Kiyoko Inui

Heparan sulfate of perlecan is involved in glomerular filtration.

Perlecan is a heparan sulfate proteoglycan and a major component of the glomerular basement membrane. To understand the role of heparan sulfate chains of perlecan in glomerular filtration, detailed analyses were performed of the kidneys of Hspg2(Delta)(3/)(Delta)(3) mice, whose perlecan lacks heparan sulfate attachment sites in N-terminal domain I. Macroscopic, histologic, and electron microscopic observations, as well as immunohistochemical and immunoelectron microscopic analyses using specific antibodies against perlecan and agrin core proteins, revealed no significant abnormalities in these mice under physiologic conditions. Polyethyleneimine staining demonstrated no significant changes in charge density in the glomerular basement membrane. Transcripts of other heparan sulfate proteoglycans, agrin, and collagen type XVIII, as well as perlecan, were expressed at similar levels to those in the wild-type littermates. Approximately 40% of the perlecan synthesized by Hspg2(Delta)(3/)(Delta)(3) fibroblasts was substituted with heparin sulfate and 60% was substituted with chondroitin sulfate. All of the perlecan synthesized by wild-type fibroblasts contained heparin sulfate, indicating an altered substitution of glycosaminoglycans on Hspg2(Delta)(3/)(Delta)(3) perlecan. Immunostaining indicated that the level of chondroitin sulfate was actually increased in the Hspg2(Delta)(3/)(Delta)(3) glomerular basement membrane. When administered intraperitoneally with BSA, Hspg2(Delta)(3/)(Delta)(3) mice exhibited remarkable proteinuria. These findings suggest that heparan sulfate chains of perlecan play an important role in glomerular filtration, especially of a large amount of protein.

Expression of epidermal growth factor and its receptor in rabbits with ischaemic acute renal failure

Urinary immunoreactive epidermal growth factor (EGF) levels decrease, and renal immunoreactive EGF levels increase in rats with ischaemic acute renal failure (ARF). We investigated the immunohistochemical localization of EGF and EGF receptor in rabbits with ischaemic ARF to clarify the significance of renal EGF. Male New Zealand White rabbits underwent right nephrectomy prior to a 60 min renal artery clamp. At 3, 6, 24, 48, 72 and 96 h after ischaemia, serum urea nitrogen and serum creatinine were determined. Guinea pig anti-rabbit EGF antibody and monoclonal anti-EGF receptor antibody were used for the primary incubation. EGF was immunolocalized to the ascending limb of Henle and the distal convoluted tubule in the normal right kidneys. However, in the post ischaemic left kidneys at 6, 24, 48 and 72 h, immunoreactivity of EGF was associated with proximal tubules. In the normal kidneys, antibody to EGF receptor reacted with distal tubules and collecting ducts. In the ischaemic kidneys, EGF receptor was localized in the basolateral membrane in the proximal tubules. The expression of EGF and EGF receptor in renal tubules may play an important role in repair following ischaemic renal damage.

Mesangial Proliferative Nephritis in Man Is Associated with Increased Expression of the Cell Survival Factor, Bcl-2

Although most studies suggest that the hypercellularity in mesangial proliferative nephritis is due to increased cell proliferation, we hypothesized that it may also be due to increased expression of survival factors that may block their removal (apoptosis). We therefore studied the expression of apoptosis preventing/delaying the bcl-2 gene product in the glomerulus with various human glomerulonephritides. Immunohistochemistry for Bcl-2, proliferating cell associated protein (Ki-67) and α-smooth muscle actin (α-SMA) was performed on 55 biopsied kidney tissues: 6 cases of orthostatic proteinuria as a control (OP); 6 cases of diffuse proliferative lupus nephritis (WHO type IV, LN-MPGN); 24 cases of IgA nephropathy (IgA); 9 cases of minimal change nephrosis and 10 cases of idiopathic membranous nephropathy. The number of Ki-67-positive cells and the expression of α-SMA in the glomerulus were significantly higher in LN-MPGN and IgA. There was a significant positive correlation between glomerular Bcl-2 expression and glomerular cell proliferation evaluated by the number of Ki-67-positive cells (r = 0.605, p < 0.01) or glomerular α-SMA expression (r = 0.674, p < 0.01). Glomerular expression of Bcl-2 in IgA or LN-MPGN was significantly higher than that in OP (p < 0.01 and p < 0.05 vs. OP, respectively). The Bcl-2-positive cells were present in mesangial locations and demonstrated a perinuclear pattern. These results suggest that maintenance of glomerular hypercellularity in human glomerular diseases is partly due to the prevention of mesangial cell death via Bcl-2 expression.

