Production of polyclonal antibody against kidney antigens: a model for studying autoantibody in feline chronic kidney diseases

Chronic kidney disease is considered to be most common in geriatric domestic cats. It has been reported that the feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccine prepared from the Crandell-Rees feline kidney (CRFK) cell line can induce cross-reactions of antibodies with feline kidney tissues. As an anti-cat kidney antibody was not available commercially for this study of autoantibody in cats, the purpose of this study was to produce anti-cat kidney antibody in rabbits for further study of autoantibody in cats after FVRCP vaccination. Kidney proteins from cadaveric cats were extracted and immunized into rabbits using Montanide as the adjuvant. Based on enzyme-linked immunosorbent assay measurement, all immunized rabbits produced high levels of anti-cat kidney antibodies and some began to produce antibodies as early as 2 weeks after immunization. Immunofluorescence staining of rabbit sera showed kidney-bound antibodies in glomerulus, Bowman's capsule, apical surface of the proximal convoluted tubule, peritubular surface, and interstitial cells. Western blot analysis of cat kidney proteins revealed molecular weights (M.W.) of 72, 55, 47, and 31 kDa, while binding to the CRFK cell proteins was observed at M.W. of 43 and 26 kDa. The antibody that recognized the 47 kDa protein was similarly detected in cats with autoantibody presence after FVRCP vaccination. The kidney-bound antibody profile at different time points and its patterns in rabbits could be used as a model for the study of autoantibody to cat kidney in feline chronic kidney diseases.

Aberrant Blood Vessel Formation Connecting the Glomerular Capillary Tuft and the Interstitium Is a Characteristic Feature of Focal Segmental Glomerulosclerosis-like IgA Nephropathy

BACKGROUND: Segmental glomerulosclerosis without significant mesangial or endocapillary proliferation is rarely seen in IgA nephropathy (IgAN), which simulates idiopathic focal segmental glomerulosclerosis (FSGS). We recently recognized aberrant blood vessels running through the adhesion sites of sclerosed tufts and Bowman's capsule in IgAN cases with mild glomerular histologic change. METHODS: To characterize aberrant blood vessels in relation to segmental sclerosis, we retrospectively reviewed the clinical and histologic features of 51 cases of FSGS-like IgAN and compared them with 51 age and gender-matched idiopathic FSGS cases. RESULTS: In FSGS-like IgAN, aberrant blood vessel formation was observed in 15.7% of cases, 1.0% of the total glomeruli, and 7.3% of the segmentally sclerosed glomeruli, significantly more frequently than in the idiopathic FSGS cases (p = .009). Aberrant blood vessels occasionally accompanied mild cellular proliferation surrounding penetrating neovessels. Clinically, all FSGS-like IgAN cases had hematuria; however, nephrotic range proteinuria was significantly less frequent than idiopathic FSGS. CONCLUSIONS: Aberrant blood vessels in IgAN are related to glomerular capillary injury and may indicate abnormal repair processes in IgAN.

Fenugreek seeds reduce aluminum toxicity associated with renal failure in rats

Despite the reports on safety concerns regarding the relationship between aluminum salts and neurological and bone disease, many countries continue to use aluminum as phosphate binders among patients with renal failure. In search for a diet supplement that could reduce aluminum toxicity related to renal failure, we carried out this prospective animal study in which the fenugreek seeds were assessed for their effects on rats nephrotoxicity induced by aluminum chloride (AlCl3). Oral AlCl3 administration during 5 months (500 mg/kg bw i.g for one month then 1600 ppm via drinking water) led to plasma biochemical changes, an inhibition of alkaline phosphatase (ALP), a decrease of total antioxidant status (TAS), and an induction of lipid peroxidation (LPO) in the blood and brain, in addition to kidney atrophy and morphological alterations at the level of Bowman's capsule, the glomerulus and different sorts of tubules, reminiscent of some known kidney disease. The treatment with the whole fenugreek seed powder (FSP) (5% in the diet) during the last 2 months showed its effectiveness in restoring normal plasma values of urea, creatinine, ALP and glucose, as well as re-increasing the TAS, inhibiting LPO and alleviating histopathological changes in the injured kidneys. This study highlights the induced nephrotoxicicity, as well as the related toxicity in the brain and bone, by chronic oral ingestion of the aluminum salts. However, the maintenance of a diet supplemented with fenugreek seeds could offer protection for the kidney, bone and brain, at the same time.

The Role of PEC Progenitors in ADPKD Progression

BACKGROUND AND OBJECTIVES: Autosomal dominant polycystic kidney disease is a pathology mainly characterized by the progressive development and enlargement of cysts in each kidneys. Such as many adult epithelial tissue, renal tubule replaces damaged or death cells through the presence of stem/progenitor cells CD133+CD24+ Obviously, in ADPKD the repair of damages is insufficient to block the disease, but renal stem cells could have a role in the pathology. In this study we investigate the localization and the involvement of cells CD133+CD24+ in ADPKD progression. METHODS AND RESULTS: Two normal kidneys and two ADPKD kidneys were examined. CD133 and CD24 expression was investigated by confocal microscopy and immunoblotting. Renal tissue and cells were analyzed. CD133 and CD24 have the same localization in ADPKD tissues and in normal kidneys: a subset of epithelial cells (PEC) of Bowman's capsule and luminal side of tubules. It is interesting that CD133+CD24+ cells are statistically more represented in ADPKD tubules (p<0.001) and in healthy glomeruli (p=0.0016). Cysts express CD133 and CD24. CONCLUSIONS: Renal epithelial progenitors demonstrate to be involved in ADPKD pathogenesis but their role will have to be clarified and possibly managed to obtain improvement, or at least stabilization, of disease.

