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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.

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.

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.

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.

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.

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.

A low-protein (LP) diet has been associated with amelioration of renal function in glomerulosclerosis (GS). However, the mechanisms involved are still unclear. We have used a mouse transgenic for bovine growth hormone (GH), which develops progressive GS and exhibits consistently elevated levels of circulating GH and insulin-like growth factor (IGF)-1, to study the effect of dietary protein restriction. LP (6% protein) and normal-protein (NP, 20% protein) diets were maintained for 30 weeks in mice with established GS of mild/moderate degree. The degree of GS was markedly attenuated in LP compared to NP mice. Quantitative analysis revealed a significantly lower GS index (1.4 +/- 0.9 in LP vs. 2.8 +/- 0.8 in NP) and glomerular volume (0.8 x 10(6) +/- 0.1 x 10(6) microm(3) in LP vs. 1.2 x 10(6) +/- 0.1 x 10(6) microm(3) in NP) in mice with restricted protein intake. These morphologic changes were accompanied by a significant reduction in renal expression of alpha(1) type-IV collagen (2.4-fold) and tenascin (1.4-fold) in LP mice. Serum IGF-1 decreased by 40% and showed a significant correlation with alpha(1) type-IV collagen expression with the LP diet. The present finding supports the use of the LP diet to decelerate the progression of GS and furthermore suggests that one of the mechanisms involved in this process is the GH/IGF-1 regulation by protein intake., (Copyright 2001 S. Karger AG, Basel)

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

Background: The growth hormone (GH)/insulin-like growth factor (IGF) system is thought to participate in the glomerulosclerosis process. Because IGF-binding proteins (IGFBPs) modulate IGF actions and hence GH secretion, this study assessed whether mice transgenic for human IGFBP-1 have altered susceptibility to glomerulosclerosis., Methods: A line of transgenic mice that express human IGFBP-1 mRNA in the liver under the control of the alpha1-antitrypsin promoter has been obtained, and morphological changes in the kidney tissue were assessed. Glomerulosclerosis was identified using light microscopy, light microscopic morphometry, and electron microscopy. Extracellular matrix components were analyzed by immunohistochemistry., Results: There was a marked increase in mesangial extracellular matrix area in homozygous transgenic mice at three months of age as compared with heterozygous transgenic mice and nontransgenic littermates. These changes were not associated with alterations in glomerular volume or cellularity. The expansion of extracellular matrix area was related to a marked increase in laminin and type IV collagen and to the appearance of type I collagen., Conclusions: These observations indicate that the enhanced expression of IGFBP-1 may result in the development of glomerulosclerosis without glomerular hypertrophy. The changes are potentially related to a decrease in IGF-I availability and/or to an IGF-I-independent role of IGFBP-1.

Background: Glomerulosclerosis, the common terminal event in chronic glomerular diseases such as diabetic nephropathy or IgA nephropathy, leads to end-stage renal disease. The considerable variation in both the risk of developing glomerulosclerosis and the rate of progression in individual patients suggest a role for genetic factors which have not been identified so far. In this study we sought to examine the mode of inheritance of glomerulosclerosis in mice., Methods: F1 animals of a mating between glomerulosclerosis-prone ROP-Os/+ male and non-sclerotic C3H female mice were backcrossed to the ROP strain. We took advantage of the radiation-induced mutation oligosyndactylism (Os) to identify glomerulosclerosis at the age of 3 months. Kidneys were perfused in situ with PBS/Formalin 10%. The extent of glomerulosclerotic lesions was evaluated on PAS stained paraffin sections using computer-aided morphometry., Results: F1 mice did not show any glomerulosclerosis. In the backcross offspring, we found a wide distribution of glomerular lesions between individual animals, ranging from normal to very severe. We calculated that at least 8-10 loci determine the severity of glomerulosclerosis in mice., Conclusions: Our data show that glomerulosclerosis is inherited in a recessive fashion involving at least 8-10 loci.

