Reduction of globotriaosylceramide in Fabry disease mice by substrate deprivation.

We used a potent inhibitor of glucosylceramide synthase to test whether substrate deprivation could lower globotriaosylceramide levels in alpha-galactosidase A (alpha-gal A) knockout mice, a model of Fabry disease. C57BL/6 mice treated twice daily for 3 days with D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidi no-propanol (D-t-EtDO-P4) showed a concentration-dependent decrement in glucosylceramide levels in kidney, liver, and spleen. A single intraperitoneal injection of D-t-EtDO-P4 resulted in a 55% reduction in renal glucosylceramide, consistent with rapid renal glucosylceramide metabolism. A concentration-dependent decrement in renal and hepatic globotriaosylceramide levels was observed in alpha-Gal A(-) males treated for 4 weeks with D-t-EtDO-P4. When 8-week-old alpha-Gal A(-) males were treated for 8 weeks with 10 mg/kg twice daily, renal globotriaosylceramide fell to below starting levels, consistent with an alpha-galactosidase A-independent salvage pathway for globotriaosylceramide degradation. Complications observed with another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, including weight loss and acellularity of lymphatic organs, were not observed with D-t-EtDO-P4. These data suggest that Fabry disease may be amenable to substrate deprivation therapy.

Differential expression of collagen type IV alpha-chains in the tubulointerstitial compartment in experimental chronic serum sickness nephritis.

The expression of collagen type IV chains in the renal tubulointerstitium was investigated during the development of chronic serum sickness (CSS) in rats, a model for immune complex-mediated renal disease. Immunohistochemical studies showed increased expression of alpha4(IV) collagen early during disease development, followed by an increase in alpha1(IV) through alpha3(IV) collagen subchain expression, especially in the tubular basement membrane. Dot-blot and in situ hybridization analysis showed a transient increase in steady-state mRNA levels for all collagen IV subchains during the development of CSS, which was most abundant for alpha1(IV), alpha2(IV), and alpha4(IV). Statistical correlations were found between the mRNA levels of alpha1(IV) and alpha2(IV) collagen and between alpha3(IV) and alpha4(IV), in line with the results of others which showed that these chains are co-distributed as heterotrimer collagen type IV molecules. However, additional correlations were found between the mRNA levels coding for alpha1(IV) and alpha3(IV) collagen, and between alpha1(IV) and alpha4(IV) mRNAs in the course of CSS. These abnormal correlations support the hypothesis that changes occur in the co-expression of the collagen IV subchains during the development of CSS. In addition, a strong correlation was found between the presence in the tubulointerstitium of alpha1(IV) and alpha2(IV) collagen chains, on the one hand, and the tubulointerstitial influx of R73+ and ED1+ cells, on the other, suggesting the involvement of inflammatory cells in the observed alterations in matrix production. Changes in the relative abundance of collagen IV chains in disease states may perturb the collagen IV network in the tubulointerstitial compartment and thereby play a role in the development of renal failure.

Insertional mutation of the collagen genes Col4a3 and Col4a4 in a mouse model of Alport syndrome.

Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.

Alternate splicing in human Na+-MI cotransporter gene yields differentially regulated transport isoforms.

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+-myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent approximately 1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3'-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3' exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.

Glomerular endothelial cells synthesize collagens but little gelatinase A and B.

