Glomerular morphometry in biopsy evaluation of minimal change disease, membranous glomerulonephritis, thin basement membrane disease and Alport's syndrome

OBJECTIVE: To assess the role of glomerular morphometry in biopsy evaluation in renal disorders in addition to conventional diagnostic procedures. STUDY DESIGN: The study includes 10 cases each of minimal change disease (MCD), idiopathic membranous glomerulonephritis (idiopathic MGN), thin basement membrane disease (TBMD) and Alport's syndrome. Renal biopsies for normal study were obtained from age- and sex-matched autopsy cases without any renal disorder, confirmed histologically and ultrastructurally. Glomerular morphometry was performed by semiautomatic procedure using Quantimet-600 image analysis system (Leica, Cambridge, United Kingdom). RESULTS: Morphometric findings revealed significant increase in glomerular "diameter and area" and "tuft diameter and area" in patients of idiopathic MGN, but no significant difference was found in patients of MCD, TBMD and Alport's syndrome. Evaluation of glomerular volume fractions revealed a decrease in capillary space volume fraction and an increase in "membranes and mesangial matrix" volume fraction in patients with idiopathic MGN. Significant decrease in capillary space volume fraction was also observed in patients of MCD. Patients with Alport's syndrome showed variable changes. CONCLUSION: Glomerular morphometry could be considered as an adjunct to the diagnostic armamentarium of light microscopy, immunofluorescence and electron microscopy because it provides deep insight into quantitative parameters.     

Glomerular extracellular matrices and anionic sites in aging ddY mice: a morphometric study

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.     

A model of strain-dependent glomerular basement membrane maintenance and its potential ramifications in health and disease

A model is developed and analyzed for type IV collagen turnover in the kidney glomerular basement membrane (GBM), which is the primary structural element in the glomerular capillary wall. The model incorporates strain dependence in both deposition and removal of the GBM, leading to an equilibrium tissue strain at which deposition and removal are balanced. The GBM thickening decreases tissue strain per unit of transcapillary pressure drop according to the law of Laplace, but increases the transcapillary pressure drop required to maintain glomerular filtration. The model results are in agreement with the observed GBM alterations in Alport syndrome and thin basement membrane disease, and the model-predicted linear relation between the inverse capillary radius and inverse capillary thickness at equilibrium is consistent with published data on different mammals. In addition, the model predicts a minimum achievable strain in the GBM based on the geometry, properties, and mechanical environment; that is, an infinitely thick GBM would still experience a finite strain. Although the model assumptions would be invalid for an extremely thick GBM, the minimum achievable strain could be significant in diseases, such as Alport syndrome, characterized by focal GBM thickening. Finally, an examination of reasonable values for the model parameters suggests that the oncotic pressure drop-the osmotic pressure difference between the plasma and the filtrate due to large molecules-plays an important role in setting the GBM strain and, thus, leakage of protein into the urine may be protective against some GBM damage.     

Estimating physical splenic filtration of Plasmodium falciparum‐infected red blood cells in malaria patients

Splenic filtration of Plasmodium falciparum‐infected red blood cells has been hypothesized to influence malaria pathogenesis. We have developed a minimum cylindrical diameter (MCD) filtration model which estimates physical splenic filtration during malaria infection. The key parameter in the model is the MCD, the smallest tube or cylinder that a red blood cell (RBC) can traverse without lysing. The MCD is defined by a relationship between the RBC surface area and volume. In the MCD filtration model, the MCD filtration function represents the probability of a cell becoming physically removed from circulation. This modelling approach was implemented at a field site in Blantyre, Malawi. We analysed peripheral blood samples from 120 study participants in four clinically defined groups (30 subjects each): cerebral malaria, uncomplicated malaria, aparasitaemic coma and healthy controls. We found statistically significant differences in the surface area and volumes of uninfected RBCs when healthy controls were compared with malaria patients. The estimated filtration rates generated by the MCD model corresponded to previous observations in ex vivo spleen experiments and models of red blood cell loss during acute malaria anaemia.There were no differences in the estimated splenic filtration rates between cerebral malaria and uncomplicated malaria patients. The MCD filtration model estimates that at time of admission, one ring‐stage infected RBC is physically filtered by the spleen for each parasite that remains in peripheral circulation. This field study is the first to use microfluidic devices to identify rheological diversity in RBC populations associated with malaria infection and illness in well‐characterized groups of children living in a malaria endemic area.     

Thickness of renal glomerular capillary basement membrane in the offspring of diabetic rats fed a regular or high-sucrose diet

Numerous animal model studies of diabetes mellitus have been reported. Diabetes-induced vascular damage is a common cause of systemic organ damage in humans and animals. Many investigations have been made of human and animal offspring of diabetic mothers. The present report documents the sequential glomerular basement membrane (GBM) thickness in fetuses and infants of diabetic rats. The postnatal increase in GBM thickness was similar in the offspring of control and diabetic rats, and was not related to the sucrose concentration in the diet.     

