Clinicopathological study of vesicoureteral reflux (VUR)-associated pyelonephritis in renal transplantation.

We retrospectively studied the occurrence of vesicoureteral reflux (VUR)-associated pyelonephritis using renal biopsies obtained from the transplanted kidneys, and correlated the histological changes with clinical parameters. Out of a total of 131 renal biopsies performed between 1990 and 2001 on renal transplant patients at the department of Urology of Nagasaki University Graduate School of Biomedical Sciences, 12 patients showed pyuria more than twice in a single year. Seven of these 12 patients were available for determining VUR by voiding cystourethrography (VCUG). Cystoureterography demonstrated VUR in three of seven studied patients with pyuria. A histopathological examination revealed dilatation of both proximal and distal tubules in renal biopsies of transplant patients with VUR, compared to renal biopsies of transplant patients without VUR, or non-transplanted patients with thin membrane disease. One of the patients with VUR showed advanced features of chronic pyelonephritis in four consecutive biopsies at different time points, suggesting a late stage of reflux nephropathy in the transplanted kidney. We conclude from our study that the occurrence of VUR-related pyelonephritis may be one of the important long-term complications in the survival of renal allografts.

Tissue and molecular events in human conjunctival scarring in ocular cicatricial pemphigoid.

Detailed histomorphometric analysis of human conjunctival biopsy specimens has convincingly demonstrated that tissue remodeling of the extracellular matrix (ECM) is an essential and dynamic process associated with conjunctival scarring in ocular cicatricial pemphigoid (OCP). The conjunctival scarring often eventually results in impaired vision and/or blindness. The molecular mechanisms of conjunctival scarring are not completely understood. Accumulating evidence indicates that the early phase of conjunctival fibrosis is linked with an immuno-inflammatory process mediated by cytokines released by activated conjunctival cells and/or by infiltrating cells. Fibrogenic cytokines secreted by inflammatory cells and fibroblasts might actively be involved in remodeling of the matrix within the conjunctival stroma, possibly by regulating the altered metabolism of matrix proteins.

Role of glomerular epithelial cell-derived heat shock protein 47 in experimental lipid nephropathy.

BACKGROUND: Heat shock protein 47 (hsp47) is a collagen-specific stress protein and is shown to be involved in the synthesis/assembly of various collagens as a molecular chaperone. This study was undertaken to investigate the possible role of hsp47 in dietary-induced hypercholesterolemic rat kidneys, which showed glomerular hypercellularity with expansion of mesangial matrix. METHODS: Dietary-induced hypercholesterolemia was induced in male Wistar rats by giving 2% cholesterol diet for four months. Immunohistochemistry was used for localization of protein products for collagens (types I, III, and IV). alpha-smooth muscle actin, vimentin, desmin, and ED-1, a macrophage/monocyte marker, and hsp47 in control and hypercholesterolemic rat kidneys. RESULTS: Compared with the control, increased accumulation of collagens was accompanied with increased expression of hsp47 in hypercholesterolemic rat kidneys, with predominant expression in the glomeruli. By double immunostaining, desmin-positive glomerular epithelial cells were found to be the main source of hsp47 in hypercholesterolemic rat kidneys. CONCLUSION: From these results, it is concluded that induced expression of hsp47 by phenotypically altered glomerular epithelial cells might play a role in the excessive assembly/synthesis of collagens and could thereby contribute to the glomerulosclerosis found in dietary-induced hypercholesterolemic rat kidneys.

Cisplatin-induced apoptosis in human proximal tubular epithelial cells is associated with the activation of the Fas/Fas ligand system.

The role of Fas and Fas ligand (Fas-L) in the apoptotic cell death process in cisplatin (CP)-treated human proximal tubular epithelial cells (PTECs) was examined. The human PTECs were treated with various concentrations (20-80 microM) of CP for 24 h, and the incidence of apoptosis in CP-treated cells was assessed by trypan blue staining, propidium iodide staining, in situ end labeling, and electron microscopy. The expression of Fas and Fas-L was detected by immunofluorescence microscopy. The results showed that: (1) CP-treatment resulted in a decreased number of live human PTECs and an increased number of dead cells, (2) CP-treated human PTECs showed an increased rate of apoptosis with its typical morphological features, and (3) expression of both Fas and Fas-L was upregulated in CP-treated human PTECs. These results indicate that CP treatment induces apoptosis in human PTECs and the activation of the Fas/Fas-L system may play an active role in the induction of the apoptotic cell death process.

Life-long caloric restriction suppresses age-associated Fas expression in the Fischer 344 rat kidney.

