin converting enzyme inhibitor (ACEi) therapy delays the onset of renal failure in diabetic nephropathy and inhibits or delays the onset of proteinuria in several animal models., Materials and Methods: We examined this question using a transgenic model of chronic glomerulosclerosis caused by an excess production of growth hormone (GH) in which there is progressive glomerular scarring leading to uremia. In addition, since GH mice do not have systemic hypertension or an elevated glomerular filtration rate, we could address the question of whether ACEi or angiotensin II receptor antagonists (AII RA) had an effect on the development of glomerulosclerosis under these conditions. Since excess matrix accumulates in glomerulosclerosis because of alterations in the balance between its synthesis and degradation, we examined the effect of ACEi and AII RA on these parameters., Results: Systemic blood pressure was unaffected by ACEi treatment, but the glomerular filtration rate decreased 85%. ACEi-treated mice had increased mesangial deposition of type I collagen and decreased 105 kD complex collagenase activity. In addition, ACEi-treated GH mice had increased glomerular alpha 1 type I collagen, alpha 1 type IV collagen, and alpha-smooth muscle cell actin mRNAs. No changes were noted in beta actin, or 72 kD metalloproteinase mRNAs. The result of these changes was a net increase in sclerosis. Surprisingly, GH mice treated with ACEi or AngII RA developed marked renal arteriolar lesions., Conclusions: In some forms of glomerulosclerosis, the lesions develop independently of angiotensin II. Pharmacological inhibition of angiotensin II, in this circumstance, may aggravate the lesions through disregulation of the levels and the balance between glomerular matrix synthesis and degradation.

Transgenic pigs were created that harboured a phosphoenolpyruvate carboxykinase-bovine growth hormone construct (PEPCK-bGH). Four founder animals and two transgenic offspring from one line were evaluated between 6 1/2 and 12 months of age. There was no evidence of severe hepatic or renal lesions in these pigs, which characterised transgenic PEPCK-bGH mice previously described. While glomerular and tubular lesions in kidney sections were not identified in the transgenic pigs, mesangial cell proliferation was observed in two transgenic offspring from a single line. Additionally, glomerular size was significantly increased in four of four puberal transgenic swine when compared to age- and sex-matched controls (28.30 +/- 4.1 vs. 14.2 +/- 2.7 x 10(5) microns 3; representing 3 transgenic lines, p < 0.05). Surprisingly, no mature adipocytes were observed in subcutaneous sections obtained in transgenic GH pigs. Histological evaluation of these transgenic pigs further illustrates the requirement for precise control of growth-related genes and their protein products.

Several lines of evidence suggest that the excessive accumulation of extracellular matrix in the glomeruli of diabetic kidneys may be due to reactive intermediates forming between glucose and matrix proteins called advanced glycation end products (AGEs). Normal mice received AGE-modified mouse serum albumin i.p. for 4 weeks, and glomerular extracellular matrix, growth factor mRNA levels, and morphology were examined. We found that AGE induced an increase in glomerular extracellular matrix alpha 1(IV) collagen, laminin B1, and transforming growth factor beta 1 mRNA levels, as measured by competitive PCR, as well as glomerular hypertrophy. The AGE response was specific because the coadministration of an AGE inhibitor, aminoguanidine, reduced all these changes. We conclude that AGEs affected expression of genes implicated in diabetic kidney disease and may play a major role in nephropathy.

The nature and slope of progression of glomerular diseases is the subject of considerable discussion. The use of renal biopsies has been accompanied by considerable improvement in the classification of glomerular disease. However, the clinical and histological tools available to assess prognosis in individual patients lack accuracy, especially in slowly progressive glomerular diseases such as diabetes mellitus. The development of molecular biology tools provides a new approach to the analysis of glomerulosclerosis. The accumulation of extracellular matrix in the glomerulus results from an exaggerated synthesis of collagens and other molecules forming the basement membranes, and is accompanied by an increase in the corresponding mRNAs. The measure of local glomerular gene activation can therefore provide a dynamic view of glomerular scarring. Utilizing a method combining microdissection of the glomeruli, reverse transcription in situ and a competitive polymerase-chain-reaction assay we were able to measure minute amounts of mRNAs in single mouse and human glomeruli. Both in mouse models and in human glomerulosclerosis we found upregulation of basement membrane collagen genes in the glomeruli. The increase appeared to be parallel to the slope of progression of glomerulosclerosis in experimental animals. This new approach may therefore provide a quantitative and sensitive method to define the propensity to sclerosis in human disease.

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.