A proton nuclear magnetic resonance-based metabolomic approach in IgA nephropathy urinary profiles

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. Both one-dimensional NOESY and transverse-relaxation filter CPMG NMR spectra were recorded to investigate the urine metabolome of 24 IgAN patients and to detect altered metabolic profiles in comparison with 68 healthy matched controls. The spectral data were analyzed using multivariate statistical techniques. The analysis revealed that the NMR spectra of IgAN patients were statistically different from those of the controls (P = 4 × 10^?7 for 1D-NOESY and P = 2 × 10^?7 for CPMG). The robustness of the determined statistical model was confirmed by its predictive performance (for the 1D-NOESY dataset: sensitivity = 67 %, specificity = 95 %; for the CPMG dataset sensitivity = 60 %, specificity = 94 %). For the first time we found metabolites, including betaine and citrate, that are differentially modulated in IgAN patients compared to controls and that may be directly involved in the pathogenesis of IgAN. These metabolites may influence, directly or indirectly, the TNF-α, a regulating factor of the Th1/Th2 cell balance that is relevant in the pathology. The involvement of metabolites such as betaine and citrate in TNF-α regulation supports the power of the identified metabolic profiles to discern IgAN from controls.     

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.     

Chemical composition and solubility of human glomerular and tubular basement membranes of adult and senescent men

The effect of aging on the composition of human renal basement membranes was studied in persons aged 22-90 years. The relative proportion of cortex in kidney appears to decrease with aging. Twenty pairs of GBM and TBM preparations were isolated using the detergent method. Protein content of the basement membrane preparations amounts to about 66% and is independent of type of membrane or age. Amino acid and carbohydrate analyses of both GBM and TBM revealed that the extents of hydroxylation of proline at the C4 position and of lysine decrease with aging. In the case of lysine this occurred from the seventh of life onwards. The decreases are probably not caused by a change of the collagen content. The extent of glycosylation of hydroxylysine is similar for all basement membrane preparations. An adapted protein assay is presented for solutions containing SDS and dithiothreitol. In the presence of these two compounds, solubility of GBM and TBM from adult and aged persons is similar and amounts to about 55 and 85% after 10 and 60 min, respectively, of heating at 95 degrees C. In SDS-polyacrylamide gels, no differences were observed for the major peptide bands between the preparations, irrespective of basement membrane type or age.     

Ultrastructural analyses of acellular glomerular basement membranes and mesangial matrix in a spontaneously diabetic rhesus monkey

Renal changes similar to those considered diagnostic for diabetes in humans are infrequently observed in spontaneous noninsulin-dependent (NID) diabetic monkeys. In the current study, renal cortical tissue blocks are rendered acellular to demonstrate glomerular basement membrane (GBM) and mesangial matrix (MM) changes in a naturally occurring NID diabetic rhesus monkey (Macaca mulatta). Transmission electron micrographs of these specimens show axial MM accumulations with numerous striated collagen fibrils that frequently extend onto internal (endothelial) surfaces of peripheral GBM. The outer (epithelial) component, although compact, appears bilaminar due to folded external surfaces not coinciding with similar irregularities on internal surfaces. By scanning electron microscopy, external surfaces of sclerotic GBMs are extensively wrinkled and, following cryofracture, show congestion and expanded MM. A fenestrated meshwork of MM, which appears less dense and compact than epithelial BM, extends from axial regions onto GBM internal surfaces. The true thickness of randomly sampled peripheral GBM thickness (approximately 400 nm) is approximately double that of normal rhesus GBM. Diabetic GBMs exhibit ruthenium red positivity for surface polyanions with linear site densities not significantly different from normal. These observations indicate that sclerotic GBMs in diabetic rhesus monkeys closely resemble those seen in human end-stage diabetic glomerulopathy and suggest that this nonhuman primate may offer an excellent model for studies of chronic diabetic BM disease.     

Periodontal Disease Bacteria Specific to Tonsil in IgA Nephropathy Patients Predicts the Remission by the Treatment

Background

Immunoglobulin (Ig)A nephropathy (IgAN) is the most common form of primary glomerulonephritis in the world. Some bacteria were reported to be the candidate of the antigen or the pathogenesis of IgAN, but systematic analysis of bacterial flora in tonsil with IgAN has not been reported. Moreover, these bacteria specific to IgAN might be candidate for the indicator which can predict the remission of IgAN treated by the combination of tonsillectomy and steroid pulse.