It has been shown that the expression of Fas is substantially increased in the aging process in various organs, but its role in the aging kidney is not yet clear. In this study, the expression of Fas in the kidneys of 6- and 24-month-old male Fischer 344 rats fed ad libitum was studied by using quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. In addition, possible effects of life-long caloric restriction (30% as those of ad libitum fed group) in the expression of Fas were also studied in 6- and 24-month-old rat kidneys. Kidneys obtained from 24-month-old ad libitum fed rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while in the kidneys of 24-month-old calorie-restricted rats, renal damage was remarkedly less than that noted in 24-month-old ad libitum fed rats kidneys. RT-PCR and immunohistochemical analysis showed an increased expression of Fas in both mRNA and protein level in 24-month-old rat kidneys; life-long caloric restriction significantly reduces renal expression of Fas. Our results suggest that increased expression of Fas is associated with age-related renal damage and that life-long diet-restricted alteration of its expression is associated with the modulation of age-associated renal structural damage.

Coadministration of basic fibroblast growth factor and sucrose octasulfate (sucralfate) facilitates the rat dorsal flap survival and viability.

The effective use of local growth factors and cytokines may replace the lengthy staged surgical delay process. We tested the efficacy of basic fibroblast growth factor (bFGF) coadministered with sucralfate (sucrose octasulfate) on the rat dorsal flap model. A total of 76 male Wistar rats were used in this experiment. Four groups of the animals were divided. Group 1 (n = 5) was the vehicle control (saline soaked), group 2 (n = 5) was sucrose octasulfate soaked (100 microg/ml, 1 ml), group 3 (n = 5) was bFGF soaked (1 microg/ml, 1 ml), and group 4 (n = 5) was both bFGF and sucrose octasulfate soaked. All agents were soaked equally in Gelfoam. The flap survival measured by the quantitative computer-assisted morphologic analysis was significantly improved by day 5 postoperatively in the combined administration group compared with the vehicle control (81 and 53 percent, respectively; p < 0.05). In lead oxide-gelatin microangiography, there was enhanced pedicle vessel formation observed as well as the extended vessel sprouting up to very close to the distal end in combined group on day 5. The endogenous bFGF mRNA expressions shown by reverse transcriptase-polymerase chain reaction were detected in all four groups. The angiogenesis indicated by alpha-smooth muscle actin immunopositivity was significantly more enhanced in the combined group than the vehicle control (37.3 and 19.4, respectively; p < 0.01). In the combined group, there was stronger immunopositivity for bFGF in epidermis and hair follicles observed, and more notably bFGF-immunopositive dermal fibroblasts were evident. Thus, coadministration of bFGF and sucralfate markedly facilitates the rat dorsal flap survivability by enhancing the bFGF expression and angiogenesis.

Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections.

Extracellular matrix accumulation is crucial in the pathogenesis of glomerulosclerosis in mesangial proliferative glomerulonephritis (GN). In an attempt to explore the distribution of type VI collagen and its synthesizing cells in normal and diseased glomeruli, we investigated mRNA and protein expression of type VI collagen in renal biopsy sections, histologically diagnosed as mesangial proliferative GN. Five renal biopsies from patients diagnosed as having minor glomerular abnormalities and one surgical renal tissue were also simultaneously examined as controls. Immunohistochemical studies revealed type VI collagen immunostaining in the mesangium and glomerular basement membrane of the control glomeruli. Compared to the control, increased deposition of type VI collagen was noted in the mesangial proliferative and sclerotic lesions in GN. To identify the cells responsible for the synthesis of type VI collagen mRNA, renal sections were hybridized in situ with digoxigenin-labeled antisense oligo-DNA probe complementary to a part of alpha1 (VI) mRNA. Occasionally intraglomerular cells hybridized with digoxigenin-labeled antisense pro alpha1 (VI) oligo-DNA in control glomeruli. An increased number of intraglomerular cells (mostly epithelial cells) were, however, positive for alpha1 (VI) mRNA expression in GN sections. The present study documents the distribution of type VI collagen in the normal glomeruli and provides further evidence of accelerated synthesis of this collagen in mesangial proliferative GN.

Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy.

