Toshihiko Nishino

Immunohistochemical detection of advanced glycosylation end products within the vascular lesions and glomeruli in diabetic nephropathy

Numerous studies over the years have implicated advanced glycosylation end products (AGEs) in the pathogenesis of many of the complications of diabetes and normal aging. The recent development of specific antibodies against AGE-modified proteins has facilitated investigations on the formation and tissue distribution of AGEs. We used anti-AGE antibodies to localize AGEs within kidney specimens obtained from both diabetic and nondiabetic individuals. Immunohistochemical staining using anti-AGE antibody showed a high level of AGE accumulation in diabetic and aged vascular intima, particularly along the inner elastic layer of arteries. Positive staining also was observed within nodular and severe diffuse lesions of glomeruli as well as in hyaline deposits of arterioles. These data support a pathogenic role for advanced glycosylation in the renal complications of diabetes and aging.

Cell proliferation and apoptosis of the glomerular epithelial cells in rats with puromycin aminonucleoside nephrosis.

Injury and repair of the glomerular epithelial cells (GECs) play an important role in the pathogenesis of focal segmental glomerulosclerosis (FSGS). To obtain a better understanding of proliferation and apoptosis of GECs, we examined immunohistochemical and in situ hybridization findings in puromycin aminonucleoside nephrosis (PAN) of rats. The minimal-change nephrotic syndrome model (PAN-MCNS) was induced by administering 5 subcutaneous injections of puromycin aminonucleoside (PA; each 1.5 mg/100 g B/W to one group of rats), whereas the FSGS model (PAN-FSGS) was induced by administering an additional 5 injections of PA to another group of rats. The cell kinetics of the GECs were assessed with labeling 5-bromo 2′-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). To investigate regulation of apoptosis in rats with PAN, we evaluated the expression of p53, Fas antigen, Fas ligand and Bcl-2. Rats with PAN-MCNS exhibited a significantly greater number of BrdU- and PCNA-labeled GECs as compared with control rats. In rats with PAN-FSGS, the number of PCNA-labeled GECs was greater than in rats with PAN-MCNS, but the number of BrdU-labeled GECs was lower. Apoptotic cells were occasionally observed in the sclerotic lesions, with the number being significantly higher in rats with PAN-FSGS than in rats with PAN-MCNS and control. Apoptotic cells were observed in the GECs of PAN-FSGS rats. However, they were negative for p53, Fas antigen, and Fas ligand. Immunohistochemical and in situ hybridization studies revealed a greater intraglomerular overexpression of Bcl-2 protein and bcl-2 mRNA in the PAN-FSGS rats as compared with control rats. These results suggest that insufficient proliferation and apoptosis in GECs may be involved in the progression of FSGS.

Measurement of Plasma and Urinary Adrenomedullin in Patients with IgA Nephropathy

In this study, we measured plasma and urinary adrenomedullin (AM) concentrations in 47 patients with IgA nephropathy. Controls were 39 healthy volunteers. Plasma and urinary AM values were measured by specific radioimmunoassay. The plasma AM concentrations were higher, and the urinary AM levels were lower in patients with IgA nephropathy than in healthy volunteers. Plasma AM concentrations showed a positive correlation with serum creatinine and blood urea nitrogen, whereas urinary AM levels correlated negatively with serum creatinine and blood urea nitrogen. The plasma AM concentrations showed a positive correlation with fractional excretions of sodium and potassium. Renal biopsy specimens of patients without renal failure were scored for activity (percentage of glomeruli demonstrating cellular crescent formation, degree of mesangial proliferation and interstitial infiltration; total score = 9). Urinary AM levels were shown to be lower in the group with a high activity (score 3–9) as compared with the group with a low activity (score 0–2) based on renal biopsy. Thus, urinary levels of AM are affected by the degree of the activity in IgA nephropathy, and AM may participate in the pathophysiology of IgA nephropathy.