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

SummaryThe ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously.This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.

Actin -related protein 3 (Arp3) is mutated in proteinuric BUF/Mna rats

The BUF/Mna strain of rat is a model of focal and segmental glomerulosclerosis (FSGS) in which a quantitative trait locus (QTL) for proteinuria, Pur1, has been identified. The aim of the present study was to identify candidates for the Pur1 gene. To narrow the Pur1 QTL, we performed fine QTL mapping and single nucleotide polymorphism (SNP) genotyping. To identify candidate genes, sequencing and gene-expression analyses of all genes contained in the narrowed locus were conducted. The narrowed Pur1 region contained 25 genes. Among these genes, only the Arp3 gene was mutated in the BUF/Mna strain; it contained a missense mutation that caused an L111F substitution. This leucine is conserved across species. Gene-expression analysis failed to identify any other candidate genes for Pur1. Arp3-mediated actin assembly abnormalities were visible in immunohistochemical and electron microscopic examinations of podocytes in old BUF/Mna rats. Taken together, these data suggest that Arp3 is a candidate for the Pur1 gene. This observation is consistent with our growing recognition that abnormal signaling-induced assembly of actin in podocytes leads to the development of FSGS.

Immunochemical Study of Epidermal Growth Factor in Rats with Mercuric Chloride-Induced Acute Renal Failure

Urine contains high concentrations of epidermal growth factor (EGF), and EGF is a potent growth promoter for proximal tubular cells. In the present study, urinary and whole-kidney EGF levels were investigated in rats with mercuric chloride (HgCl2)-induced acute renal failure (ARF) using a specific radioimmunoassay for rat EGF to clarify changes in EGF after toxic injury. Male Wistar rats were given HgCl2 (2 mg/kg, subcutaneously) or saline. Serum urea nitrogen and creatinine levels were measured for 4 days after toxin administration. Forty-eight hours after toxic injury, urinary immunoreactive EGF levels in HgCl2-treated rats decreased significantly compared with control rats (1.62 +/- 0.15 versus 3.78 +/- 0.21 ng/mg creatinine; p < 0.01). Urinary immunoreactive EGF was at its lowest level 96 h after toxic injury (0.64 +/- 0.06 ng/mg creatinine; p < 0.001). Twenty-four hours after toxic injury, renal immunoreactive EGF levels increased significantly compared with control rats (22.04 +/- 2.12 versus 4.84 +/- 0.70 ng/g wet weight tissue; p < 0.001), and the increase persisted for as long as 48 h (13.36 +/- 1.61 ng/g wet weight tissue; p < 0.05). In summary, urinary immunoreactive EGF levels decreased and renal EGF levels increased in rats with HgCl2-induced ARF. These findings suggest that there is an impairment in the excretion of EGF in rats with HgCl2-induced ARF and that local paracrine production of EGF continues in ARF.

Aldosterone mediates glomerular inflammation in experimental mesangial proliferative glomerulonephritis

BACKGROUND The renoprotection of the mineralocorticoid receptor antagonist (MRA) is considered to be mainly via its antifibrotic activity, and the possibility that it may also have antiinflammatory effects has not been studied. We tested the hypothesis that MRA might influence the inflammatory changes that accompany experimental glomerular injury. METHODS Administration of vehicle (control) or a selective MRA, eplerenone (50 mg/kg x 2 times/day) was started 7 days (-7d) before induction of anti-Thy-1.1 glomerulonephritis. Kidney samples were evaluated serially over a 12-day period for the presence of cell proliferation, macrophage infiltration, mesangial cell phenotypic activation and expression of the chemokine monocyte chemoattractant protein-1 (MCP-1). RESULTS MRA did not prevent the mesangiolysis associated with anti-Thy-1 antibody. However, MRA significantly inhibited MCP-1 expression, glomerular macrophage infiltration and mesangial phenotypic activation (alpha-smooth muscle actin expression). CONCLUSION MRA alters glomerular inflammation and mesangial cell activation in experimental glomerular injury. MRA may be a novel way to treat acute glomerular diseases.

Losartan preserves glomerular basement membrane anionic charge sites in a rat model of nephropathy.