The mechanism of structural changes of the kidney in human diabetic nephropathy (DN) and IgA nephropathy (IgAN) is not yet completely known, but excessive deposition of extracellular matrix (ECM), including various collagens, may be crucial to this process. Heat shock protein (HSP) 47 has been identified as collagen-binding stress protein, shown to have a specific role in the intracellular processing of procollagen molecules during collagen assembly. To determine whether increased deposition of collagens in human DN and IgAN is related to HSP47, we investigated the expression of HSP47 in renal biopsy and autopsy sections obtained from 22 DN and 45 IgAN patients. Five renal biopsy specimens, diagnosed as minor glomerular abnormalities, were simultaneously studied as controls. Monoclonal antibodies specific for HSP47, type III collagen and type IV collagen were used to assess the relative expression of their proteins in paraffin-embedded renal sections by immunohistochemistry. Increased deposition of collagens was closely related to the sclerotic activity of the disease process in DN and IgAN; increased deposition of collagens was often present in relation to a strong expression of HSP47, a stress protein known to regulate collagen synthesis/assembly. By double immunostaining, we found colocalization of collagens and their molecular chaperone HSP47 in the sclerotic glomeruli and tubulointerstitium in DN and IgAN. Our results strongly support a pathologic role for HSP47 in both these diseases and that increased levels of HSP47 may play an important role in the excessive assembly of collagens resulting in glomerulosclerosis and interstitial fibrosis found in DN and IgAN patients.

Expression of vascular endothelial growth factor and its receptors in rats with protein-overload nephrosis.

BACKGROUND: Based on the fact that vascular endothelial growth factor (VEGF) increases vascular permeability, it is speculated that VEGF might be involved in the development of proteinuria, although this remains unconfirmed. The production and site of action of VEGF remains unclear in nephrotic renal diseases. METHODS: Non-radioactive in situ hybridization was performed to examine the expression of VEGF mRNA and its receptors, flt-1 and KDR/flk-1, in a rat model of nephrosis induced by intraperitoneal injection of bovine serum albumin (BSA). Saline injected rats were served as control animals. RESULTS: Neither morphological changes nor deposition of immunoglobulin or complement were observed in our model. Proteinuria developed, reaching a maximum level in rats injected with BSA for 3 days, followed by persistent proteinuria until day 14. The expression of mRNA for VEGF and the two receptors was markedly upregulated in glomeruli of BSA-induced nephritis compared with the control group. VEGF mRNA was localized in glomerular cells, including cells in mesangium, visceral and parietal epithelial cells. In contrast, flt-1 mRNA and KDR/flk-1 mRNA were expressed on glomerular endothelial cells and cells in mesangium. The ratio of glomerular cells positive for VEGF mRNA and its receptors mRNA increased proportionately with the severity of proteinuria. Immunohistochemistry for ED-1 and proliferating cell nuclear antigen showed no significant increase in infiltrating macrophage or cellular proliferation. CONCLUSIONS: Our results suggest that altered glomerular expression of VEGF and its receptors is not associated with proliferation of endothelial cells, but rather with proteinuria in BSA-induced nephritis in rats. VEGF may play a different role in different renal diseases.

[A case of systemic lupus erythematosus associated with minimal change nephrotic syndrome].

A case of systemic lupus erythematosus (SLE) associated with minimal change nephrotic syndrome (MCNS) in a 25-year-old female is described. The patient suddenly manifested butterfly rash and proteinuria was first pointed out on March, 1994. On admission, her skin biopsy indicated SLE. Subsequently, she developed nephrotic syndrome. Urinalysis showed heavy proteinuria (4.1 g/day), with no other abnormalities in the urinary sediment. Immunological examination revealed positive antinuclear antibody at a titer of 1:80 with a speckled pattern. Anti-ssDNA and anti-SS-A antibodies were positive, but other antibodies were negative. Serum complement (CH50) was within the normal range (30.5 U/ml). The renal biopsy showed no apparent cellular proliferation or increase of extracellular matrices in glomeruli by light microscopy. Slight deposition of IgG, IgM, C3 and C1q was focally seen in the mesangium and capillary wall by immunofluorescence. Electron microscopic examination revealed small and scattered dense deposits in the mesangium, subepithelium and subendothelium, associated with diffuse fusion of the foot processes of epithelial cells along the glomerular basement membrane. According to the WHO classification, the histological features were compatible with those of lupus nephritis (LN), class Ib. The patient was treated with PREDNISOLONE, Mizorbine and Dilazep, resulting in the disappearance of proteinuria and a normal serum level of total protein. The association of LN and MCNS is very rare. We also investigated the relationship between the intensity of proteinuria and histological types of 53 cases with LN examined in our laboratory. The cases with heavy proteinuria were mostly classified as WHO-Class IV and Class V. We report here a case of LN associated with MCNS and also review the literatures.

Identification of type VI collagen synthesizing cells in human diabetic glomerulosclerosis using renal biopsy sections.