Podocyte Expression of Osteopontin and FSP-1/S100A4 in Human Crescentic Glomerulonephritis

BACKGROUND: Osteopontin (OPN) is a cytokine associated with a cell-matrix via integrins. Fibroblast specific protein-1 (FSP-1), known as S100A4, has been implicated in cell migration by non-muscle myosin. We investigated whether the role of OPN and FSP-1/S100A4 expression in their contribution to the podocyte phenotype change to form podocyte bridge and cellular crescent. METHODS: Glomerular expression of OPN and FSP-1/S100A4 in renal biopsies of 16 patients with crescentic glomerulonephritis (CrGN) and 13 normal renal biopsies were studied by immunohistochemistry. RESULTS: The expression of OPN and FSP-1/S100A4 was increased in the podocytes of glomeruli, with and without crescents, in patients with CrGN. Neither OPN nor FSP-1/S100A4 was expressed in glomeruli from the normal controls (p<0.01). A significant positive correlation was found between the expression of OPN in glomerular tufts and cellular crescents, and the expression of OPN and FSP-1/S100A4 in glomerular tufts (p<0.05). CONCLUSIONS: The results suggest that OPN plays a role in early podocyte attachment to Bowman's capsule, and FSP-1/S100A4 potentiate podocyte contribution to cellular crescent formation by inducing cellular migration and growth.

Efeito do herbicida 2, 4-D (Ácido 2, 4-Diclorofenoxiacético) na morfologia e função renal de Ratos Wistar / Efect of the herbicide 2, 4-D (2, 4-Dichlorophenoxyaceticacid) on the renal morphology and function of Wistar Rats

O herbicida 2,4-D (ácido 2,4-diclorofenoxiacético) tem elevado potencial tóxico podendo causar efeitos indesejáveis a curto e longo prazo por seu emprego prolongado em diversas culturas de frutas e cereais. O objetivo deste estudo foi observar o efeito do 2,4-D na função e estrutura renal de ratos Wistar através da avaliação da taxa de filtração glomerular (TFG), proteinúria e análise histológica. Foram utilizados 34 ratos Wistar (220-260 g) mantidos em gaiolas individuais, que foram separados em 2 lotes. No primeiro lote: G-I(n=5), G-II(n=4) e G-III(n=5) os animais receberam, respectivamente, por via oral, água, 2,5 e 5,0 mg/kg/dia de 2,4-D diluído em água durante 15 dias. No segundo lote: G-I(n=8), G-II(n=8) receberam, respectivamente, via oral, 2,5 e 5,0 mg/kg/dia de 2,4-D diluído em água e G-III(n=4) que recebeu água por 42 dias. Após o período de tratamento, fez-se coleta de sangue por punção cardíaca, urina de 24 horas e dos rins. Para análise histológica os rins foram fixados em “Bouin”, cortados em 5mm e corados pelo Tricrômico de Masson. Não foi observada alteração significativa (p<0,05) na TFG (G-I=7,3±0,8; G-II=7,0±0,9; G-III=7,6±0,6 mg/min/kg). Houve presença significativa de proteína na urina (p<0,05) nos animais que receberam o 2,4-D por 15 dias (G-I=3,7±07; G-II=21,3±2,9; G-III=22,9±7,9 mg/24 h). Entretanto, a análise histológica dos animais que receberam 2,4-D por 42 dias não mostrou presença de esclerose glomerular e fibrose tubulointersticial. Portanto, a proteinúria provocada pelo 2,4-D no período estudado não foi suficiente para provocar alterações na região tubulointersticial e nem na região glomerular dos rins destes animais

