Hideto Sakai

Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy.

Advanced glycation end products (AGEs) include a variety of protein adducts whose accumulation alters the structure and function of tissue proteins and stimulates cellular responses. They have been implicated in tissue damage associated with diabetic complications. To assess the possible link between AGE accumulation and the development of diabetic nephropathy (DN), we have examined the immunohistochemical localization of various AGE structures postulated to date, i.e., pentosidine, Nepsilon-(carboxymethyl)lysine (CML), and pyrraline, in diabetic and control kidneys. CML and pentosidine accumulate in the expanded mesangial matrix and thickened glomerular capillary walls of early DN and in nodular lesions and arterial walls of advanced DN, but were absent in control kidneys. By contrast, pyrraline was not found within diabetic glomeruli but was detected in the interstitial connective tissue of both normal and diabetic kidneys. Although the distribution of pyrraline was topographically identical to type III collagen, distribution of pentosidine and CML was not specific for collagen type, suggesting that difference in matrix protein composition per se could not explain heterogeneous AGE localization. Since oxidation is linked closely to the formation of pentosidine and CML, we also immunostained malondialdehyde (MDA), a lipid peroxidation product whose formation is accelerated by oxidative stress, assuming that local oxidative stress may serve as a mechanism of pentosidine and CML accumulation. Consistent with our assumption, diabetic nodular lesions were stained positive for MDA. These findings show that AGE localization in DN varies according to AGE structure, and suggest that the colocalization of markers of glycoxidation (pentosidine and CML) with a marker of lipid peroxidation reflects a local oxidative stress in association with the pathogenesis of diabetic glomerular lesions. Thus, glycoxidation markers may serve as useful biomarkers of oxidative damage in DN.

Generation of protein carbonyls by glycoxidation and lipoxidation reactions with autoxidation products of ascorbic acid and polyunsaturated fatty acids.

Accumulation of carbonyl derivatives of proteins (protein carbonyl) is taken as a biomarker of oxidative protein damage in aging and in various diseases. We detected protein carbonyls in situ in human diabetic arteriosclerotic tissues and characterized the formation of protein carbonyls. Protein carbonyls were identified in the thickened intima of arterial walls and co‐localized with protein adducts formed by carbonyl amine chemistry between protein and carbonyl compounds derived from autoxidation of carbohydrates, lipids, and ascorbate, i.e. advanced glycation end products or glycoxidation products, such as carboxymethyllysine (CML) and pentosidine, and lipoxidation products, such as malondialdehyde (MDA) and 4‐hydroxy‐nonenal (HNE). In vitro incubation of proteins with ascorbic acid accelerated the production of protein carbonyls as well as CML and pentosidine, and incubation with arachidonate accelerated the production of protein carbonyls as well as CML, MDA, and HNE. By contrast, incubation of proteins with glucose resulted in the production of CML and pentosidine, but not protein carbonyls. Schiff base inhibitors, (±)‐2‐isopropylidenehydrazono‐4‐oxo‐thiazolidin‐5‐ylacetanilide and aminoguanidine, inhibited the production of protein carbonyls after incubation with ascorbate and arachidonate. The present study suggests that ascorbate and polyunsaturated fatty acids, but not glucose, represent potential sources of protein carbonyls, and that both the glycoxidation and lipoxidation reactions contribute to protein carbonyl formation in aging and various diseases.

Acute Hydrothorax in Continuous Ambulatory Peritoneal Dialysis – A Collaborative Study of 161 Centers

Follow-up studies on 3,195 patients from 161 centers in Japan undergoing continuous ambulatory peritoneal dialysis (CAPD) were performed for 1-104 months to clarify the incidence as well as the clinical features of acute hydrothorax. In these studies, 50 patients (1.6%) developed this complication. Twenty-seven (54%) were men, and 23 (46%) were women, ranging in age from 6 to 79 (average 49) years. The interval between onset of CAPD and hydrothorax ranged from 1 day to 8 years. Four had left-sided, and 2 had bilateral hydrothorax, but the majority (88%) were right-sided. Dyspnea was experienced by 37 of these 50 patients, but the remaining 13 (26%) patients were asymptomatic. Hydrothorax was fully resolved in 27 of them following a brief interruption of CAPD or the combined use of small exchange volumes in a semi-sitting position and pleurodesis with tetracycline or other agents. The remaining 23 patients (46%) were switched to hemodialysis permanently. Despite recurrence, 1 patient continued successfully on CAPD. It was concluded that acute hydrothorax is one important possible complication, although the risk may be low. Constant surveillance is necessary to detect pleural effusions in patients during CAPD.

