Jun Soma

The role of macrophages in diabetic glomerulosclerosis

To elucidate the role of macrophages in diabetic glomerulosclerosis (DGS), an immunohistologic study was performed using monoclonal antibodies to common leukocyte antigen (DAKO-LC), T cells (T3), B cells (CD22), and macrophages (MAC 387, Leu-M5, and EBM-11). Kidney biopsy specimens were obtained from 28 patients with non-insulin-dependent diabetes mellitus. Cells were identified by a three-layer immunoperoxidase technique applied to cold ethanol-fixed, paraffin-embedded sections and quantitated as the number of cells per glomerular cross-sections and number of cells per square millimeter of glomerulus. The severity of the diffuse lesions in each glomerulus was graded semiquantitatively. The average grades for all the glomeruli were calculated and registered as an index of DGS for a biopsy specimen. There was no relationship between the index of DGS and the number of T or B cells. However, the number of macrophages and common leukocyte-positive cells increased significantly in the moderate stage of glomerulosclerosis compared with the mild or advanced stage. The results suggest that macrophages may transiently infiltrate during the moderate stage of diffuse DGS, contributing to irreversible structural damage.

Cytochrome P450 4A expression and arachidonic acid omega-hydroxylation in the kidney of the spontaneously hypertensive rat.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a major arachidonate metabolite in the kidney of the spontaneously hypertensive rat (SHR). The increase in its synthesis has been associated with the elevation of blood pressure in the SHR. The omega-hydroxylation of arachidonic acid is an activity associated with members of the CYP4A gene family which, in the rat, comprises three major isoforms: 4A1, 4A2 and 4A3. 20-HETE displays potent and diverse biological activities which can affect pro- and anti-hypertensive mechanisms dependent upon where, when and by which isoform it has been produced. Therefore, it is important to identify and characterize its biosynthetic system. We compared CYP4A mRNA and protein expression to patterns of 20-HETE synthesis in the SHR kidney. The reverse transcription/polymerase chain reaction (RT/PCR) technique was used to amplify CYP4A mRNA in microdissected nephron segments. Southern blot hybridization of PCR products obtained from nephron segments with the CYP4A1 cDNA probe demonstrated strong signals in S2 and S3 segments of the proximal tubule. Immunoblots of nephron segments using a polyclonal anti-rat liver CYP4A1 antibody which cross-reacts with CYP4A2 and CYP4A3, and 14C-arachidonic acid metabolism, confirmed that arachidonic acid omega-hydroxylation, i.e., 14C-20HETE formation, and CYP4A proteins were also localized mainly in the S2 and S3 segments. Correlation also existed between the age-dependent increase in arachidonate omega-hydroxylation in the kidney and CYP4A mRNA levels as measured by Northern hybridization of total RNA using the CYP4A1 cDNA probe. Immunoblot analysis revealed that at 7 weeks, where 20-HETE production is at its maximum, all three proteins are expressed. CYP4A3 and 4A1 immunoreactive proteins appeared at 3 weeks, showed maximum levels at 5 and 7 weeks, respectively, and gradually decreased to lower levels at 13 and 20 weeks, whereas CYP4A2 levels were undetectable at 3, 5 and 7 weeks but appeared at 13-20 weeks. Additional immunoblots indicated that renal cortical CYP4A1 protein levels were higher in SHR compared to Sprague-Dawley and Wistar-Kyoto rats. The increased levels of CYP4A1-immunoreactive band at 7 weeks corresponded to the maximal activity of arachidonate omega-hydroxylation. Thus, CYP4A1 might play a significant role in contributing to the increased cortical/proximal production of 20-HETE seen in 7-week-old SHR. However, given the high homology among members of the CYP4A gene family and the lack of specific tools to discern among these isoforms, additional studies have to be carried out to substantiate our findings.

A multicenter randomized controlled trial of tonsillectomy combined with steroid pulse therapy in patients with immunoglobulin A nephropathy

Background The study aim was, for the first time, to conduct a multicenter randomized controlled trial to evaluate the effect of tonsillectomy in patients with IgA nephropathy (IgAN). Methods Patients with biopsy-proven IgAN, proteinuria and low serum creatinine were randomly allocated to receive tonsillectomy combined with steroid pulses (Group A; n = 33) or steroid pulses alone (Group B; n = 39). The primary end points were urinary protein excretion and the disappearance of proteinuria and/or hematuria. Results During 12 months from baseline, the percentage decrease in urinary protein excretion was significantly larger in Group A than that in Group B (P < 0.05). However, the frequency of the disappearance of proteinuria, hematuria, or both (clinical remission) at 12 months was not statistically different between the groups. Logistic regression analyses revealed the assigned treatment was a significant, independent factor contributing to the disappearance of proteinuria (odds ratio 2.98, 95% CI 1.01–8.83, P = 0.049), but did not identify an independent factor in achieving the disappearance of hematuria or clinical remission. Conclusions The results indicate tonsillectomy combined with steroid pulse therapy has no beneficial effect over steroid pulses alone to attenuate hematuria and to increase the incidence of clinical remission. Although the antiproteinuric effect was significantly greater in combined therapy, the difference was marginal, and its impact on the renal functional outcome remains to be clarified.