Plasma and Urinary Levels of Adrenomedullin in Chronic Glomerulonephritis Patients with Proteinuria

In this study, we measured levels of plasma and urinary adrenomedullin (AM) in 37 patients with chronic glomerulonephritis including minimal change nephrotic syndrome, focal segmental glomerulosclerosis or membranous nephropathy that can induce severe proteinuria. Thirty-nine healthy volunteers were enrolled as controls. Plasma and urinary AM levels were measured by an AM-specific radioimmunoassay. Plasma AM concentrations were higher and urinary AM levels were lower in patients with chronic glomerulonephritis than in healthy volunteers. Patients were divided into two groups according to urinary excretion of protein for 24 h (UPro, g/day) which reflects the disease activity or glomerular damage of the glomerulonephritis (group I: Upro < 1, group II: Upro ≥ 1). Plasma AM levels positively and urinary AM-levels negatively correlated with the degree of proteinuria. These results suggest that plasma and urinary AM levels in patients with chronic glomerulonephritis reflect the disease activity or glomerular damage represented by the degree of proteinuria.

Adrenomedullin Gene Transcription Is Decreased in Peripheral Blood Mononuclear Cells of Patients with IgA Nephropathy

We measured mRNA levels of adrenomedullin (AM), C-type natriuretic peptide (CNP), vascular endothelial growth factor (VEGF), interleukin 1β (IL-1β) and interleukin 6 (IL-6) in peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy. To evaluate these mRNA levels, we employed a real-time quantitative PCR method which was performed using a hybridization probe labeled with two fluorescence dyes. This strategy was found to afford the standard curves with a high correlation, suggesting that this method is useful for evaluations of mRNA levels. By this method, levels of AM, CNP, VEGF, IL-1β and IL-6 mRNA in PBMC of 49 IgA nephropathy patients and 35 healthy volunteers were evaluated. Among the mRNAs examined, AM mRNA levels were significantly lower in severe-grade than in mild-grade IgA nephropathy patients. Furthermore, AM mRNA levels correlated with CNP mRNA levels in PBMC of patients with IgA nephropathy, and each peptide generated from these mRNAs has antiproliferative effects on mesangial cells. These data indicate that gene expression of AM in PBMC is regulated according to the pathophysiological states of IgA nephropathy and that decreased AM production may contribute to the progression of IgA nephropathy.

Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis

In this study, we measured the mRNA levels of adrenomedullin (AM), C‐type natriuretic peptide, vascular endothelial growth factor, interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real‐time quantitative PCR method. Compared with healthy volunteers, IL‐6 mRNA levels were elevated in LN patients (P < 0·005), while AM mRNA levels were decreased (P < 0·05). Also, IL‐6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL‐6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL‐6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL‐6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL‐6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL‐6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL‐6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.

Enhanced Expression of Plasminogen Activator Inhibitor 1 in Patients with Nephrotic Syndrome

Hypercoagulability is present in patients with nephrotic syndrome. However, alterations in coagulation and fibrinolysis reflected in the glomeruli and urine are not fully understood. We examined plasma and urine concentrations of tissue-type plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) in 33 patients with nephrotic syndrome (nephrotic group). We compared these concentrations with the concentrations in 30 nonnephrotic patients with chronic glomerulonephritis (nonnephrotic group) and with the concentrations in 30 healthy volunteers (control group). We also examined fibrin/fibrinogen degradation products in serum and urine and plasma D-dimers. The expression of tPA and PAI-1 was examined in isolated glomeruli using RT-PCR methods. Deposition of fibrinogen/fibrin-related antigen was observed by direct immunofluorescence. The incidence of fibrinogen/fibrin-related antigen deposition in the nephrotic group was significantly higher than that in the nonnephrotic group. The concentrations of fibrin/fibrinogen degradation products in serum and urine and of plasma D-dimers were significantly elevated in the nephrotic group as compared with the nonnephrotic and control groups. The plasma concentrations of tPA in the nephrotic group were significantly higher than those in the control group. The urinary excretion of tPA in the nephrotic group was also significantly higher than in the nonnephrotic and control groups. The urinary excretion of PAI-1 in the nephrotic group was higher than that in the control group. The ratio of PAI-1 mRNA to tPA mRNA in glomeruli was increased in the nephrotic group as compared with the nonnephrotic group. These results indicate that the fibrinolytic activity is increased in patients with nephrotic syndrome despite urinary losses of tPA. However, a relatively enhanced expression of PAI-1 may be involved in the intraglomerular fibrinogen/fibrin-related antigen deposition seen in nephrotic syndrome.