In situ hybridization of interleukin 6 in diabetic nephropathy

Increased mesangial expansion is one of the most characteristic histological changes in diabetic nephropathy (DN). Although the pathogenesis of DN remains unclear, recent studies associate interleukin (IL) 6 with mesangial proliferative glomerulonephritis. To elucidate the expression and localization of IL-6 mRNA in renal tissues of patients with DN, a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide was performed. Patients were divided into three groups based on light microscopy findings: mild (group 1), moderate (group 2), and severe (group 3) mesangial expansion. The relationship between the expression of IL-6 mRNA and the degree of glomerular mesangial expansion in DN was examined. Individual cells positive for IL-6 mRNA were observed in glomeruli. These cells were mesangial cells, glomerular epithelial cells, and Bowmans capsule. The signal intensity was strongest in tissues from group 2 but was weak in those from groups 1 and 3. Most cells in the area of mesangial proliferation were strongly stained for IL-6 mRNA, and few positive cells were found in the Kimmelstiel-Wilson nodular lesion. In the interstitium, some tubules, particularly atrophic tubules, and some infiltrating cells were positively stained for IL-6 mRNA. The interstitial expression of IL-6 mRNA correlated significantly with the degree of interstitial injury and was remarkable in tissues from groups 2 and 3. We conclude that IL-6 mRNA is expressed by glomerular resident cells and interstitial cells in the renal tissue of patients with DN and that its expression may be associated with mesangial proliferation and may be involved in the tissue injury of DN.

Clinical guidelines for immunoglobulin A (IgA) nephropathy in Japan, second version

In 1995, clinical guidelines for the diagnosis and treatment of patients with immunoglobulin A (IgA) nephropathy in Japan were published by a joint committee of the Special Study Group on Progressive Glomerular Disease, Ministry of Health and Welfare of Japan (Chairman, Kiyoshi Kurokawa, MD), and the Japanese Society of Nephrology (President, Toshihiko Nagasawa, MD).1 The purpose of this new committee of the Special Study Group on Progressive Glomerular Disease, Ministry of Health and Welfare of Japan (Chairman, Hideto Sakai, MD), is to provide new clinical guidelines for the diagnosis and treatment of patients with IgA nephropathy in Japan. Although this document addresses current clinical practice in Japan, it is hoped that this English version will be helpful to the international community and will facilitate further discussion on this increasingly important disease.

Prevalence and Renal Prognosis of Diagnosed Autosomal Dominant Polycystic Kidney Disease in Japan

The prevalence and renal prognosis of diagnosed autosomal dominant polycystic kidney disease (ADPKD) in Japan were estimated. Hospital-based nationwide surveys were conducted in 1995. The number of ADPKD patients who visited hospitals but were not on chronic dialysis was estimated to be 10,000 (95% confidence interval: 8,200–11,900) and that of ADPKD patients on dialysis was 4,590, yielding a prevalence of ADPKD of 117 per million population at the end of 1994 (95% confidence interval: 102–132). The prevalence increased with age and reached a peak value of 261 per million population at the age group of 55–59 years. The rate of end-stage renal disease among living patients was calculated based on the assumption that the prevalence of ADPKD in the population under the age of 55 years was 261 per million population. The rate of end-stage renal disease increased with the progression of the patients’ age, reaching 49% at the age of 65–69 years and declining thereafter. Conclusion: The hospital-based prevalence of ADPKD is lower than the autopsy-based prevalence, suggesting that a fairly large number of these patients do not receive medical care in their lifetime. The probability of end-stage renal disease is at most 50% among ADPKD patients who visit a hospital.

Prognosis of parathyroid function after successful percutaneous ethanol injection therapy guided by color doppler flow mapping in chronic dialysis patients

Selective percutaneous ethanol injection therapy (PEIT) of the parathyroid glands has been shown to be effective in chronic dialysis patients with severe secondary hyperparathyroidism. In this study, we examined whether it was possible to maintain parathyroid function within target range (intact parathyroid hormone [iPTH], 160 to 360 pg/mL) in the long term after successful destruction of hyperplastic tissue. PEIT was performed in 46 patients resistant to calcitriol pulse therapy, and all glands larger than 5 mm in diameter were destroyed by ethanol, guided by power Doppler flow mapping. Serum iPTH levels decreased from 633.3 +/- 359.9 to 226.3 +/- 204.7 pg/mL at 3 weeks and were maintained at 289.9 +/- 222.4 pg/mL at 1 year after PEIT. Total alkaline phosphatase activity decreased from 384.9 +/- 160.1 to 234.0 +/- 110.5 IU/L at 1 year after PEIT. In 19 patients, iPTH levels decreased into relative hypoparathyroidism (iPTH < 160 pg/mL) at 3 weeks after PEIT; however, they recovered at 1 year after PEIT (191.1 +/- 29.6 pg/mL). Parathyroid function was maintained within target range in 80.4% of the patients at 1 year after PEIT with appropriate medical therapy. Surgical parathyroidectomy was not required in any patient. Conversely, in eight other patients with recurrent hyperparathyroidism after subtotal parathyroidectomy, iPTH levels recovered in only 50% of the patients at 1 year after PEIT. Thus, destruction of hyperplastic tissue should be optimized in such patients. Recurrent nerve palsy was observed in only one patient, but was reversible. In conclusion, selective PEIT guided by color Doppler flow mapping is an effective and safe adjunct to medical therapy with a low risk for hypoparathyroidism.