Tranilast Slows the Progression of Advanced Diabetic Nephropathy

Background: Tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, suppresses collagen synthesis by various cells, including macrophages and fibroblasts, by interfering with the actions of transforming growth factor-beta 1. We investigated the effect of tranilast on progression of diabetic nephropathy (DN), since this process is associated with accumulation of collagens in the glomerulus and interstitium. Methods: Tranilast (100 mg, 3 times daily) was administered to 9 outpatients with advanced DN who were receiving an angiotensin-converting enzyme inhibitor or an angiotensin II receptor antagonist and who exhibited a progressive decline in renal function. The decline in renal function before and during tranilast treatment was evaluated for each patient on the basis of the slope in reciprocal serum creatinine (1/SCr) over time. Urinary type IV collagen (U-IV·C) and protein (U-P) excretions were measured just before commencement of tranilast treatment and every 2 months during the treatment. Results: One male patient dropped out soon after commencement of tranilast treatment due to development of lung cancer, and hemodialysis was introduced in one female patient 6 months after the start of treatment. In the 8 patients who did not drop out, 1/SCr was significantly less steep during tranilast treatment than before treatment (–0.00748 ± 0.00700 vs. –0.01348 ± 0.00636 dl/mg/month, respectively; p = 0.0374). U-IV·C and U-P tended to decrease with time, although the decrease was statistically insignificant. Conclusions: Our data suggest that tranilast treatment may suppress accumulation of collagens in renal tissue and may be therapeutically useful for reducing the progression of advanced DN.

Quenching the Thirst in Dialysis Patients

In a double-blind cross-over trial, 22 stable end-stage renal failure patients on maintenance haemodialysis were subjected to conventional dialysis with dialysate containing 137 mEq/l sodium and constant ultrafiltration (UF) and to a different dialysis therapy, in which, by linear sodium modelling, the dialysate sodium was reduced from 137 to 128 mEq/l. A computerized UF program was used to gradually reduce the UF to a minimum towards the end of the session. Severity of thirst, interdialytic weight gain and intradialytic complications were less with low sodium dialysate. It allowed adequate UF with absolute hemodynamic stability. The reduced incidence of complication with low sodium dialysate therapy was probably because they required less UF.

Contribution of cellular infiltration to the progression of IgA nephropathy: A longitudinal, immunocytochemical study on repeated renal biopsy specimens

Summary: A retrospective immunocytochemical study was performed on repeated renal biopsy specimens from 47 patients with IgA nephropathy, 23 of whom received steroid therapy after the initial biopsy. Immune cells in renal tissues were detected by the immunoperoxidase method using monoclonal antibodies against common leukocyte antigens, T cells, B cells and monocytes/macrophages.

Effect of a Nonpeptide Vasopressin V1 Antagonist (OPC-21268) on Experimental Accelerated Focal Glomerulosclerosis

The effects of the nonpeptide orally effective vasopressin V1 receptor antagonist OPC-21268 were studied in progressive focal glomerulosclerosis (FGS) which developed in spontaneously hypercholesterolemic (SHC) rats with manifestations of hypercholesterolemia and proteinuria. Unilateral nephrectomy was performed at 7 weeks of age to accelerate spontaneous FGS. After nephrectomy, OPC-administered rats were fed chow containing 1% OPC-21268 for 9 weeks. Treatment with vasopressin V1 antagonist significantly reduced the rate of increase in the levels of triglyceride, systolic blood pressure, serum creatinine and BUN, and prevented a significant deterioration in creatinine clearance. Rats were sacrificed at 16 weeks of age. Histologically, the index of glomerular sclerosis in the OPC group showed a significant decrease compared to that in the control group (2.2 +/- 0.1 vs. 2.6 +/- 0.1, p < 0.01). Relative interstitial volume and glomerular volume in the OPC group showed a tendency to decrease compared to those in the control group. These results indicate that vasopressin plays an important role through V1 receptors in the development of glomerulosclerosis, and vasopressin V1 antagonist may prevent the progression of renal injury in glomerulosclerosis.

Intercellular adhesion molecule- 1/leukocyte function associated antigen-1-mediated and complement receptor type 4—mediated infiltration and activation of glomerular immune cells in immunoglobulin a nephropathy

