Effect of tonsillectomy with steroid pulse therapy on circulating tumor necrosis factor receptors 1 and 2 in IgA nephropathy.

BACKGROUND: High circulating levels of soluble tumor necrosis factor receptors (TNFRs: TNFR1, TNFR2) predict renal function decline in a variety of kidney diseases. Tonsillectomy with steroid pulse (TSP) therapy has been reported as a remission induction therapy in IgA nephropathy (IgAN), mainly in Japan. However, little is known about whether TNFR levels change after TSP therapy in patients with IgAN. METHODS: Two hundred twenty-three patients with IgAN were stratified according to the estimated glomerular filtration rate (eGFR): Group I (eGFR ≥ 60 mL/min/1.73 m2, n = 172) and Group II (eGFR < 60 mL/min/1.73 m2, n = 51). We measured serum TNFR levels with immunoassay in all patients at the time of renal biopsy, and also in patients whose samples just before the first (after tonsillectomy) (n = 34) and/or the third steroid pulse therapy (n = 77) were available. RESULTS: The TNFR levels were significantly higher in Group II than in Group I. A significant negative correlation was observed between TNFR levels and eGFR at baseline (TNFRs: r > -0.50). In multivariate logistic regression analysis, both TNFRs were associated with renal function decline, independent of age and uric acid levels. Proteinuria and hematuria remarkably improved after TSP therapy, as expected. In comparison with baseline TNFR levels, the levels of TNFR2, but not TNFR1, decreased significantly just before the third steroid pulse therapy, although both levels did not change after tonsillectomy. CONCLUSIONS: The TNFR2 level did not change after tonsillectomy alone but decreased significantly after steroid pulse therapy in patients with IgAN.

Beneficial effects of tonsillectomy plus steroid pulse therapy on inflammatory and tubular markers in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. Tonsillectomy plus steroid pulse therapy has been able to induce clinical remission in early-stage IgAN. However, its possible effect on systemic and local cytokines and tubular markers has not been fully investigated. METHODS: We obtained serum and urine samples from 38 patients just before renal biopsy and third steroid pulse therapy. Markers of tubular damage such as N-acetyl-ß-d-glucosaminidase, and kidney injury molecule-1 and inflammation such as interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 were measured by immunoassay. RESULTS: Before renal biopsy, only urinary inflammatory markers, except MCP-1, were associated with glomerular (proteinuria) and/or tubular damage markers. Proteinuria, hematuria, and estimated glomerular filtration rate dramatically improved after therapy. In addition, levels of serum IL-6 and ICAM-1 and all urinary markers declined significantly; however, serum MCP-1 and VCAM-1 levels did not. None of the urinary markers correlated with the serum inflammatory markers. CONCLUSION: Tonsillectomy plus steroid pulse therapy for patients with IgAN might be useful for improving not only glomerular damage marker but also tubular damage markers through the improvement of local renal inflammation.

Podocyte-specific deletion of Rac1 leads to aggravation of renal injury in STZ-induced diabetic mice.

Rac1, a GTPase of the Rho subfamily, has a crucial role in cytoskeletal architecture, as well as the regulation of cell migration and growth. However, renal injury in mice with podocyte-specific deletion of Rac1 has yet to be elucidated fully due to conflicting findings. Herein, we identified a possible role for Rac1 in podocytes of streptozotocin- (STZ) induced diabetic mice. The urinary albumin/creatinine ratio (ACR) in the knockout (KO) group was significantly higher than that in the wild type (WT) group at any week of age. A more marked ACR increase was observed in STZ/KO group than STZ/WT group, although ACR did increase with weeks of age in both diabetic groups. The kidney sections from diabetic mice revealed a glomerular hypertrophy with mesangial expansion, but there was no appreciable difference in glomerular findings under a light microscope between STZ/WT and STZ/KO mice. However, an electron microscopy analysis revealed that regardless of the presence or absence of diabetes, both KO (KO and STZ/KO) groups had a higher rate of foot process effacement compared with both WT (WT and STZ/WT) groups. The expression levels of the slit diaphragm protein, podocin, was reduced with the induction of diabetes, and the levels in the STZ/KO group experienced a further reduction compared with the STZ/WT group. The number of WT1-positive cells in the STZ/KO group was more significantly decreased than that in the other three groups. In contrast, the numbers of cleaved caspase 3- and TUNEL-positive cells in the glomeruli of the STZ/KO group were more increased than those in the STZ/WT group. Thus, this study provides evidence that podocyte-specific deletion of Rac1 results in morphological alteration in podocytes, and that the induction of apoptosis or decreased expression of the slit diaphragm proteins by hyperglycemic stimuli are associated with the progression of diabetic nephropathy.