Mesangial sclerosis is a major feature of progressive renal disease. The mesangium contains mesangial cells and is bounded by the peripheral glomerular basement membrane and endothelial cells. Mesangial cells synthesize and degrade extracellular matrix. Whereas both mesangial and endothelial cells synthesize extracellular matrix components, the degradative pathway, well studied in the former, has not been investigated in endothelial cells. This study examines lines of all three glomerular cell types derived from female B6SJLF1/J mice, as well as mRNA levels for collagens alpha1(I), alpha1(IV), alpha3 (IV), alpha5 (IV), and alpha1 (VI), laminin, tenascin, matrix metalloproteinase-2 (MMP-2), and MMP-9. Type I and IV collagen synthesis was confirmed by enzyme-linked immunosorbent assay. MMP-2 and MMP-9 enzyme activity was measured by zymography. It was found that glomerular endothelial cells are a significant source of collagens, laminin, and tenascin. However, they express only low levels of MMP-2 and no detectable MMP-9. Stimulation with exogenous transforming growth factor-beta1 leads to a significant increase in collagen I, tissue inhibitors of metalloproteinase-1, and MMP-9 in conditioned media. These data suggest that glomerular endothelial cells may play an active role in extracellular matrix remodeling in glomerular disease.

Augmented expression of glomerular basement membrane specific type IV collagen isoforms (alpha3-alpha5) in experimental membranous nephropathy.

In human and experimental membranous nephropathy, new extracellular matrix accumulates between, and eventually surrounds, immune deposits on the subepithelial aspect of the glomerular basement membrane (GBM). To define the nature and source of this newly deposited matrix, we studied by in situ hybridization and immunohistology the production and tissue deposition of the recently defined basement membrane type IV collagen chain isoforms alpha3, alpha4, and alpha5, the mesangium-specific alpha1 and alpha2 isoforms of type IV collagen, and the fibrillar interstitial type I collagen during the development of immunological injury in passive Heymann nephritis (PHN), a rodent model of membranous nephropathy. Our results show that steady-state mRNA levels of alpha3-alpha5 (IV) but not alpha1 (IV) are significantly increased in the glomeruli of rats with PHN at the peak of immunological injury after 14 days. Increased signal for alpha4 (IV) and the new appearance of alpha1 (I) could be clearly localized to glomerular podocytes, the target of injury in this model. In addition, increased levels of immunoreactive alpha3-alpha5 were visible in the peripheral and paramesangial GBM together with de novo deposits of type I collagen. A modest increase in mesangial staining for alpha1/alpha2 (IV) was present in PHN glomeruli. In rats depleted of complement for 5 days after PHN induction, the peak of alpha4 (IV) mRNA expression on day 14 was blunted. In conclusion, we have shown increased production of the intrinsic GBM type IV collagen isoforms alpha3-alpha5 and ectopic production of type I collagen by injured podocytes in PHN. These changes may contribute to the formation of an expanded and disorganized GBM, as seen in experimental and human membranous nephropathy.

Human Na(+)-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts.

Na(+)-myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na(+)-myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na(+)-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and approximately 1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.

Differential expression of collagen IV isoforms in experimental glomerulosclerosis.

Expansion of the glomerular mesangial matrix (MM), thickening of the glomerular basement membrane (GBM), and eventually the development of glomerulosclerosis are often seen in immunologically mediated kidney diseases. In addition to quantitative changes in the extracellular matrix (ECM), qualitative changes in ECM molecules may contribute to alterations in the composition of the glomerular matrix. The expression of collagen IV, alpha 1-5(IV) mRNA, and polypeptides was therefore investigated during the development of chronic graft-versus-host disease (GvHD) in mice, a model for lupus nephritis, and in chronic serum sickness (CSS) in rats, a model for membranous nephropathy. Immunohistochemical studies showed increased mesangial expression of alpha 1 and alpha 2 early in the disease, but only late in the GBM. In contrast, alpha 3 and alpha 4 increased in the GBM during disease, but not in the MM. The mRNA levels for all collagen IV chains were increased in isolated glomeruli before morphological alterations were detectable. The mRNA increase was earlier and more profound for alpha 3, alpha 4 and alpha 5 than for alpha 1 and alpha 2. Expression of alpha 3(IV) was greatest in GvHD, whereas expression of alpha 4 was greatest in CSS. As determined by in situ hybridization (ISH), alpha 1 mRNA was observed dispersed in the glomerulus, but alpha 3, alpha 4, and alpha 5 mRNAs were mainly located in cells at the periphery of the glomerular tuft. The changes in the relative abundance of collagen IV mRNA in disease states may perturb the collagen IV network, altering glomerular structure and function, and may thereby play a central role in the development of glomerulonephritis and glomerulosclerosis.