BACKGROUND Disruption of the size and charge selectivity of the glomerular basement membrane (GBM) leads to proteinuria. Blood pressure medications suppress proteinuria by preserving GBM function. We investigated the mechanism of losartan, an angiotensin II receptor blocker (ARB), in a rat model of nephropathy. METHODS Male Wistar rats were given 25 mg/kg per day of losartan or the same volume of saline (control) from 5 days before, to 14 days after, induction of nephropathy by injection of puromycin aminonucleoside (PAN). Serum blood urea nitrogen (BUN) and creatinine, blood pressure, urinary protein, glomerular morphology and the number of GBM anionic sites were measured in the 2 groups on days 0, 7 and 14. RESULTS The losartan group had significantly lower urinary protein on days 7 and 14, and higher BUN on day 14, but there were no significant differences between the losartan and control groups in serum creatinine or blood pressure. Light microscopy indicated reduced mesangial cell proliferation and expansion of the mesangial area in the losartan group relative to controls. There were more GBM anionic sites in the losartan group on days 7 and 14. In addition, all anionic sites on the surface of foot processes of epithelial cells disappeared in the control group but remained in the losartan group. CONCLUSIONS A rat model of nephropathy indicates that losartan reduces urinary protein and preserves the number of anionic sites on the GBM, but has no apparent effect on hemodynamics.

A mutation in transcription factor MAFB causes Focal Segmental Glomerulosclerosis with Duane Retraction Syndrome

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease in children and adults. Genetic factors significantly contribute to early-onset FSGS, but the etiologies of most adult cases remain unknown. Genetic studies of monogenic syndromic FSGS exhibiting extra-renal manifestations have uncovered an unexpected biological role for genes in the development of both podocytes and other cellular lineages. To help define these roles, we studied two unrelated families with FSGS associated with Duane Retraction Syndrome, characterized by impaired horizontal eye movement due to cranial nerve malformation. All four affected individuals developed FSGS and Duane Retraction Syndrome in their first to second decade of life, manifested as restricted abduction together with globe retraction and narrowed palpebral fissure on attempted adduction. Hypoplasia of the abducens nerves and hearing impairment occurred in severely affected individuals. Genetic analyses revealed that affected individuals harbor a rare heterozygous substitution (p.Leu239Pro) in MAFB, a leucine zipper transcription factor. Luciferase assays with cultured monocytes indicated that the substitution significantly reduced transactivation of the F4/80 promoter, the known MAFB recognition element. Additionally, immunohistochemistry indicated reduced MAFB expression in the podocytes of patients. Structural modeling suggested that the p.Leu239Pro substitution in the DNA-binding domain possibly interferes with the stability of the adjacent zinc finger. Lastly, podocytes in neonatal mice with p.Leu239Pro displayed impaired differentiation. Thus, MAFB mutations impair development and/or maintenance of podocytes, abducens neurons and the inner ear. The interactions between MAFB and regulatory elements in these developing organs are likely highly specific based on spatiotemporal requirements.

Comparison of Renal Outcome among Japanese Patients with or without Microangiopathic Hemolysis in Malignant Phase Hypertension: A Single-Center Retrospective Study

Background/Aims: Although microangiopathic hemolysis (MAH) is a well-known complication of malignant phase hypertension (MPH), only less data on whether MAH in MPH predicts renal outcome exist. Therefore, we evaluated whether MAH was associated with the renal outcome in patients with MPH. Methods: We conducted a single-center, retrospective, cohort study. Data from 35 patients diagnosed with MPH between October 1998 and January 2015 were analyzed. MPH was defined as the presence of a diastolic blood pressure of ≥120 mm Hg and grades III/IV hypertensive retinopathy according to the Keith-Wagener-Barker classification. MAH was defined as the presence of a low platelet count (<150 × 109/L) together with either an elevated level of lactate dehydrogenase (LDH; >220 U/L), or the presence of schistocytes, or both and the normalization of platelet and LDH level or schistocyte levels after adequate blood pressure control was achieved. The primary outcome was dialysis induction. Results: Fifteen patients had MAH. Those with MAH had significantly severe renal dysfunction at the onset of MPH. The length of follow-up (median, interquartile range) of patients with MAH and those without MAH were 30 (16-94) and 48 (25-115) months, respectively. Dialysis was induced in 9 of 15 patients with MAH and in 6 of 20 patients without MAH. Renal survival in patients with MAH was worse than that in those without, but this was not statistically significant (p = 0.08). By multivariate Cox regression analysis, MAH was not shown to contribute to dialysis induction. Conclusion: MAH did not predict renal outcome in MPH.