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.

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.

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.

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 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.

Extensive aetiological investigations in acute pancreatitis: results of a 1-year prospective study.

BACKGROUND: Epidemiological data on acute pancreatitis are poorly defined. AIMS: To prospectively evaluate the aetiology of acute pancreatitis and to assess the benefits of intensive investigations. METHODS: In a prospective, 1-year study all cases of acute pancreatitis in the Nice catchment area were enrolled. Subjects underwent routine (serum calcium, phosphate and triglycerides; abdominal ultrasonography and CT scan) and additional, delayed intensive investigations (ERCP with bile sampling and/or endoscopy ultrasonography). RESULTS: One hundred and twenty-one cases were included. After routine investigations, a biliary, alcoholic, miscellaneous or unknown origin was diagnosed in 43%, 31.4%, 9.9% and 15.7%, respectively. In subjects with biliary pancreatitis, 43% had no previous history of biliary disease. In the alcohol-related subgroup, pancreatitis recurred in 18.5% during 114.5 days mean follow-up. In subjects with a first episode of alcoholic pancreatitis, delayed supplemental investigations revealed underlying chronic pancreatitis in 92.8%. After routine investigations, a diagnosis of pancreatitis of unknown origin was made in 15.7% (n = 19) of subjects. Additional investigations revealed an underlying cause in 57.8% of these patients (n = 11), including malignancy (n = 3) and biliary disease (n = 4), reducing the overall rate of pancreatitis with no apparent cause to 6.6%. CONCLUSIONS: Investigative techniques, particularly ERCP, will reveal the underlying aetiology of pancreatitis in the majority of patients presenting with 'idiopathic' pancreatitis and should be considered when routine tests are negative.

Inferior vena cava hypoplasia with intrahepatic venous continuation: sonographic, angiographic and MR features including MR angiography.

In cases of inborn or acquired obstacles on the inferior vena cava (IVC), the derived blood flow usually goes through collaterals in the azygos or the hemiazygos venous systems. Exceptionally, a collateral pathway through the portal system or through an anastomosis in between hepatic veins, shunting the IVC interruption, is encountered. In the present paper, the authors describe the fortuitous discovery of a IVC hypoplasia in its retrohepatic segment. MR venography, correlated with fluoroscopic angiography, clearly depicted an intrahepatic collateral circulation consisting of a double aneurysmal communication between an inferior right hepatic vein and the main right hepatic vein.

[Palliative endoscopic treatment of malignant duodenal stenosis by metal prosthesis]. / Traitement endoscopique palliatif des sténoses malignes duodénales par prothèse métallique.

PURPOSE: To evaluate the efficacy of through-the-scope metal stents for palliation of malignant duodenal stenosis. MATERIAL AND METHODS: Fourty two patients with malignant primary or secondary duodenal stenoses who were treated with a through-the-scope metal stent were analysed. When obstructive jaundice occurred either before, during, or after the initial episode of gastrointestinal luminal obstruction, a biliary stent was inserted. RESULTS: Duodenal metal stents were deployed in 40 patients. Endoprosthesis insertion led to restoration of oral intake in 39 patients. The procedure was not associated with morbidity or mortality. During a mean follow-up of 9.7 weeks, adequate oral intake was maintained in 38/39 cases. Tumour in-growth led to stent occlusion in 4 cases and re-cannulation was obtained by placement of another stent within the original stent. Obstructive jaundice occurred during the course of the illness in 32 patients and was successfully treated with a biliary metal stent in all cases. CONCLUSIONS: Endoscopically placed metal stents offer an effective, well-tolerated alternative to surgical palliation in case of incurable malignant obstruction to gastric outflow.

Endoscopic argon plasma trimming of biliary and gastrointestinal metallic stents.