Histopathologic Studies on Crescentic Glomerulonephritis / 대한신장학회잡지

BACKGROUND: Crescentic glomerulonephritis is expressed pathologically by crescent formation in Bowman's capsule and clinically by rapidly progressive loss of renal function. The pathologic experience of crescentic glomerulonephritis in one institution was analyzed here. METHODS: We classified 25 cases of crescentic glomerulonephritis patients into 4 categories and reviewed the cases pathologically and clinically. RESULTS: We found no case with group I (anti- GBM disease), 8 cases in group II (immune complex glomerulonephritis) including 3 patients with IgA nephropathy, 2 patients with Henoch-Sch nlein purpura and 3 patients with APSGN, 12 cases in group III (ANCA-associated glomerulonephritis) including 7 patients with microscopic polyangitis, 4 patients with Wegener's granulmatosis and 1 patient with ANCA GN, and 5 cases in group IV (idiopathic crescentic glomerulonephritis). The mean ages of patients with group II, III, and IV were 32.0, 59.3 and 39.0 years old, respectively, and mean serum creatinine levels at the time of biopsy were measured as 9.1, 5.2, 8.8 mg/dL in each group. On light microscopic findings, the frequency of crescents in glomeruli was 64.4% in group II, 43.7% in group III, and 51.2% in group IV. The score of infiltration into tubules of inflammatory cells was 0.8 in group II, 0.4 in group III, and 0.6 in group IV and the score of fibrosis in interstitium was 1.0 in group II, 0.8 in group III and 1.2 in group IV. The score of atherosclerosis in arteries was 1.4, 0.9 and 1.6 in each group. CONCLUSION: We conclude that the precise diagnosis and classification of crescentic glomerulonephritis by an early renal biopsy and clinical assessments are important in the management of rapidly progressive (crescentic) glomerulonephritis. Since the number of the cases was not so enough, we could not analyze the statistical significance between morphologic differences of each group of crescentic glomerulonephritis, but if more cases were collected, acknowledgements of differences and prognostic factors in pathologic findings could be possible.

Effect of lead toxicity on ultrastructure of bowman's capsule of adult rats

This study was designed to evaluate the lead induced toxic effects on the ultrastructure of adult rat Bowman's capsule, and comprised 18 rats. The animals were divided into two equal groups, [n=9]: control group given pure distilled water and study group given a 0.13% lead acetate solution in drinking water. After 8 weeks exposure, rats were sacrificed and their kidneys were removed and a small piece of renal cortex was fixed and sections were prepared for electron microscopic examination. The parietal cells of Bowman's capsule of the experimenatal group showed signs of damage to nucleus and organelles, with abundance of cytoplasmic vacuoles and phagolysosomes. We can conclude that exposure to lead can induce damage to the parietal cells of Bowman's capsule in rats

Expression of Transforming Growth Factor-beta1 in Cyclosporine-Induced Nephropathy in Rats

Cyclosporine nephropathy was induced by intraperitoneal injection of cyclosporine 25 mg/kg in Sprague-Dawley rats daily for 1, 4, 8, and 12 weeks to clarify the relationship between cyclosporine nephropathy and the expression of TGF-beta1 with extracellular matrix deposition. On light microscopic examination, the kidneys in the 12 week cyclosporine-treated rats showed focal or striped fibrosis, vacuolization of tubular cells, and injury of endothelial cells. Immunohistochemically, TGF-beta1 protein was strongly expressed in the cyclosporine-treated rat kidneys, especially in the glomerular endothelial cells, interstitial endothelial cells, tubular epithelial cells, and parietal cells in the Bowman's capsule of the glomerulus as well as the periglomerular arterioles. The amount of TGF-beta1 expression was correlated with the morphological change in the cyclosporine-treated rats. Extracellular matrix, such as fibronectin and collagen IV, was also expressed in the endothelial cells of the glomerulus and the interstitium. It can be concluded, therefore that TGF-beta1 protein is probably involved in the early stage of fibrogenesis in cyclosporine nephropathy. It can be postulated that cyclosporine nephropathy results from the accumulation of extracellular matrix associated with the increase of TGF-beta1 transcription. Therefore, these results could be used in reducing fibrosis in cyclosporine nephropathy.

Sequential Studies of Glomerular Crescent Formation in Rabbits with Anti-Glomerular Basement Membrane(GBM) Antibody Induced Glomerulonephritis(GN)

To investigate the mechanism of crescent formation, sequential pathologic changes from the New Zealand White rabbits with anti-GBM antibody induced GN by administration of guinea pig anti-GBM IgG were studied by light (LM), immunofluorescent (IF) and electron (EM) microscopy. Although no glomerular changes were observed in LM, swelling of the endothelial cells and the epithelial cells were noted in EM by day 2. By day 7, early and cellular crescents were evident. Proteinaceous materials and fibrins were noted in the glomerular capillary lumina (GCL) and Bowman's space (BS) associated with segmental hypercellularity. The GBM damage became progressively severe, followed by focal detachment of the visceral epithelial cells from the GBM. At day 14, fibrin strands, mononuclear cells and collagen fibrils were present between the proliferating extracapillary cells. At day 31, fibrocellular crescents were predominated. Elongated spindle cells, morphologically resembling myofibroblasts, were noted near the Bowman's capsule (BC). A degree of tubular atrophy, interstitial fibrosis, and inflammatory infiltrates increased as it did with fibrous organization of crescent. Intense linear IF staining for IgG and C3 were seen throughout the experiments along the GBM. In conclusion, the progression of crescent from an early "proteinaceous" stage through cellular, fibrocellular and fibrous stages was well documented in this study. Inflammatory cells and coagulation mechanism may activate the initiation of the GBM damage at the early stage. Activated periglomerular mononuclear cells may also cause disruption of BC which facilitates entry of activated periglomerular cells and fibroblasts into BS leading to progressive fibrous crescent formation.