Background: We reported that ROP, but not C57, mice were prone to glomerulosclerosis (GS) after nephron reduction (J Clin Invest 97:1242, 1996)., Methods: In this study, we induced diabetes in ROP and C57 mice to determine if the glomerulosclerotic response was stimulus specific. We used the oligosyndactyly mutation (Os), to produce a congenital 50% reduction in nephron number. Stable hyperglycemia was induced by streptozotocin and mice were maintained for 12 weeks without insulin treatment., Results: Glomerular hypertrophy occurred in diabetic ROP +/+ and C57 +/+ mice, but glomeruli of diabetic ROP +/+ mice had 1.92-fold higher laminin B1 and 1.5-fold higher tenascin mRNA levels than diabetic C57 +/+ mice. Diabetic ROP Os/+ mice had severe glomerulosclerosis with arteriolar and tubulointerstitial lesions while there was only moderate mesangial sclerosis in diabetic C57 Os/+ mice. Glomerular size was increased in all non-diabetic Os/+ mice. It was further increased in diabetic ROP Os/+ mice, but not in diabetic C57 Os/+ mice. Glomerular mRNA levels were higher in diabetic ROP OS/+ than in diabetic C57 OS/+ mice [alpha 1 (i.v.) collagen 3.2-fold, laminin B1 2.1-fold, and tenascin 1.6-fold]., Conclusion: Overall, our data further support the hypothesis that the susceptibility to glomerulosclerosis is inherited, and suggest that hyperglycemia serves principally as a triggering event in the development of diabetic nephropathy. Since the acceleration of diabetic nephropathy by nephron reduction was also largely strain dependent, it appears that the propensity to glomerulosclerosis is a general renal response and is not stimulus specific.

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.

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.

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.

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)

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.

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.

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.

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.

We previously described the development of glomerulosclerosis in mice transgenic for large T-antigen, a gene whose in vitro expression markedly increases proliferation of cultured cells. In the current study we sought to determine the effect of unilateral nephrectomy on these sclerosis-prone animals which have a genetically defined potential for increased renal growth. In comparison with sham nephrectomy animals, nephrectomized transgenic female mice had significantly larger kidneys, larger glomeruli (5886 mu2 npx vs. 3796 mu2 sham), more cells per glomerulus and more severe glomerulosclerosis. Nephrectomized transgenic male animals had variable increases in kidney size, no significant increase in glomerular size (4750 mu2 npx vs. 4502 mu2 sham) or cellularity, and no worsening of glomerulosclerosis. In non-transgenic female animals nephrectomy induced an increase in kidney size but not in glomerular size (2640 mu2 npx vs. 2625 mu2 sham) and failed to induce glomerular lesions. A close correlation (r = 0.91) was found between glomerular size and severity of glomerulosclerosis in these animals. This finding supports the hypothesis that a pathophysiologic link exists between glomerular enlargement and glomerulosclerosis. We also found that increases in total kidney size and glomerular size did not consistently parallel each other, that is, renal hypertrophy may occur without an increase in glomerular size. This finding suggests that total kidney growth and glomerular growth may be independently regulated or may have different thresholds for activation following unilateral nephrectomy.

An increase in glomerular size occurs in normal maturation after subtotal renal ablation and disease states such as diabetes mellitus. The role that growth hormone (GH), growth hormone releasing factor (GHRF), and insulinlike growth factor-1 (IGF-1) play in these processes has been investigated using transgenic mice chronically expressing these hormones. The glomeruli were enlarged in all 3 strains of mice. Mesangial proliferation followed by progressive glomerulosclerosis was observed in the GH and GHRF animals only. In the IGF-1 mice the large glomeruli remained morphologically normal except for the enlargement. These data suggest that the glomerulosclerosis was due, in part, to disordered mesangial cell growth in response to circulating GH. The mesangial lesions in mice with chronically high plasma GH levels mimicked those in human diabetes mellitus. These models provide a means to study the hormonal regulation of glomerular growth and the role that specific hormones might play in the pathogenesis of glomerulosclerosis.

Considerable evidence indicates that genetic determinants play a major role in the pathogenesis of a variety of human and experimentally-induced renal diseases. There are, however, no firm data to indicate which genes or types of genes can induce or promote renal disease. The recently acquired ability to make specific alterations in the genetic background of an animal affords a unique opportunity to assess the effect(s) of a given gene on the structure and function of an organ of interest. Such modifications have been carried out in the creation of transgenic mice. We examined mice transgenic for the transforming gene encoding large T-antigen which is present in the early region of simian virus 40 (SV40). Renal lesions were present in most animals. While there was some heterogeneity in the type and severity of the renal lesions observed, a majority of the older mice displayed glomerulosclerosis and/or proliferative tubular lesions which in some were associated with multiple, large tubular cysts. The appearance of these lesions in mice transgenic for a transforming gene suggests that renal expression of a gene which controls cell proliferation may be associated with the development of glomerulosclerosis and renal cysts. These findings indicate a possible role for other transforming genes, or oncogenes, in the pathogenesis of glomerulosclerosis and cystic renal disease in humans and other animal models.