Although the role of extracellular matrices in the development of glomerulosclerosis has been discussed widely, the cellular origin of type VI collagen in diabetic nephropathy (DN) has remained relatively unexplored. This study reports the distribution and cellular origin of type VI collagen in DN. Type VI collagen-specific oligonucleotide probes and monoclonal antibody were used to assess the relative expression of mRNA for alpha 1 (VI) chain and its translated protein in paraffin-embedded renal biopsy sections of DN. By immunohistochemistry, compared to the control, increased deposition of type VI collagen was noted in the diffuse and nodular lesions of diabetic glomeruli. For cellular localization of type VI collagen mRNA, paraffin-embedded renal sections of the control and DN were hybridized in situ with digoxigenin (Dig)-labeled antisense oligo-DNA probe complementary to a part of alpha 1 (VI) mRNA. In comparison to the control kidney sections, increased numbers of intraglomerular cells (both mesangial and epithelial cells) were positive for alpha 1 (VI) mRNA in renal biopsy sections of DN. From the results, we conclude that overexpression of type VI collagen by intraglomerular cells with its increased deposition might significantly contribute to the glomerulosclerosis found in DN.

Significance of increased accumulation of type VI collagen and transforming growth factor beta 1 in tubulointerstitial damage in hypertensive nephrosclerosis: an immunohistochemical study.

The distribution of type VI collagen and transforming growth factor beta 1 (TGF beta 1) was studied by immunohistochemistry in 12 renal biopsy specimens of hypertensive nephrosclerosis and five control cases. In control kidneys, the immunostaining of type VI collagen was found in the mesangium, glomerular basement membrane and tubular basement membrane. For TGF beta 1, mesangium, glomerular basement membrane, tubular basement membrane and tubular epithelial cells stained positively in the control kidneys. In contrast to the control cases, markedly increased immunostaining for both type VI collagen and TGF beta 1 was consistently observed in tubulointerstitial damage in hypertensive nephrosclerosis. These immunohistochemical findings provide the evidence for a parallel increase of both type VI collagen and TGF beta 1 during the process of tubulointerstitial injury in hypertensive nephrosclerosis. From the results of the present study, it is speculated that TGF beta 1 may contribute to the tubulointerstitial injury by stimulating increased synthesis of various extracellular matrix including type VI collagen.

Expression of type III collagen mRNA in renal biopsy specimens of patients with idiopathic membranous glomerulonephritis.

Aim-To investigate the distribution of type III collagen in membranous glomerulonephritis (MGN); to identify the cells responsible for the synthesis of alpha1 (III) mRNA.method-The distribution of type III collagen was studied by immunohistochemistry in 10 renal biopsy specimens, histologically diagnosed as MGN, and five control renal tissue samples obtained at surgery. Synthesis of alpha1 (III) mRNA was detected by non-radioactive in situ hybridisation.Results-On immunohistochemistry, type III collagen was not observed in the control glomeruli, but was present focally in the glomeruli in samples from patients with MGN. No specific hybridisation signal for alpha1 (III) mRNA was found in the control glomeruli on non-radioactive in situ hybridisation. By contrast, positive signals for alpha1 (III) chain mRNA were detected in glomerular epithelial cells and mesangial cells in MGN tissue samples.Conclusion-These data suggest that additional synthesis of type III collagen by intraglomerular cells contributes to the changes in the glomerular basement membrane characteristic of MGN.

Immunohistochemical analysis of type III and IV collagens in tubulointerstitial damage in human benign nephrosclerosis.

Prolonged hypertension causes structural changes including glomerulosclerosis and tubulointerstitial damage of the kidney, termed benign nephrosclerosis. It is generally accepted that, in benign nephrosclerosis, increased accumulation of extracellular matrix in the glomeruli results in glomerulosclerosis. Little is known, however, about the possible role of the extracellular matrix in the tubulointerstitial damage in benign nephrosclerosis. In this study, the possible roles of type IV basement-membrane collagen and type III interstitial collagen in tubulointerstitial damage caused by hypertension were explored. Immunohistochemical techniques were used to investigate the distribution of type III and type IV collagens in the kidney sections of 15 patients with benign nephrosclerosis with tubulointerstitial damage and in 10 controls. In the control renal sections strong immunostaining for type III collagen was found in the interstitium and immunostaining for type IV collagen was present in the tubular basement membrane and weakly in the interstitium. In the patients with tubulointerstitial damage there was increased immunostaining for both type III and type IV collagens in the expanded interstitium and damaged tubules than was found in the control kidney sections. These findings indicate that increased accumulation of both type III and type IV collagens might play a significant role in the tubulointerstitial damage in benign nephrosclerosis.