ROS-ERK Pathway as Dual Mediators of Cellular Injury and Autophagy-Associated Adaptive Response in Urinary Protein-Irritated Renal Tubular Epithelial Cells.

ERK, an extracellular signal-regulated protein kinase, is involved in various biological responses, such as cell proliferation and differentiation, cell morphology maintenance, cytoskeletal construction, apoptosis, and canceration of cells. In this study, we focused on ERK pathway on cellular injury and autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells and explored the potential mechanisms underlying it. By using antioxidants N-acetylcysteine and catalase, we found that ERK pathway was activated by a reactive oxygen species- (ROS-) dependent mechanism after exposure to urinary proteins. What is more, ERK inhibitor U0126 could decrease the release of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and the number of apoptotic cells induced by urinary proteins, indicating the damaging effects of ERK pathway in mediating cellular injury and apoptosis in HK-2 cells. Interestingly, we also found that the increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II (a key marker of autophagy) and the decreased expression of p62 (autophagic substrate) induced by urinary proteins were reversed by U0126, suggesting autophagy was activated by ERK pathway. Furthermore, rapamycin reduced urinary protein-induced NGAL and KIM-1 secretion and cell growth inhibition, while chloroquine played the opposite effect, indicating that autophagy activation by ERK pathway was an adaptive response in the exposure to urinary proteins. Taken together, our results indicate that activated ROS-ERK pathway can induce cellular injury and in the meantime provide an autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells.

USP40 gene knockdown disrupts glomerular permeability in zebrafish.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

Expression patterns of RelA and c-mip are associated with different glomerular diseases following anti-VEGF therapy.

Renal toxicity constitutes a dose-limiting side effect of anticancer therapies targeting vascular endothelial growth factor (VEGF). In order to study this further, we followed up 29 patients receiving this treatment, who experienced proteinuria, hypertension, and/or renal insufficiency. Eight developed minimal change nephropathy/focal segmental glomerulopathy (MCN/FSG)-like lesions and 13 developed thrombotic microangiopathy (TMA). Patients receiving receptor tyrosine kinase inhibitors (RTKIs) mainly developed MCN/FSG-like lesions, whereas TMA complicated anti-VEGF therapy. There were no mutations in factor H, factor I, or membrane cofactor protein of the complement alternative pathway, while plasma ADAMTS13 activity persisted and anti-ADAMTS13 antibodies were undetectable in patients with TMA. Glomerular VEGF expression was undetectable in TMA and decreased in MCN/FSG. Glomeruli from patients with TMA displayed a high abundance of RelA in endothelial cells and in the podocyte nuclei, but c-mip was not detected. Conversely, MCN/FSG-like lesions exhibited a high abundance of c-mip, whereas RelA was scarcely detected. RelA binds in vivo to the c-mip promoter and prevents its transcriptional activation, whereas RelA knockdown releases c-mip activation. The RTKI sorafenib inhibited RelA activity, which then promoted c-mip expression. Thus, our results suggest that c-mip and RelA define two distinct types of renal damage associated with VEGF-targeted therapies.

Selective albuminuria via podocyte albumin transport in puromycin nephrotic rats is attenuated by an inhibitor of NADPH oxidase.

The mechanism of selective albuminuria in minimal change nephrotic syndrome, in which glomerular capillaries are diffusely covered by effaced podocyte foot processes with reduced slit diaphragms, is unknown. Podocyte injury is due, in part, to NADPH-induced oxidative stress. Here we studied mechanism of selective albuminuria in puromycin aminonucleoside (PAN) nephrotic rats, a model of minimal change nephrotic syndrome. In these rats, Evans Blue-labeled human albumin was taken up by podocytes and its urinary excretion markedly increased, with retained selectivity for albumin. Immunogold scanning electron micrographic images found increased human albumin in podocyte vesicles and on the apical membrane in nephrotic compared with control rats. Apocynin, an inhibitor of NADPH oxidase, decreased superoxide production in podocytes, and inhibited endocytosis and urinary albumin excretion. Real-time confocal microscopy found an initial delay in the appearance of Evans Blue-labeled human albumin in the tubular lumen, reflecting the time needed for transcellular transport. Immunoprecipitation analysis indicated that FcRn, a receptor for albumin transport, mediated podocyte albumin transport, and treatment with anti-FcRn antibody reduced proteinuria in these nephrotic rats. Thus, podocyte albumin transport was enhanced in PAN nephrotic rats by means of FcRn, which may explain the mechanism of selective proteinuria. This was blocked by apocynin, suggesting a new therapeutic approach.