Renal biopsy collagen I mRNA predicts scarring in rabbit anti-GBM disease: comparison with conventional measures.

Progressive loss of normal structure associated with scarring is the hallmark of chronic diseases of most organs. To test the hypothesis that measurement of interstitial collagen mRNA levels would be a useful index to predict future scarring, we developed an assay to quantitate alpha 1(I) procollagen mRNA factored for GAPDH mRNA using RT-PCR (the "CI:G ratio"). We first defined conditions under which the assay could be used for analysis of renal biopsy samples. The CI:G ratio was then used to determine whether mRNA measurements performed at an early stage of inflammation (day 7) in a model of anti-GBM disease in the rabbit would predict outcome at day 30 as measured by interstitial and glomerular scarring and renal cortical hydroxyproline accumulation. The predictive value of this assay was compared to functional (serum creatinine and urine protein:creatinine ratio) and histologic (glomerular and interstitial scoring) parameters also measured at day 7. We found that the CI:G ratio alone provided a sensitive and discriminating assay over a wide range of renal injury that predicted various parameters of scarring with an average coefficient of determination (r2) of 0.69. This predictive power was higher than that found for conventional measures, which tended to have good discriminatory capacity over limited ranges of renal injury. The CI:G ratio provided significant additional predictive power over and above that available from combinations of conventional functional or histologic parameters. We conclude that measurement of the CI:G ratio in biopsy samples deserves further assessment as a potentially useful quantitative predictor of outcome that could lead to improved clinical decision-making.

Altered kidney matrix gene expression in early stages of experimental diabetes.

The expression of mRNA and distribution of alpha 1(IV), alpha 3(IV) chains of type IV collagen, matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of metalloproteinase 1 (TIMP-1) were examined in kidneys from streptozotocin-diabetic rats, 2.5 months after administration of the drug, an early time point when specific diabetic glomerular changes were still minimal. Ten age-matched Sprague-Dawley rats were assigned to control and diabetic groups. Compared to the controls, the diabetic rats had a significantly lower body weight, higher kidney weight and serum glucose levels, but no significant changes of glomerular surface area and urine albumin were observed. Northern blot analysis, using whole kidney mRNA, revealed that diabetic rat kidneys expressed 113.5% more alpha 1(IV), 46.5% more alpha 3(IV), 54.8% less MMP-2 and 246% more TIMP-1 (in all instances: p < 0.05). These results were corroborated by in situ hybridization for RNA expression. A quantitative analysis of the data indicated the following changes in glomeruli: (1) 74.6% more alpha 1(IV), (2) 103.8% more alpha 3(IV), (3) 40.7% less MMP-2 and (4) 80.9% more TIMP-1. Similar changes were observed in tubular (proximal and distal) cells. We conclude that an increased synthesis and decreased degradation of renal extracellular matrix components occur early after induction of experimental diabetes, before the onset of typical structural changes in the kidneys, and represent changes of specific gene expression at the transcriptional level. All the cell types in the glomerulus as well as the proximal and distal tubules appear to be involved in this alteration of expression, and this is a novel finding.

Synthesis and secretion of endothelin in a cortical collecting duct cell line.