Circulating TNF receptors 1 and 2 are associated with the severity of renal interstitial fibrosis in IgA nephropathy.

The current study aimed to examine whether the levels of TNF receptors 1 and 2 (TNFR1 and TNFR2) in serum and urine were associated with other markers of kidney injury and renal histological findings, including TNFR expression, in IgA nephropathy (IgAN). The levels of the parameters of interest were measured by immunoassay in 106 biopsy-proven IgAN patients using samples obtained immediately before renal biopsy and in 34 healthy subjects. Renal histological findings were evaluated using immunohistochemistry. The levels of serum TNFRs were higher in IgAN patients than in healthy subjects. The levels of both TNFRs in serum or urine were strongly correlated with each other (r > 0.9). Serum TNFR levels were positively correlated with the urinary protein to creatinine ratio (UPCR) and four markers of tubular damage of interest (N-acetyl-ß-D-glucosaminidase [NAG], ß2 microglobulin [ß2m], liver-type fatty acid-binding protein [L-FABP], and kidney injury molecule-1 [KIM-1]) and negatively correlated with estimated glomerular filtration rate (eGFR). Patients in the highest tertile of serum TNFR levels showed more severe renal interstitial fibrosis than did those in the lowest or second tertiles. The tubulointerstitial TNFR2-, but not TNFR1-, positive area was significantly correlated with the serum levels of TNFRs and eGFR. Stepwise multiple regression analysis revealed that elevated serum TNFR1 or TNFR2 levels were a significant determinant of renal interstitial fibrosis after adjusting for eGFR, UPCR, and other markers of tubular damage. In conclusion, elevated serum TNFR levels were significantly associated with the severity of renal interstitial fibrosis in IgAN patients. However, the source of TNFRs in serum and urine remains unclear.

Role of the TNF pathway in the progression of diabetic nephropathy in KK-A(y) mice.

Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNF-α remains unclear. The objectives of the present study were to examine whether TNF-α inhibition with a soluble TNF receptor (TNFR)2 fusion protein, i.e., etanercept (ETN), improves the early stage of DN in the type 2 diabetic model of the KK-A(y) mouse and to also investigate which TNF pathway, TNFR1 or TNFR2, is predominantly involved in the progression of this disease. ETN was injected intraperitoneally into mice for 8 wk. Renal damage was evaluated by immunohistochemistry, Western blot analysis, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNF-α and/or high glucose (HG) and treated with ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNF-α and TNFR1, in ETN-treated KK-A(y) mice were significantly decreased compared with untreated KK-A(y) mice. mRNA levels of ICAM-1, VCAM-1, and monocyte chemoattractant protein-1 and the number of F4/80-positive cells were all decreased after treatment. Numbers of cleaved caspase-3- and TUNEL-positive cells in untreated mice were very few and were not different from ETN-treated mice. In vitro, stimulation with TNF-α or HG markedly increased both mRNA levels of TNFRs, unlike in the in vivo case. Furthermore, ETN partly recovered TNF-α-induced but not HG-induced TNFR mRNA levels. In conclusion, it appears that ETN may improve the progression of the early stage of DN predominantly through inhibition of the anti-inflammatory action of the TNF-α-TNFR2 pathway.

Hepatic extramedullary hematopoiesis and macrophages in the adult mouse: histometrical and immunohistochemical studies.

Fetal liver hematopoiesis in mice disappears approximately 2 weeks after birth; however, under experimental acute anemia extramedullary hematopoiesis occurs in the livers of adult mice. The hematopoietic foci in the liver during extramedullary hematopoiesis contain erythroblasts and macrophages. In this study, the extent of involvement of macrophages in the development and involutional process of the hematopoietic foci in adult mice livers was clarified by experimentally inducing extramedullary hematopoiesis. Hematopoietic cells appeared in the livers 2 days after phenylhydrazine (PHZ) injections. The number and area of the foci increased rapidly, reaching peak values on the sixth day. F4/80-positive macrophages were observed in the sinusoids as well as the hematopoietic foci, and were tightly surrounded by erythroblasts. Sinusoidal macrophages in normal adult livers were positive for F4/80 but negative for ER-HR3. However, in extramedullary hematopoiesis-induced livers, sinusoidal macrophages became positive for ER-HR3 antibodies. The number of ER-HR3-positive macrophages was 9.2 ± 2.9/mm(2) on the second day after PHZ was administered, and increased to 200.3 ± 4.2/mm(2) on the sixth day. On the seventh day after the PHZ injections, the number decreased and they were no longer detected at 30 days after PHZ was injected. The present study revealed that erythroblasts accumulate around sinusoidal macrophages to form an erythroblastic island with a central macrophage similar to erythropoiesis in the fetal liver. Furthermore, in line with development and regression of extramedullary hematopoiesis, macrophages express ER-HR3, a hematopoiesis related antigen.