Alterations in extracellular matrix components and integrins in patients with preeclamptic nephropathy

The glomerular features of patients with preeclapsia consist of swelling of endothelial cells, subendothelial deposits of incompletely defined material, and thickening of the capillary walls. These abnormalities are thought to resolve in the postpartum period. The distribution of extracellular matrix (ECM) components and integrins was investigated in 10 such patients. Frozen sections and paraffin-embedded sections were stained with antibodies to type IV collagen, laminin (LN), fibronectin (FN), vitronectin (VN), tenascin (TN), fibronectin receptor (FNR), and vitronectin receptor (VNR). In preeclamptic nephropathy, the accumulation of type IV collagen, LN, FN, TN, and FNR was observed in the thickened capillary walls, particularly in the subendothelial layer and, to some extent, in the mesangium. However, deposits of VN were sparse and the distribution of VNR was similar to that in normal kidney. In segmental sclerotic lesions, the amounts of type IV collagen, LN, FN, VN, and TN were increased, whereas those of FNR and VNR were markedly decreased. These results suggest that the materials deposited in the subendothelial space consist of ECM components as well as of plasma-derived proteins, and that the deposition of ECM components and of FNR may be involved in the development and the reparative process of the characteristic glomerular lesions. The formation of sclerotic lesions was linked to the accumulation of ECM components, but not to an interaction with integrins.

Expression of Bcl-2 and Bax in Hypokalemic Nephropathy in Rats

Objectives: Potassium depletion results in hyperplasia of renal tubular and interstitial cells in humans and animals, and potassium repletion induces rapid regression of hyperplasia. Apoptosis participates importantly in this reduction of cell number, although we have observed tubular and interstitial apoptosis in rats during potassium depletion as well. Methods: To investigate mechanisms of apoptosis in this model, we assessed expression of Bcl-2 and Bax, using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Results: Cell proliferation identifiable by labeling with 5-bromo 2′-deoxyuridine was prominent in tubular and interstitial cells of the cortex and outer medulla (OM) 7 days after potassium depletion. Simultaneously present apoptotic cells identified by light microscopy, electron microscopy, and nick end labeling were located mainly in the OM. Seven days after potassium repletion, apoptotic cells increased again but proliferating cells decreased. Bcl-2 protein distributed in the tubules of the OM was significantly decreased in potassium-depleted and potassium-repleted rats compared with control rats, while immunoreactivity for Bax protein tended to increase above control levels in potassium-depleted rats. RT-PCR for bcl-2 and bax demonstrated a significant decrease in levels of bcl-2 mRNA in potassium-depleted and potassium-repleted rats relative to those in controls. Expression of bax mRNA in potassium-depleted and potassium-repleted rats tended to increase, while ratios of bcl-2 mRNA to bax mRNA significantly decreased. Conclusions: These results suggest that apoptosis is associated with progression and regression of cellular proliferation in hypokalemic nephropathy, and a decrease in Bcl-2 may be involved in promoting this apoptotic process.

Cytosine arabinoside induced acute interstitial nephritis in a patient treated for refractory anemia with excess of blasts in transformation.

Accessible online at: www.karger.com/journals/nef Dear Sir, Cytosine arabinoside (Ara-C) is an effective agent against acute nonlymphocytic leukemia, myelodysplastic syndrome, and malignant lymphoma. Low-dose Ara-C is prescribed for refractory anemia with excess of blasts (RAEB) or RAEB in transformation (RAEB-t), since this treatment is believed to suppress leukemic cell proliferation and to induce differentiation in malignant cells [1, 2]. Toxicity of Ara-C extends to various organs, including heart, lungs, gastrointestinal tract, liver, central and peripheral nervous systems, and kidneys [3]. Nephrotoxicity is not uncommon, and an elevated serum creatinine concentration has been reported to occur in 5–20% of high-dose courses of Ara-C [3]. Although the precise mechanism of kidney damage remains unknown, many factors predispose to renal dysfunction during intensive chemotherapy. These include tumor lysis with hyperuricemia, dehydration, nephrotoxic antibiotics and other drugs, and rhabdomyolysis [3, 4]. Hepatorenal syndrome also has been reported to occur [5]. However, acute interstitial nephritis (AIN) has not been reported, to our knowledge. We treated a patient with myelodysplastic syndrome in whom AIN complicated low-dose Ara-C therapy. A 68-year-old Japanese woman was admitted to our department in January 1997 Fig. 1. Clinical course.