Methods and Findings

We made a comprehensive analysis of tonsil flora in 68 IgAN patients and 28 control patients using Denaturing gradient gel electrophoresis methods. We also analyzed the relationship between several bacteria specific to the IgAN and the prognosis of the IgAN. Treponema sp. were identified in 24% IgAN patients, while in 7% control patients (P = 0.062). Haemophilus segnis were detected in 53% IgAN patients, while in 25% control patients (P = 0.012). Campylobacter rectus were identified in 49% IgAN patients, while in 14% control patients (P = 0.002). Multiple Cox proportional-hazards model revealed that Treponema sp. or Campylobactor rectus are significant for the remission of proteinuria (Hazard ratio 2.35, p = 0.019). There was significant difference in remission rates between IgAN patients with Treponema sp. and those without the bacterium (p = 0.046), and in remission rates between IgAN patients with Campylobacter rectus and those without the bacterium (p = 0.037) by Kaplan-Meier analysis. Those bacteria are well known to be related with the periodontal disease. Periodontal bacteria has known to cause immune reaction and many diseases, and also might cause IgA nephropathy.

Conclusion

This insight into IgAN might be useful for diagnosis of the IgAN patients and the decision of treatment of IgAN.     

Investigation of serum proteome alterations in human glioblastoma multiforme

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. The present study was conducted to investigate the alterations in the serum proteome in GBM patients compared to healthy controls. Comparative proteomic analysis was performed employing classical 2DE and 2D‐DIGE combined with MALDI TOF/TOF MS and results were further validated through Western blotting and immunoturbidimetric assay. Comparison of the serum proteome of GBM and healthy subjects revealed 55 differentially expressed and statistically significant (p <0.05) protein spots. Among the identified proteins, haptoglobin, plasminogen precursor, apolipoprotein A‐1 and M, and transthyretin are very significant due to their functional consequences in glioma tumor growth and migration, and could further be studied as glioma biomarkers and grade‐specific protein signatures. Analysis of the lipoprotein pattern indicated elevated serum levels of cholesterol, triacylglycerol, and low‐density lipoproteins in GBM patients. Functional pathway analysis was performed using multiple software including ingenuity pathway analysis (IPA), protein analysis through evolutionary relationships (PANTHER), database for annotation, visualization and integrated discovery (DAVID), and GeneSpring to investigate the biological context of the identified proteins, which revealed the association of candidate proteins in a few essential physiological pathways such as intrinsic prothrombin activation pathway, plasminogen activating cascade, coagulation system, glioma invasiveness signaling, and PI3K signaling in B lymphocytes. A subset of the differentially expressed proteins was applied to build statistical sample class prediction models for discrimination of GBM patients and healthy controls employing partial least squares discriminant analysis (PLS‐DA) and other machine learning methods such as support vector machine (SVM), Decision Tree and Naïve Bayes, and excellent discrimination between GBM and control groups was accomplished.     

Moderate/subclinical calcium deficiency attenuates trabecular mass,microarchitecture and bone growth in growing rats

Adequate dietary calcium (Ca) intake is essential for bone accretion, peak bone mass (PBM) attainment, bone quality and strength during the mammalian growth period. Severe Ca deficiency during growing age results in secondary hyperparathyroidism (SHPT) and poor bone quality and strength. However, the impact of moderate Ca deficiency during rats early growth period on bone health and the reversibility with supplementing calcium later in adult life remains unclear. Female Sprague-Dawley (SD) rats (postnatal 28th day, P28) were initiated either with a moderate calcium-deficient diet (MCD, 0.25% w/w Ca) or a control diet (0.8% w/w Ca, control group) till P70. Thereafter, MCD rats were continued either with MCD diet or supplemented with calcium diet (0.8% w/w Ca, calcium supplemented group, CaS) till P150. Another group (control rats) were fed 0.8% w/w Ca containing diet from P28 till P150.MCD group, as compared to the control group, had significantly reduced serum ionized Ca and procollagen type 1 N-terminal propeptide (P1NP) at P70 while no significant change was observed in serum corrected Ca, inorganic phosphate (P), alkaline phosphatase (ALP), 25-hydroxy vitamin D [25(OH)D], intact parathyroid hormone (iPTH), and urinary C-terminal telopeptide of collagen 1 (CTX-1), Ca, and P. Femoral and tibial metaphysis in MCD rats had significantly reduced linear growth, cortical and trabecular volumetric BMD (vBMD), trabecular microarchitecture (BV/TV%, trabecular thickness, separation and number, structural model index and connectivity density), cortical thickness, and bone stiffness despite the absence of secondary hyperparathyroidism (SHPT). Continued MCD at P70–P150 results in persistence of compromised bone strength while calcium supplementation (CaS group) improved all the parameters related to bone strength and microarchitecture. Our results indicate that uncorrected moderate/subclinical calcium deficiency in growing rats can result in poor bone quality and strength despite the absence of SHPT. This finding could have relevance in children with poor calcium intake in childhood and adolescence.     