Nitric oxide synthase gene polymorphisms in children with minimal change nephrotic syndrome.

AIMS: Nitric oxide (NO) attenuates many functions within the kidney, and all NO synthase (NOS) isoforms are constitutively expressed in the kidney. But the exact role of NO in renal diseases is still debatable. The aim of the present study was to investigate endothelial (eNOS), and neuronal (nNOS) NOS gene polymorphisms in children with minimal change nephrotic syndrome (MCNS). MATERIALS AND METHODS: Eighty-six Turkish children with clinical MCNS, ranging in age from 2 to 10 years, were compared with 114 healthy age- and sex-matched controls. The glu 298 Asp (G/T) polymorphism of the eNOS, and C276T (C/T) polymorphism of nNOS genes were genotyped using polymerase chain reaction. RESULTS: The distribution of GG, TG, and TT genotypes for eNOS was 52%, 33% and 15% in MCNS compared with 61%, 26% and 13% in the controls (P > 0.05). The distribution of CC, TC, and TT genotypes for nNOS was 16%, 66% and 18% in MCNS compared with 10%, 43% and 47% in the controls. TT genotype distribution of nNOS was found to be lower in patients (P = 0.003). The eNOS and nNOS gene polymorphisms were not associated with gender, positive family history, frequency of relapses, or response to steroid. CONCLUSIONS: The present study is the first to investigate eNOS and nNOS gene polymorphisms in children with MCNS. The nNOS gene polymorphism may be associated with MCNS in children, but further studies in a larger population with different glomerular diseases are needed to confirm the results.

ACE gene polymorphism in children with nephrotic syndrome in the Indonesian population.

BACKGROUND: The angiotensin converting enzyme (ACE) gene carries insertion (I) and deletion (D) polymorphism within its intron 16. The presence of D-allele in the ACE gene has been reported as a probable genetic risk factor for idiopathic nephrotic syndrome (INS), especially the subtype of focal segmental glomerulosclerosis (FSGS). The D-allele may be related to poor responsiveness to steroid therapy. To clarify the relationship between the D-allele and INS, we studied the prevalence of the D-allele in the Javanese-Indonesian patients. Additionally, we also analyzed relationship between each genotype and steroid sensitivity among the MCNS patients. METHODS: Eighty-five Javanese-Indonesian patients under 15 years of age with INS were enrolled in this study: 16 patients with FSGS and 69 patients with minimal change nephrotic syndrome (MCNS). As controls, 68 healthy adult Javanese-Indonesians with no history of kidney disease volunteered to participate in this study. Genotypes based on the polymorphisms (I/D) were determined by using a PCR method. As for the steroid responsiveness, the information of 14 out of 16 FSGS patient (87.5%) and 69 out of 69 MCNS patients (100%) was available. RESULTS: The genotype frequencies in the FSGS patients were II 37% (6/16), ID 44% (7/16) and DD 19% (3/16), and the D-allele frequency was 41% (13/32). The genotype frequencies in the MCNS patients were II 56% (39/69), ID 38% (26/69) and DD 6% (4/69), and the D-allele frequency was 25% (34/138). The genotype frequencies in the controls were II 60% (41/68), ID 31% (21/68), and DD 9% (6/68), and the D-allele frequency was 26% (33/136). None of the FSGS patients were sensitive to steroid, while almost all MCNS patients (66/69) were sensitive to steroid. The genotype frequencies among steroid-sensitive MCNS patients were consistent with those of the controls, suggesting that there was no relationship between each genotype and steroid sensitivity. CONCLUSIONS: In the Javanese-Indonesian population, none of the comparisons showed any significant differences in the genotypic distribution and allelic frequencies among the three groups, FSGS, MCNS and controls, although D-allele tended to exist more frequently in FSGS patients than in the MCNS patients and controls. In addition, the D-allele frequency was not related to steroid sensitivity in the MCNS patients.

Variants of alpha 1-proteinase inhibitor in black and white South African patients with focal glomerulosclerosis and minimal change nephrotic syndrome.