Previous experiments have shown that epithelial cells in the renal medulla produce endothelin-1 (ET-1) and possess ETB receptors. It has been suggested that medullary ET-1 may affect water and sodium absorption along the collecting ducts in an autocrine fashion. To study possible mechanisms responsible for the regulation of medullary ET-1 production, experiments were performed in M-1 cells and mIMCD-K2 cells, cell lines derived from cortical and inner medullary collecting ducts of SV40 transgenic mice, grown to confluence on collagen-coated filter inserts. Both cell lines were found to express ET-1 mRNA and to secrete ET almost exclusively into the basolateral medium as long as the transepithelial resistance was high. Inhibition of transcription with actinomycin D was followed by a decline in both ET mRNA [halftime (t1/2) = 30 min] and ET secretion (t1/2 = approximately 90 min). The addition of arginine vasopressin (AVP, 10(-8) M; 2- or 4-h exposure) or incubation of M-1 cells in hypertonic media (+50 mM NaCl, 4- or 6-h exposure) did not significantly alter ET secretion or ET-1 mRNA expression. In contrast, simultaneously increasing AVP(10(-8) M in the basolateral medium) and tonicity (+50 mM NaCl) for 4 h increased ET secretion (from 28.9 +/- 3.9 to 41.8 +/- 3.8 pg.h-1.mg protein-1; P = 0.029, n = 10) and ET-1 mRNA (control = 2,138 cpm/microliter, log of 3.33 +/- 0.048, n = 4; AVP + NaCl = 3,548.1 cpm/microliter, log of 3.55 +/- 0.09; P = 0.045, n = 5). Exposure of M-1 cells to hypertonic media (+50 mM NaCl or 100 mM mannitol) for 24 h was associated with a marked reduction of ET secretion (-83.9% with NaCl and -78.4% with mannitol; P < 0.0001). This reduction was attenuated, but not prevented, by the presence of AVP in the basolateral medium (-40%). ET-1 mRNA, in contrast, did not change with 24-h exposure to hypertonic media and increased when AVP was present. Results are compatible with the concept that generation of ET by collecting duct cells may contribute in a complex and time-dependent fashion to the paracrine control of collecting duct cell function.

Transforming growth factor-beta modulation of the alpha 1(IV) collagen gene in murine proximal tubular cells.

We have examined the expression of the alpha 1(IV) collagen gene in murine proximal tubular cells (MCT) to better understand how it is regulated in parenchymal cells. Transcriptional activity was examined using luciferase reporters driven by the alpha 1(IV) promoter and varying lengths of 5'-flanking sequences. The minimal bidirectional promoter showed low intrinsic activity in MCT cells, but addition of upstream sequences increased luciferase expression. Maximal activity resided within the first 1,200 bp upstream. A minigene construct was generated by placing a portion of the alpha 1(IV) first intron downstream from the promoter region. The intronic sequences significantly decreased activity of the promoter in MCT cells and 3T3 fibroblasts but greatly enhanced expression in murine parietal yolk sac (PYS) endodermal cells. Addition of transforming growth factor-beta (TGF-beta) to MCT cultures elevated the levels of secreted type IV collagen. Treatment of either transiently or stably transfected MCT cells with TGF-beta produced an increase in the levels of expression of all of the reporters tested. These data support the hypothesis that cell-specific regulation of alpha 1(IV) collagen is dependent upon downstream sequences, which act to decrease the expression of type IV collagen in tubular epithelium. The activity of the alpha 1(IV) collagen gene in proximal tubular cells is increased by TGF-beta, which acts on the domain(s) embedded within the intergenic bidirectional promoter.

Coordinate regulation of renal expression of nitric oxide synthase, renin, and angiotensinogen mRNA by dietary salt.