Glomerular expression of intercellular adhesion molecule-1 (ICAM1) (CD54) and membrane cofactor protein (MCP; CD46) and positive infiltrating cells in leukocyte function associated antigen-1 (LFA1)alpha (CD11a) and C3bi receptors (CR3/CD11b, CR4/CD11c) were examined by the indirect immunoperoxidase method on 43 sets of repeated renal biopsy specimens from patients with immunoglobulin A nephropathy. Twenty-four-hour urine protein at the time of renal biopsy was also evaluated. Glomerular infiltration of LFA1alpha+ cells was significantly correlated with glomerular expression of ICAM1 (r = 0.494, P < 0.0001). Glomerular complement receptor type 4 (CR4)+ cells were significantly correlated with glomerular expression of MCP (r = 0.405, P < 0.0001). The glomerular expressions of ICAM1 and MCP were significantly correlated with each other (r = 0.700, P < 0.00001). The glomerular infiltrations of LFA1alpha+ and CR4+ cells were highly correlated with each other (r = 0.884, P < 0.00001), and both cell types were significantly correlated with urine protein (respectively, r = 0.426 and 0.478, P < 0.001 and 0.0001). When the change in these parameters between the time of the initial and follow-up biopsies was evaluated, there was a significant correlation between the change in glomerular expression of ICAM1 (DeltaICAM1) and MCP (DeltaMCP) as well as between the change in glomerular infiltration of LFA1alpha+ cells (DeltaLFA1alpha+) and CR4+ cells (DeltaCR4+). Both DeltaLFA1alpha+ and DeltaCR4+ were significantly correlated with the change in urine protein. These findings suggest that ICAM1/LFA1 interaction and MCP-mediated C3bi/C3biR interaction cooperate and participate in persistent glomerular infiltration of immune cells in immunoglobulin A nephropathy, and that these LFA1alpha+ and C3biR+ cells contribute to the induction of proteinuria.

Oxyphil Cell Function in Secondary Parathyroid Hyperplasia

Oxyphil cell function in secondary parathyroid hyperplasia due to chronic renal failure was evaluated using in situ hybridization and heterotransplantation of parathyroid tissue. In situ hybridization and histologic analysis were performed on continuous frozen sections using 22 parathyroid tissues. A restricted area composed exclusively of oxyphil cells was observed in 10 specimens, and an area of only chief cells was found in 12 specimens. Silver grains demonstrating the existence of parathyroid hormone (PTH) mRNA were 18.8 +/- 7.8 (mean +/- SD) in oxyphil cells while those in chief cells were 17.2 +/- 7.5. PTH mRNA was abundant in both the oxyphil and chief cells. Further analysis of oxyphil cell function was assessed by the heterotransplantation of parathyroid nodules, consisting exclusively of oxyphil or chief cells, into nude mice. The function of these implants was assessed by measuring the concentration of human intact PTH which did not cross-react with mouse PTH. Serum PTH concentrations were correlated with the volume of implanted tissue. Elevations of PTH concentrations were similar in the mice transplanted with oxyphil or chief cells, indicating that both cell types had similar PTH secretory activity. The basic histologic characteristics of both cell types were not altered following transplantation. These results demonstrate that oxyphil cells in secondary parathyroid hyperplasia synthesize and secrete PTH, and that this secretion contributes to the pathophysiology of hyperparathyroidism.

Glomerulointerstitial interaction of adhesion molecules in IgA nephropathy and membranoproliferative glomerulonephritis.

The expression of two adhesion molecules, ICAM1 (CD54) and ICAM3 (CD50), infiltrating cells positive for their ligand, LFA1 (CD11a), and the markers of total leukocytes (CD45), T cells (CD3), granulocytes/monocytes (CD15), and macrophages (CD68) in renal interstitium were examined by an indirect immunoperoxidase method. The study was longitudinally performed on repeat renal biopsy specimens from 69 patients with two different proliferative glomerulonephritides: 43 with IgA nephropathy (IgAN) and 26 with membranoproliferative glomerulonephritis (MPGN). Interstitial ICAM1 (iICAM1) was mainly expressed on endothelium of peritubular venules and sometimes on tubular epithelium, and interstitial ICAM3 (iICAM3) on infiltrating immune cells. In IgAN, iICAM1 was significantly correlated with glomerular infiltration of LFA1+ cells (gLFA1) and CD68+ cells (gCD68) (r = 0.478/0.500; P < 0.0001) as well as CD3+ cells (gCD3) (r = 0.402; P < 0.002). In MPGN, iICAM1 was significantly correlated only with gCD68 (r = 0.382; P < 0.05). In both diseases, iICAM1 and iICAM3 were significantly correlated with interstitial infiltration of LFA1+ cells (iLFA1) and CD68+ cells (iCD68) (r = 0.616 to 0.815; P < 0.0001) and with interstitial infiltration of CD3+ cells (iCD3) (r = 0.474 to 0.816; P < 0.01). The iICAM3 was also significantly correlated with interstitial CD45+ cells (iCD45) (r = 0.672 in IgAN and 0.769 in MPGN; P < 0.00001). Interstitial infiltration of these immune cells was significantly correlated with the histologic parameters indicating renal injury, such as the index of glomerular lesion and the percent interstitial volume (r = 0.410 to 692; P < 0.05). Longitudinal analysis revealed that the parameters described above showed corresponding change with each other at the follow-up biopsy. These findings suggest that the glomeruler infiltration of T cells and macrophages influences the ICAM1/ICAM3 expression of the interstitial cells, especially In IgAN, and that ICAM1/LFA1 and ICAM3/LFA1 interactions contribute to the persistent infiltration of the interstitium by immune cells in both diseases.