OBJECTIVE: To determine the prevalence and biochemical characteristics of certain alleles of alpha 1-proteinase inhibitor in black and white South African patients with two common types of pathology causing the nephrotic syndrome. DESIGN: A cross sectional study of black and white patients with focal glomerulosclerosis (FGS) or minimal change disease (MCNS) and black and white controls. SETTING: The patients were drawn from the Paediatric Nephrology Units at the Johannesburg and Baragwanath Hospitals and the controls were drawn from the South African Blood Transfusion Service and the Paediatric Nephrology Clinic in Johannesburg. RESULTS: There was a significant increase in the prevalence of the V allele in black patients with FGS (12%) as compared to black controls (1%) (p = 0.01). None of the white patients with FGS had the V allele but two out the five coloured (mixed race) patients had the V allele (20%). An increase in the prevalence of the S allele of alpha 1PI was found in white patients with FGS and MCNS (10%) as compared to white controls (2%). The plasma elastase inhibitory capacity (EIC) associated with the phenotypes (PI) M1 (Ala213)S, M1 (Ala213) V, and M1 (Ala213) M1 (Ala213) was significantly decreased as compared to the EIC associated with PI M1 (Val213) M1 (Val213) (p = 0.006, p = 0.004, and p = 0.025, respectively). Twelve of 13 patients with FGS and infected with tuberculosis had either the M1 (Ala213) V or F alleles and required transplantation owing to the severity of the disease. All of these patients were either black or coloured. However, eight of 12 patients with FGS who had the M1 (Ala213) V or S alleles but were PPD negative did not require transplantation. CONCLUSION: It is possible that the combination of functionally less efficient alpha 1PI and an inflammatory challenge associated with an infection such as tuberculosis could predispose black and coloured nephrotic patients to more aggressive scarring in FGS.

Lymphocyte ectoenzymes in childhood idiopathic nephrotic syndrome.

Lymphocyte ectoenzymes with immunomodulatory function were investigated in 11 children with minimal change disease (MCD), 9 with primary focal segmental glomerulosclerosis (FSGS), and 17 age- and sex-matched healthy children. Basal, concanavalin A (Con A)-, and pokeweed mitogen (PWM)-stimulated lymphocyte ecto-5'-nucleotidase (5'-Nu), dipeptidyl peptidase IV (DPP IV), and alkaline phosphodiesterase I (APD) activities were determined. In MCD relapse ecto-APD activity of unstimulated lymphocytes was higher than controls. Ecto-APD of Con A-stimulated lymphocytes was below controls (23.0, range 7.2-48.7 nmol/min per 10(6) lymphocytes) in all active MCD (18.7, range 7.6-32.6), during corticosteroid treatment (14.6, range 4.5-54), and in remission (13.1, range 6.1-19.6), but was significant only in remission. Con A-stimulated DPP IV was significantly lower from controls (53.8, range 19.3-85.7 nmol/min per 10(6) lymphocytes) in all active MCD (38.1, range 10.8-82.1), during treatment (37.5, range 20.2-58.7), and in remission (39.4, range 24.3-69.6). In FSGS, unstimulated lymphocyte ecto-APD activity was greater than controls. However, Con A-stimulated lymphocyte ecto-APD and DPP IV activities were not significantly different from controls. Con A stimulation of lymphocyte ecto-APD and DPP IV activity was significantly reduced in MCD relapse and in remission, but not in FSGS. Basal, Con A-, and PWM-stimulated ecto-5'-Nu in MCD and FSGS were not different from controls. These results suggest a role for abnormal T cell function in MCD but not in FSGS. The difference in mitogen-stimulated expression of these ectoenzymes suggests a different pathogenesis of childhood MCD and primary FSGS.

Clearance ratios of amylase isoenzymes and IgG subclasses: do they reflect glomerular charge selectivity?