Experiments were performed to examine the effect of changes in dietary salt intake on the neuronal form of the constitutive nitric oxide synthase (ncNOS, type I NOS), renin, and angiotensinogen mRNA expression in the kidney. Three groups of Sprague-Dawley rats were studied as follows: rats maintained on a 3% Na diet plus 0.45% NaCl in the drinking fluid for 7 days (high salt), rats given a single injection of furosemide (2 mg/kg i.p.) and a 0.03% Na diet for 7 days (low salt), and rats on a diet containing 0.2% Na (control). mRNA expression was assessed with reverse transcription-polymerase chain reaction (RT-PCR) methods using cDNA prepared from samples of renal cortex and microdissected tubular segments. ncNOS PCR products were quantified by comparison with a dilution series of a mutant deletion template. Compared with their respective control, ncNOS mRNA levels in renal cortical tissue were elevated in rats on a low-salt diet and reduced in rats on a high-salt diet. Similar changes were seen in the expression of renin and angiotensinogen mRNA. Dietary salt intake did not alter the mRNA levels for ncNOS from the inner medulla or for endothelial constitutive NOS (ecNOS, type III NOS) and inducible NOS (iNOS, type II NOS) in the renal cortex. ncNOS mRNA was found in glomeruli dissected with the macula densa-containing segment (MDCS), but only at marginal levels in glomeruli without MDCS. Furthermore, a low-salt diet stimulated ncNOS mRNA in glomeruli with MDCS by 6.2-fold compared with a high-salt diet. There was no effect of salt diet on ncNOS mRNA in glomeruli without MDCS or in inner medullary collecting ducts. These results suggest that ncNOS expression in macula densa cells is inversely regulated by salt intake, thus following the known response of the renin-angiotensin system to changes in salt balance.

Cloning of the mouse fibronectin V-region and variation of its splicing pattern in experimental immune complex glomerulonephritis.

Increased mRNA and protein expression of extracellular matrix (ECM) components, including fibronectin, occurs during the development of glomerulonephritis and glomerulosclerosis in immunologically mediated kidney diseases. However, in addition to these quantitative changes in ECM expression, qualitative changes in these molecules may contribute to malformations in the composition of the glomerular matrix. These qualitative changes may include alterations in the splicing pattern of the V-region of fibronectin, since this region plays a role in its accumulation. The splicing patterns of this region have been studied in chronic graft-versus-host disease (GvHD) in mice, a model of lupus nephritis, and in chronic serum sickness (CSS) in rats, a model of immune complex nephritis. Cloning of the mouse fibronectin V-region from kidney tissue revealed 96.1 per cent homology with the corresponding domain in rat fibronectin. PCR (polymerase chain reaction) analysis of RNA from isolated glomeruli revealed three isoforms of this region in both mouse and rat fibronectin, namely inclusion or exclusion of the whole region, or exclusion of only the CS1 domain. In both models, increased exclusion of the V-region was observed early in the disease. However, in GvHD the splicing pattern returned to normal, whereas in CSS the shift persisted during the course of the experiment. Differentiated expression of fibronectin isoforms may exert an important effect on the structure and biological function of the glomerulus and may thus play a role in the development of glomerulonephritis and glomerulosclerosis.

Developmental changes in seminiferous tubule extracellular matrix components of the mouse testis: alpha 3(IV) collagen chain expressed at the initiation of spermatogenesis.

The temporal expression of type IV collagen, laminin, and entactin in the basal laminae of the seminiferous tubule during development of the mouse testis was determined. Northern blot analysis was used to examine changes in testicular mRNA for alpha 1-alpha 5 type IV collagen (IV) chains in mice ranging in age from newborn to adult (60 days). Levels for mRNA alpha 1(IV) and alpha 2(IV) chains were highest in newborns through Day 5 and remained elevated through Day 10, but then sharply declined to adult values by Day 30. In sharp contrast, alpha 3(IV) and alpha 4(IV) chain levels were low in newborns, peaked at Day 10, and then declined to adult values by Day 30. 5(IV) mRNA was elevated in newborns and at Days 5 and 10 before dropping to adult levels by Day 30. Changes in the deposition of alpha 1, alpha 2, and alpha 3(IV) collagen chains, laminin, and entactin into the inner and outer basal laminae of the seminiferous tubule were determined from the beginning of tubule formation (embryonic Day 12.5) through adulthood by immunofluorescence microscopy using polyclonal antibodies for these constituents. The alpha 1 and alpha 2(IV) chains, laminin, and entactin were deposited into the inner basal lamina at embyronic Day 12.5 and into the newly formed outer basal lamina at Day 5 after birth. The alpha 3(IV) chains were deposited into both the inner and outer basal laminae at Day 5. Thus, testicular alpha 1-alpha 3(IV) mRNA levels coincide with the incorporation of detectable collagen chains into the seminiferous basal laminae, suggesting transcriptional control of these alpha (IV) chains. Expression of of the alpha 3(IV) chain coincides with the initiation of spermatogenesis, suggesting a functional role of this chain in spermatogonial proliferation.