Endothelial cells isolated from a variety of vascular beds bind and transport insulin but exhibit relatively low insulin degrading activity. Because endothelial cells exhibit heterogeneity and since kidney is a major site of insulin degradation, we studied the processing of insulin by glomerular endothelial cells (GEC). When exposed to 2 x 10(-10) M 125I-labeled insulin, GEC associated with the hormone in a specific manner. This interaction was inhibited by insulin but not by a number of unrelated peptide hormones. Over a 90-min period, GEC degraded 42 +/- 3% of the 125I-insulin, as measured by solubility in trichloroacetic acid (TCA). Degradation was inhibited 90% by an excess of insulin or adrenocorticotropic hormone (10(-6) M) and 57% by glucagon, whereas growth hormone and calcitonin were without effect. Separation of plasma membrane bound from internalized insulin was achieved by decreasing extracellular pH. In the steady state, 43% of cell-associated insulin was membrane bound and 57% internalized. The fate of the internalized 125I-insulin was examined by incubating acid-washed cells at 37 degrees C for 60 min. Over this time 18% of the radioactivity was released as TCA insoluble- and 72% as TCA-soluble radioactivity. Release was increased by insulin (10(-6) M) but not by unrelated peptide hormones. In the presence of chloroquine, 125I-insulin release increased by one third while degradation fell. High-performance liquid chromatography revealed that GEC released both intact insulin and large intermediates and that chloroquine inhibited intermediate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the balance between synthesis and degradation of extracellular matrix may result in glomerulosclerosis. The interaction between metalloproteinases and their inhibitors presumably modulates the rate of glomerular matrix degradation. We examined the gene expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in human glomeruli and TIMP-2 protein in tissue sections. Kidney tissue was obtained from adults undergoing nephrectomy for renal tumor (n = 9) or biopsy for nephrosis and renal failure (n = 1). Glomeruli were microdissected and subjected to reverse transcription. TIMP cDNAs were quantitated by competitive polymerase chain reaction assays. Five nephrectomy specimens had normal glomeruli and four had diffuse glomerulosclerosis. TIMP-1 and TIMP-2 cDNA levels, detected in glomeruli from all patients, were increased fourfold and threefold, respectively, in patients with glomerulosclerosis. The elevated TIMP cDNA levels could not be attributed to an increased number of glomerular cells. TIMP-2 protein was detected within normal and sclerotic glomeruli. In conclusion, both TIMP genes were expressed in normal glomeruli, and their level of expression was increased in glomerulosclerosis associated with renal carcinoma, suggesting that expression of these inhibitors may correlate with the development of sclerosis.

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.

Background: Mice transgenic for bovine growth hormone (bGH) gene have increased body weight and severe glomerulosclerosis leading to death in uremia., Experimental Design: The aim of this study was to determine if body growth and glomerulosclerosis were mediated by different bGH regions. Amino acid substitutions in the bGH alpha-helix III were generated, and lines of transgenic mice that expressed these products were developed. Female transgenic mice carrying the native bGH gene (bGH mice), a mutated bGH gene that encodes a destabilized alpha-helix III (bGH-L121P, E126G; bGH-m11 mice), or a mutated bGH gene that encodes a perfect amphiphilic alpha-helix III (bGH-E117L, G119R, A122D; bGH-m8 mice) were examined at 2-3 months and 6-9 months of age. Body, kidney, and heart weights were measured. Urinary glucose, albumin, creatinine, and serum glucose were measured in all mice. Serum levels of insulin-like growth factor I (IGF-I) were measured in the 2-3 month group. Whole blood hemoglobin A1 was measured in some mice of the 6-9 month group. Kidney sections were examined by light and immunofluorescence microscopy. Glomerular volume was measured and related to body weight by allometry., Results: The bGH-m11 mice developed glomerulosclerosis indistinguishable from that seen in bGH transgenic mice, even though they had normal body size. Glomerular growth exceeded body growth by allometry in both bGH and bGH-m11 strains. bGH-m8 mice had glomeruli of appropriate size and normal histologic appearance; however, they were dwarfs. IGF-I was increased in bGH mice; they also had an increased albumin/creatinine ratio at 6-9 months. None of the mice were hyperglycemic., Conclusions: These data indicated that development of glomerulosclerosis and body growth promotion were mediated by different regions of the growth hormone molecule. The glomerular response to bGH was unique and consisted of increased size and glomerulosclerosis.

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.

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.