The clearance ratios of endogenous plasma proteins with the same size but a different charge, such as the amylase isoenzymes and the immunoglobulin (Ig) G subclasses, have been used to assess glomerular charge selectivity in man. These proteins are, however, subject to tubular reabsorption. In this study we measured the IgG subclass/IgG clearance ratios for IgG1 (pI 8.0-9.5), IgG2 (pI 7.0-7.5) and IgG4 (pI < 6) in 6 healthy volunteers. Our results suggested a selective influence of tubular reabsorption: the IgG1/IgG clearance ratio was 0.68 +/- 0.14 (mean +/- SD) and lower than IgG2/IgG (2.02 +/- 1.06, p < or = 0.01). IgG4/IgG was 0.89 +/- 0.39. In addition, we studied the clearance ratios of pancreatic (PA, pI 7.0) and salivary amylase (SA, pI 5.9-6.4) and of IgG1 and IgG2 in 8 patients with minimal change nephrotic syndrome (MCNS), 11 patients recovering from acute tubular necrosis (ATN) and 9 healthy volunteers (controls). In MCNS glomerular charge selectivity is lost, while in recovering ATN tubular function is severely disturbed. The PA/SA clearance ratio was 3.25 +/- 0.89 in controls, reflecting intact glomerular charge selectivity. In MCNS patients the PA/SA clearance ratio had decreased to 1.21 +/- 0.23 (p < or = 0.001). In ATN patients the PA/SA clearance ratio was reduced as well: 1.55 +/- 0.41 (p < or = 0.001), although the aselective nature of the proteinuria and the modest albuminuria indicated intact glomerular charge selectivity. The IgG1/ IgG2 clearance ratio was 0.54 +/- 0.15 in controls, again suggesting preferential tubular reabsorption of IgG1. In MCNS patients the IgG1/IgG2 clearance ratio was 0.16 +/- 0.10 (p < or = 0.001); this probably reflects the relatively increased glomerular sieving of IgG2 when glomerular charge selectivity is lost. In ATN patients the IgG1/IgG2 clearance ratio was 1.07 +/- 0.47 (p < or = 0.001), which suggests a partial loss of preferential reabsorption of IgG1. It was concluded that the PA/SA clearance ratio is influenced by loss of tubular function and therefore does not reflect glomerular charge selectivity specifically. The IgG1/IgG2 ratio cannot be used to assess glomerular charge selectivity either because of the interference of selective tubular reabsorption of the subclasses. These findings put the assessment of glomerular charge using endogenous proteins in a new light and bring forward the necessity to interpret these ratios with the utmost cautiousness.

Increased mRNA expression of metalloproteinase-9 in peripheral blood monocytes from patients with immunoglobulin A nephropathy.

We examined metalloproteinase (MMP)-1, -2, -3, and -9 mRNA expression by peripheral blood monocytes from 50 patients with immunoglobulin A (IgA) nephropathy, 20 with membranous nephropathy, 10 with minimal-change nephrotic syndrome, five with focal glomerulosclerosis, 30 with non-IgA proliferative glomerulonephritis, and 40 healthy normal controls who were comparable with regard to age and sex. Monocytes from patients with IgA nephropathy expressed a higher level of MMP-9 mRNA than those from patients with other forms of glomerulonephritis or from healthy controls (MMP-9 to glyceraldehyde-3-phosphate dehydrogenase ratio: IgA nephropathy, 1.68 +/- 0.24; membranous nephropathy, 0.22 +/- 0.08; minimal-change nephrotic syndrome, 0.24 +/- 0.06; focal glomerulosclerosis, 0.32 +/- 0.08; non-IgA proliferative glomerulonephritis, 0.30 +/- 0.12; and healthy controls, 0.16 +/- 0.04). When the biopsy specimens were classified into four grades according to the severity of glomerular and interstitial pathology, highly significant differences were observed among MMP-9 mRNA levels in monocytes from all four groups of patients with IgA nephropathy (grade I, 0.44 +/- 0.09; grade II, 1.06 +/- 0.26; grade III, 2.22 +/- 0.68; grade IV, 2.86 +/- 0.88). In addition, MMP-9 mRNA levels from patients with IgA nephropathy correlated with urinary protein excretion (P < 0.001). However, we detected minimal mRNA expression of MMP-1, -2, and -3 by peripheral blood monocytes from patients with IgA nephropathy or other forms of glomerulonephritis and from normal healthy controls. Our results suggest that increased MMP-9 mRNA expression in circulating monocytes may contribute to the progression of IgA nephropathy.

[Study of serum levels of alpha 1-antitrypsin and alpha macroglobulins in children with glomerulonephritis]. / Issledovanie alpha 1-ingibitora proteinaz i alpha 2-makroglobulina v syvorotke krovi pri glomerulonefrite u detei.

To define the clinico-pathogenetic importance of alpha 1-inhibitor of proteinases and alpha 2-macroglobulin of the blood in children with glomerulonephritis, a study was made of the phenotype of alpha 1-inhibitor of proteinases and its concentration in the blood serum of 156 patients with different clinical forms of glomerulonephritis. Overall 1290 practically healthy children were examined as control. The patients suffering from glomerulonephritis did not demonstrate phenotypes responsible for acute deficiency of alpha 1-inhibitor of proteinases (PISS, PISZ). A relationship was established between the amount of alpha 1-inhibitor of proteinases in the blood serum in children with different clinical forms of glomerulonephritis: the patients with the nephrotic form manifested a significant decrease of the inhibitor concentration in the blood serum, whereas in the hematuric form, a significant rise of it was recorded. All the patients suffering from glomerulonephritis showed a significant increase of the content of alpha 2-macroglobulin, particularly in the nephrotic form, which is likely to be determined by the enhanced output of the given protein and its negligible loss with urine in connection with a high molecular weight.