Role of the extracellular matrix in the development of glomerulosclerosis in experimental chronic serum sickness.

Glomerulosclerosis is a severe complication of many immunologically mediated kidney diseases and is associated with a poor prognosis with respect to renal function. The aim of this study was to elucidate the role of the extracellular matrix (ECM) in the development of glomerulosclerosis in experimental immune complex glomerulonephritis. Induction of chronic serum sickness by repetitive injections of human IgG into preimmunized Wistar rats leads to the development of immune complex nephritis and glomerulosclerosis. At an early stage of the disease fibrinogen accumulation was observed along the endothelial cells, presumably related to damage of the endothelial lining. mRNA levels for several collagen types, laminin B1 and B2, and fibronectin were increased in both whole-kidney tissue and in isolated glomeruli, but morphological changes were not observed. In situ hybridization experiments demonstrated increased ECM mRNA levels in glomerular and tubular cells. Starting at week 15, glomerular mesangial matrix expansion and thickening of the glomerular basement membrane (GBM) was observed. ECM components were abundantly present. Coagulation factors were not observed at this point. ECM mRNA levels were decreased as compared to week 0, but were still above normal. Focal and segmental end-stage sclerotic lesions developed at weeks 25-30, in which fibronectin and fibrinogen were the major constituents. Other ECM components were found peripherally from these lesions in the remnants of the mesangial matrix and GBM. Sclerotic matrices did not demonstrate an increase of cellular-fibronectin, and other constituents from the circulation were not present in the lesions. Glomerular ECM mRNA was decreased to normal levels. However, a dramatic increase of ECM mRNA expression was observed at sites of inflammatory infiltrate in the perivascular, interstitial, and periglomerular regions. In conclusion, the development of glomerulosclerosis in chronic serum sickness rats is preceded by mesangial matrix expansion in which several ECM components are increasingly expressed. Steady state mRNA levels for these components are increased before morphological changes are detectable. In the final stage there is a specific accumulation of exogenous fibronectin in the glomerular end-stage sclerotic lesions. Simultaneously, an interstitial inflammatory reaction takes place leading to increased ECM production in the tissue surrounding the damaged glomeruli.

Cholesterol emboli presenting as acute allograft dysfunction after renal transplantation.

Cholesterol emboli are a common complication of atherosclerotic vascular disease. A 40-yr-old renal transplant recipient who developed acute allograft dysfunction 1 day after the initiation of cyclosporine therapy and 6 days after transplantation is described. A renal allograft biopsy revealed cholesterol emboli in interlobular arteries and in glomeruli. Four previously reported cases of cholesterol emboli in renal allografts are described, and the cause and pathogenesis of atheroembolic disease are reviewed. Atheroemboli causing injury to the renal allograft may arise from either donor or recipient vessels. Vigilance for the occurrence of these emboli needs to be maintained when donor or recipient vessels demonstrate evidence of significant atherosclerotic vascular disease.

Specific accumulation of exogenous fibronectin in experimental glomerulosclerosis.