Studies of age-related changes in glomerular extracellular matrix (ECM) synthesis in normal mice have been hampered by the difficulty of isolating sufficient numbers of intact glomeruli and by the inability to quantify different mRNA species. The purpose of this study was to identify and quantitate the individual mRNAs coding for alpha 1- and alpha 2-chains of type IV collagen in isolated, single glomeruli of normal mice at different ages. These data on normal ECM synthesis were necessary for the understanding of glomerulosclerosis, a condition characterized by excess deposition of collagen. Pools of freshly microdissected adult mouse glomeruli were reverse transcribed in situ, and alpha 1-IV and alpha 2-IV collagen mRNAs were individually amplified by means of specific primers and the polymerase chain reaction (PCR), according to a previously published method. A competitive PCR assay, based on utilization of mutated cDNAs, allowed the reproducible, quantitative, and separate determination of the absolute amounts of both alpha 1-IV and alpha 2-IV mRNAs measured, as their respective cDNAs, in one-tenth of one glomerulus. The levels of alpha 1-IV and alpha 2-IV collagen mRNA were 208 +/- 36.0 x 10(-4) and 161.2 +/- 18.6 x 10(-4) amol/glomerulus in 5-wk-old mice. There were no significant age-related differences at 8, 12, and 24 wk. The mean levels over this period were 60.2 +/- 4.9 x 10(-4) for alpha 1-IV collagen mRNA and 63.9 +/- 5.8 x 10(-4) amol/glomerulus for alpha 2-IV collagen mRNA. Two of three 24-wk-old mice had mild glomerulosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The mean glomerular volume, glomerular fraction of cortical volume, and percentage of obsolescent glomeruli were calculated in kidney specimens from autopsies on 34 Pima Indians, of whom 15 had non-insulin dependent diabetes mellitus and kidney disease of diabetes mellitus. These values were compared with those of black, white, and non-Pima native American individuals without diabetes mellitus. Glomerular volume in the Pima Indians was similar in the diabetic and nondiabetic subjects and significantly greater than in the white subjects. Black and non-Pima native American individuals had glomerular volumes intermediate between white individuals and Pima Indians. The mean glomerular volume was not affected by the number of obsolescent glomeruli in diabetic Pima Indians. The glomerular volume fraction was greater in the Pimas than in the other groups. These data showed that glomerular volume in the Pima Indians was significantly greater than that in white subjects. There was no difference between diabetic and nondiabetics Pimas, and glomerular size was not correlated with the presence or degree of glomerulosclerosis in this population.

Renal disease is one of the most common and severe complications of diabetes mellitus. The hallmark of the disease, glomerulosclerosis, is characterized by an accumulation of extracellular matrix in the mesangial areas, leading to progressive obliteration of the vascular spaces. The role of the metabolic derangements of diabetes mellitus in the development of these lesions is incompletely understood. One of the consequences of hyperglycemia is the formation of advanced glycosylation end products (AGEs), which result from a series of rearrangements secondary to nonenzymatic reaction of glucose with proteins. Specific receptors for proteins modified by AGEs, present in several cell types, were recently described in human and rat mesangial cells. Furthermore, exposure of mesangial cells to AGEs was followed by an increase in fibronectin production. In the present study we show evidence that mouse mesangial cells exhibit an increase in collagen type IV mRNA and peptide synthesis after exposure to AGEs. Antibodies to AGE receptors prevent this increase, indicating that the response is AGE-receptor-mediated. In addition, anti-platelet-derived growth factor abrogates the AGE response, suggesting that platelet-derived growth factor acts as an intermediate factor. Transcription assay reveals that the elevated mRNA levels are due to an increase in the transcription rate, rather than to an increase in the stability of the message. Finally, the mRNAs coding for laminin and heparan sulfate proteoglycan are also increased after exposure to AGE, whereas glyceraldehyde 3-phosphate dehydrogenase mRNA levels remain constant. The increase in extracellular matrix mRNAs seen in the current study suggests that AGE formation in vivo may be one of the metabolic events leading to the development of diabetic glomerulosclerosis.

The evolution of glomerulosclerosis consists of a progressive increase in mesangial matrix with gradual glomerular obliteration. The sclerotic process is thought to be irreversible and include a progressive loss of glomerular cells. To investigate this process, we selected mice transgenic for bovine growth hormone because they develop progressive glomerulosclerosis and renal failure. The sequence of histologic events in the growth hormone mice consists initially of an increase in the number of centrolobular glomerular cells, followed by an accumulation of extracellular matrix. This is accompanied by an increase in glomerular size which is disproportionate to the overall increment in kidney or body weight. The [3H]thymidine labeling index of the cells of the glomerular tuft was assessed before the development of recognizable sclerosis and at a time when the sclerosis was far advanced. The labeling index was more than five-fold increased over controls at the early time point. Contrary to what was expected, the labeling index remained at the same high levels in densely sclerotic glomeruli at the late time point. In conclusion, increased cell turnover is a significant component of the sclerotic process both at the onset and in the late stages of this model.

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.