Phenotypic Characterization of Cytokine Expression in Patients With IgA Nephropathy

To identify the cytokines that play a relevant role in the pathogenesis of IgA nephropathy, we analyzed and compared the gene expression of proinflammatory cytokines, immuno-regulatory cytokines, and growth factors in peripheral blood mononuclear cells (PBMC). Expression of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF-α, and PDGF was examined in 28 patients with IgA nephropathy (IgAN), 20 patients with non-IgA mesangial proliferative glomerulo-nephritis (mesPGN), and 19 healthy controls. Compared with healthy controls, a significant number of IgAN and mesPGN patients showed increased expression of IL-1β, IL-4, IL-10, IL-12, and IFN-γ. The cytokine profile of renal tissue of 10 IgAN and 5 mesPGN biopsies was simultaneously analyzed and compared with that of PBMC. The proinflammatory IL-1α and growth factor PDGF-B were expressed more in renal tissues than in PBMC. Furthermore, the renal profile of IL-α, IFN-γ, and TNF-α expression was associated with the expression of IFN-γ PBMC. The serum level of IFN-γ of IgAN correlated significantly (P = 0.0003) with that of IL-12, suggesting a potential role for cross-stimulation. More importantly, expression of IFN-γ in PBMC and the elevated serum level correlated with the decline in glomerular filtration rate (P = 0.0012) and severity of renal histopathologic grade. To elucidate the role of leukocytes in renal cytokine expression, surface markers of T cells (CD3), monocytes (CD14), natural killer cells (CD16), and B cells (CD19) were also examined in renal tissues. The prominent renal expression of CD3, CD14, and CD16 implicates the leukocytes as the major source of proinflammatory cytokines in IgAN. Collectively, these findings indicate that IFN-γ plays a prominent role in an interactive network of cytokines that contribute to the pathogenesis and progression of IgA nephropathy.

Detection of immune complexes in polymorphonuclear leukocytes by double immunofluorescence in patients with IgA nephropathy.

Abstract The amounts of cytoplasmic inclusion bodies in peripheral blood polymorphonuclear cells (PMN) were determined in patients with IgA nephropathy and other glomerular diseases to elucidate whether or not IgA-dominant immune complexes were phagocytized by PMN in these patients. Fifteen patients with IgA nephropathy, 8 patients with other glomerular diseases, and 10 healthy adults were examined. The amounts of cytoplasmic inclusion bodies were measured by a double immunofluorescence technique. It was demonstrated that the percentages of IgA with C3 and IgA without C3 cytoplasmic inclusion bodies were significantly increased in patients with IgA nephropathy compared with those obtained in other glomerular diseases and healthy adults. There was a significant correlation between the levels of serum IgA and the percentage of IgA with C3 cytoplasmic inclusion bodies in PMN from patients with IgA nephropathy. It was suggested that IgA-dominant immune complexes are phagocytized by peripheral blood PMN in patients with IgA nephropathy.

Expression of E-cadherin in bone and soft tissue sarcomas: A possible role in epithelial differentiation

The cadherin-mediated cell-cell adhesion system is now known to play a critical role in both the morphogenesis of cancer cells and suppression of their invasion. However, the pattern of expression of E-cadherin, the major cadherin of epithelial cells in bone and soft tissue sarcomas, remains unclear. This prompted us to study E-cadherin expression in a variety of bone and soft tissue sarcomas. Using the monoclonal antibody HECD-1, raised against the extracellular domain of E-cadherin, we observed immunoreactivity in 1 pleomorphic rhabdomyosarcoma, 2 of 5 diffuse mesotheliomas, 4 of 5 clear cell sarcomas, 1 of 5 epithelioid sarcomas, and 10 synovial sarcomas. Other types of bone and soft tissue sarcoma (4 osteosarcomas, 4 chondrosarcomas, 3 primitive neuroectodermal tumors, 1 fibrosarcoma, 4 malignant fibrous histiocytomas, 5 liposarcomas, 4 leiomyosarcomas, 6 alveolar and 5 embryonal rhabdomyosarcomas, 4 angiosarcomas, 4 malignant peripheral nerve sheath tumors, 2 extraskeletal myxoid chondrosarcomas, 2 extraskeletal osteosarcomas, and 3 alveolar soft part sarcomas) were completely negative for E-cadherin. Our findings indicate that E-cadherin is expressed in certain kinds of soft tissue sarcomas, especially those with epithelioid features, suggesting that E-cadherin plays a role in the constitution of their architecture.