The prognosis of patients showing glomerulosclerosis as a complication of an immunologically mediated kidney disease is poor. To improve the diagnosis and treatment of these patients, it is important to understand the processes involved in the development of glomerulosclerosis. In this study, we investigated the molecular composition of experimental end-stage glomerular sclerotic lesions and their pathogenesis in chronic graft-versus-host disease (GvHD) in the mouse and chronic serum sickness in the rat. Accumulation studies were performed to determine the degree of specific trapping of constituents from the circulation. Two different models were investigated to determine whether differences in disease initiation resulted in different compositions of the glomerulosclerotic lesions. In both models, glomerulosclerosis was preceded by expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM). The end-stage sclerotic lesions consisted mainly of fibronectin, which appeared to displace the other extracellular matrix (ECM) components peripherally in the mesangial matrix and GBM. The abundance of fibronectin in the lesions was not reflected in the mRNA levels for this component. Indeed, antibodies directed against the cellular form of fibronectin did not stain positive in the end-stage lesions. These findings, together with accumulation studies, suggest that specific accumulation rather than de novo synthesis of fibronectin plays a major role in the development of experimental glomerulosclerosis, which appears to be independent of the pathway of induction.

Kidney and retinal defects (Krd), a transgene-induced mutation with a deletion of mouse chromosome 19 that includes the Pax2 locus.

The semidominant mutation Krd (kidney and retinal defects) was identified in transgenic line Tg8052. Krd/+ mice have a high incidence of kidney defects including aplastic, hypoplastic, and cystic kidneys. Retinal defects in Krd/+ mice include abnormal electroretinograms and a reduction of cell numbers that is most extreme in the inner cell and ganglion layers. Viability of Krd/+ mice is strongly influenced by genetic background, and growth retardation is observed in young animals. Homozygosity results in early embryonic lethality. Fluorescence in situ hybridization of a transgene-specific probe localized the insertion site to the distal region of mouse Chromosome 19. The sequence of the insertion site revealed transgene insertion into a LINE element with deletion of a single nucleotide from the 3' terminus of the transgene. A polymorphic microsatellite, D19Umi1, was identified in a junction clone and mapped in several large crosses. D19Umi1 is located 1.7 +/- 1.0 cM distal to Pax2, which encodes a paired type transcription factor expressed in embryonic kidney and eye. Deletion of Pax2 from the transgenic chromosome was demonstrated by Southern analysis of genomic DNA from (Krd/+ x SPRET/Ei)F1 mice. Additional genetic and molecular data are consistent with an approximately 7-cM deletion that includes the loci stearoyl CoA desaturase (Scd1), pale ear (ep), D19Mit17, D19Mit24, D19Mit27, D19Mit11, and Pax2. This deletion, Del(19)TgN8052Mm, will be useful for genetic and functional studies of this region of mouse Chromosome 19.

Lipopolysaccharide induces monocyte chemoattractant protein production by rat mesangial cells.

Lipopolysaccharide, a potent pro-inflammatory constituent of bacterial cell walls, is capable of promoting glomerular inflammation, by both activating circulating inflammatory cells and local interactions with renal parenchymal cells. We sought to determine whether lipopolysaccharide was capable of promoting glomerular inflammation by directly stimulating mesangial cell production of monocyte chemoattractant protein 1, a recently described cytokine capable of eliciting recruitment of mononuclear phagocytes into inflammatory foci. Northern hybridization analysis revealed dose and time-dependent induction of mRNA coding for monocyte chemoattractant protein 1 in quiescent rat mesangial cells treated with lipopolysaccharide. Lipopolysaccharide-elicited induction of monocyte chemoattractant protein mRNA was detectable after 1 hour and persisted for at least 30 hours. Media isolated from rat mesangial cell cultures stimulated by lipopolysaccharide possessed monocyte chemotactic activity that was detectable at 8 hours and peaked at 24 hours; an antimonocyte chemoattractant protein antibody blocked 87% of this chemotactic activity. We suggest that lipopolysaccharide, released from bacterial cell walls, promotes glomerular inflammation by stimulating mesangial cell production of monocyte chemoattractant protein 1.