Surface morphology of the central macrophages of erythroblastic islets in the spleen of aged and pregnant mice: an immunohistochemical light microscopic study.

This study used 100-mum thick paraffin sections stained by the ER-HR3 antibody to examine the three-dimensional surface morphology of the central macrophages of erythroblastic islets in the splenic red pulp of aged and pregnant mice. The ER-HR3-positive cells were the macrophages located at the center of the erythroblastic islets, and the number per unit of splenic area was almost constant until 30 days of age, thereafter showing a marked decrease. In pregnant females, the ER-HR3-positive macrophage number significantly increased and became approximately eight times higher than the control value. In aged virgin females, the islet macrophages were generally ovoid in cell profile, and shallow cup-shaped dents were formed on their cell surface. However, in pregnant females, the macrophages became larger in size, and cell socket structures, formed by long finger-like cytoplasmic processes, became prominent on their cell surface. The 3-D images, obtained from 100-mum thick paraffin sections, provided the clear morphological evidence of the activity of the islet macrophages in spleen erythropoiesis.

[A histological study and three-dimensional reconstruction of F4/80-positive reticular cells and macrophages at the onset of murine bone marrow hematopoiesis].

Adult bone marrow consists of two different compartments, a vascular compartment of sinusoid and a hematopoietic compartment consisting of stromal cells and hematopoietic cells. In the hematopoietic compartment, stromal cells play an important role in the formation of the microenvironment for hematopoiesis. To clarify the relationship between hematopoietic cells and stromal cells, particularly reticular cells and macrophages, we examined the femur bone marrow of ICR mouse fetuses and neonates using F4/80 immunostaining and three-dimensional reconstruction under light and electron microscopy. In the fetal femurs, the marrow cavity formed early from 15 days of gestation, and it showed a marked increase in volume thereafter. On the basis of the appearance of hematopoietic cells, marrow development could be classified into two stages, a pre-hematopoietic stage from 15 days of gestation to two days of age, and a beginning stage of hematopoiesis thereafter. The pre-hematopoietic bone marrow contains not only stromal reticular cells but also macrophages, and both types of stromal cells were strongly positive to F4/80 monoclonal antibody. These F4/80-positive reticular cells had a triangular cell profile with long and slender cytoplasmic processes. Reticular cells often contained large lysosomes of not only dying neutrophils but also erythroblast nuclei. A few erythroblasts accumulated around the processes, and the number of erythroblasts around reticular cells increased with bone marrow development. On the other hand, macrophages were located either close to sinusoids or in sinusoid lumen, and a close relationship to hematopoietic cells was hardly noticeable. At the beginning stage of hematopoiesis, F4/80-positive reticular cells extended their long and slender cytoplasmic processes, and the number and length of the processes appeared markedly increased. The three-dimensional cell surface of the F4/80-positive reticular cells became very complex. Numerous erythroblasts accumulated around the processes, and erythroblastic islands could gradually be recognized after four days of age. In the erythroblastic islands, central reticular cells were F4/80-positive and contained numerous large phagosomes originating from the expelled nuclei of erythroblasts. Although macrophages contained large phagosomes, the relationship between macrophages and hematopoietic cells could not clearly be elucidated even at the beginning stage of hematopoiesis. At the onset of bone marrow hematopoiesis, the hematopoietic compartment contained two kinds of F4/80-positive phagocytes, i.e., reticular cells and macrophages. In marrow erythroblastic islands, not macrophages but F4/80-positive reticular cells were located at the center of each island.

Three-dimensional surface structure of macrophages in fetal and adult mouse liver: an immunohistochemical light microscopic study.

Using 100-microm-thick paraffin sections stained by F4/80 antibody, the three-dimensional surface morphology of macrophages in fetal and adult livers was examined by conventional light microscope equipped with a computer-controlled z-axis stepping motor. Hematopoietic macrophages in fetal livers were located in the center of erythroid cell clusters, forming cell sockets which consisted of two different kinds of projections. The primary cytoplasmic processes were membranous projections and the secondary processes were finger-like projections extending from the primary processes. Erythroids in the cell sockets were linearly arranged on the macrophage surface. Adult sinusoidal macrophages possessed a few pseudopod-like processes, ridge-like profiles and numerous ruffle or spike-like processes, and cell contact with neighboring macrophages could be recognized. Compared to confocal laser scanning microscopy and scanning electron microscopy, this study provided information about cell surface structures at reasonably low cost, although the resolution was limited in z axis due to the stepping interval.