BACKGROUND AND STUDY AIMS: The aim of this study was to assess the feasibility and efficiency of plasma argon trimming of gastrointestinal and biliary metallic stents. PATIENTS AND METHODS: A total of 31 patients underwent plasma argon trimming of their metallic stents (14 women, 17 men; mean +/- SD age 73 +/- 12.2 years, range 46 - 96 years). Of these 31 patients, 24 had had covered or noncovered Unistep Wallstents placed in the biliary tract (13 patients with pancreatic neoplasms, five patients with Vater ampulloma, five patients with biliary tract carcinoma and one patient with chronic calcifiying pancreatitis); three patients had noncovered Enteral Unistep Wallstents (pyloroduodenal); two patients with obstructive colorectal carcinoma had a noncovered Bard Memotherm stent inserted; and two patients had noncovered Ultraflex stents placed for esophageal carcinoma. Endoscopic trimming of the stents was performed under propofol-induced general anesthesia, with the power set at 70 - 80 watts and an argon flow of 0.8 liters/minute. RESULTS: Complete and satisfactory trimming of the stents was possible, without complications (mean follow-up 15.8 months), in all patients except one, a patient with a covered biliary Wallstent. In 13 patients with biliary or Enteral Wallstents the trimming procedure was preventive. In eight patients with ulceration and/or hemorrhage (duodenal or rectal), healing was achieved after stent trimming and epinephrine (adrenaline) injection followed by electrocoagulation. Stent trimming restored patency of the duodenal lumen in six patients and of the esophageal lumen in two patients, and was done to allow insertion of a biliary stent in one patient whose duodenal stent was covering the papilla. In one patient with rectal tenesmus, stent shortening resulted in complete resolution of symptoms. CONCLUSIONS: Endsocopic plasma argon trimming of metallic stents is an efficient procedure which allows easy, reproducible and well-tolerated correction of complications that arise due to these prostheses.

Progression of glomerular diseases.

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.

Argon plasma endoscopic section of biliary metallic prostheses.

We report our recent experience of using argon plasma to endoscopically cut biliary Wallstent prostheses in these patients. The first patient had a bleeding duodenal ulceration caused by the impaction of the prosthesis meshes whereas the second patient had an ill-positioned biliary stent with impaction into the opposite duodenal wall. Both prostheses were shortened using argon plasma. In the third patient, the lower extremity of a obstructed biliary Wallstent was positioned in the third duodenum preventing its endoscopic catheterization. After shortening using argon plasma, a new plastic stent could be inserted to allow drainage. The outcomes in these cases demonstrate the feasibility of endoscopically shortening metallic Wallstents after release using argon plasma.

Agenesis of the horizontal segment of the left portal vein demonstrated by magnetic resonance imaging including phase-contrast magnetic resonance venography.

Two cases of agenesis of the horizontal segment of the left portal vein are reported. This very rare vascular anomaly probably corresponds to an embryological variation rather than to an obstruction of the left portal vein. In almost all cases liver ultrasonography is sufficient for identifying such vascular abnormalities. It shows a large aberrant vessel emerging from a right anterior segmental portal branch and running transversely in the quadrate lobe towards the teres ligamentum from which the portal supply to the left lobe arises. It is important to be able to recognize the magnetic resonance imaging features of this vascular variation, as magnetic resonance imaging may be the initial imaging study, and ultrasound may be technically challenging. To our knowledge, we present the first description of these features, including an enhanced gradient-echo T1-weighted sequence, a turbo spin-echo T2-weighted sequence with fat saturation, and a three-dimensional phase-contrast magnetic resonance portography.

Long term treatment of biliary stricture due to chronic pancreatitis with a metallic stent.

The exact role of endoprostheses in the management of chronic pancreatitis-associated biliary strictures has not yet been clearly established. We report an unusual case of a patient with this condition who was treated for an unexpectedly long term with a self-expanding metallic endoprosthesis. There has only been one previous report of the use of metallic stents in this situation. It appears that metallic endoprostheses may have a role to play in the management of selected patients who have chronic pancreatitis-associated bile duct stricture.

Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease.

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 molecular basis of increased glomerulosclerosis after blockade of the renin angiotensin system in growth hormone transgenic mice.

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.

Relationships between mesangial cell proliferation and types I and IV collagen mRNA levels in vitro.

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.

Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA.

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.

Age-related changes in alpha 1- and alpha 2-chain type IV collagen mRNAs in adult mouse glomeruli: competitive PCR.

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)