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4–5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.     

Molecular Predictors of Long-Term Survival in Glioblastoma Multiforme Patients

Glioblastoma multiforme (GBM) is the most common and aggressive adult primary brain cancer, with <10% of patients surviving for more than 3 years. Demographic and clinical factors (e.g. age) and individual molecular biomarkers have been associated with prolonged survival in GBM patients. However, comprehensive systems-level analyses of molecular profiles associated with long-term survival (LTS) in GBM patients are still lacking. We present an integrative study of molecular data and clinical variables in these long-term survivors (LTSs, patients surviving >3 years) to identify biomarkers associated with prolonged survival, and to assess the possible similarity of molecular characteristics between LGG and LTS GBM. We analyzed the relationship between multivariable molecular data and LTS in GBM patients from the Cancer Genome Atlas (TCGA), including germline and somatic point mutation, gene expression, DNA methylation, copy number variation (CNV) and microRNA (miRNA) expression using logistic regression models. The molecular relationship between GBM LTS and LGG tumors was examined through cluster analysis. We identified 13, 94, 43, 29, and 1 significant predictors of LTS using Lasso logistic regression from the somatic point mutation, gene expression, DNA methylation, CNV, and miRNA expression data sets, respectively. Individually, DNA methylation provided the best prediction performance (AUC = 0.84). Combining multiple classes of molecular data into joint regression models did not improve prediction accuracy, but did identify additional genes that were not significantly predictive in individual models. PCA and clustering analyses showed that GBM LTS typically had gene expression profiles similar to non-LTS GBM. Furthermore, cluster analysis did not identify a close affinity between LTS GBM and LGG, nor did we find a significant association between LTS and secondary GBM. The absence of unique LTS profiles and the lack of similarity between LTS GBM and LGG, indicates that there are multiple genetic and epigenetic pathways to LTS in GBM patients.     

Assessment of genetic markers and glioblastoma stem-like cells in activation of dendritic cells

Glioblastoma (GBM) is the most common and aggressive intraparenchymal primary brain tumor in adults. The principal reasons for the poor outcomes of GBM are the high rates of recurrence and resistance to chemotherapy. The aim of this study was to determine the role of tailored cellular therapy for GBM with a poor prognosis and compare the activity of dendritic cells (DCs) that have encountered GBM cells. Detecting the correlations between methylation and expression of MGMT and PTEN genes and GBM cancer stem cells (CSCs) markers after co-cultures with a mononuclear cell cocktail are also aims for this study. Allogenic umbilical cord blood (UCB)-derived DCs were labeled with the CD11a and CD123 for immature DCs, and CD80 and CD11c for mature DCs. CD34, CD45, and CD56 cells were isolated from allogenic UCB for using in DCs maturation. GBM CSCs were detected with CD133/1 and CD111 antibodies after co-culture studies. DC activation was carried out via GBM cells including CD133 and CD111 cells and a mononuclear cells cocktail including CD34, CD45, and CD56 natural killer cells. Real-time PCR was performed to detect the expression and promoter methylation status of PTEN and MGMT genes. The expression of CSCs markers was found in all GBM cases, and a statistically significant correlation was found among them after co-culture studies. The most pronounced affinity of DCs to GBM cells was observed at dilutions between 1/4 and 1/256 in co-cultures. There was a statistically significant correlation between cellularity and granularity ratios for CD123 and CD11c. PTEN and MGMT gene expression and methylation values were evaluated with respect to CSCs expression and no statistical significance was found. Activation of DCs might associate with CSCs and the mononuclear cells cocktail including CD34, CD45, and CD56 cells which were obtained from allogenic UCB.     

Study of temporomandibular joint disorder in older patients by magnetic resonance imaging

doi: 10.1111/j.1741‐2358.2011.00552.x Study of temporomandibular joint disorder in older patients by magnetic resonance imaging (MRI) Objectives: To compare characteristics in older patients in a sample of the general population of those with temporomandibular joint disorder (TMJD). Materials and methods: A prospective study was carried out between 2001 and 2008 in patients with TMJD. The whole sample consisted of 141 patients divided in two groups: 31 patients aged over 60 (median age 67.9, ranging from 60 to 82) and the remaining 110 patients (median age 36.3, ranging from 12 to 59) who were seeking treatment. Clinical diagnostics was confirmed by MRI. Pain intensity was rated on a visual analogue scale (VAS 0‐10). Results: There was no statistical difference between average pain in older patients (6.2) and patients aged up to 59 (5.7) evaluated by VAS. There was a statistically significant difference (p = 0.002) in pain duration: older patients reported shorter duration of experienced pain (7.8 months) than patients aged up to 59 (12.2 months). Conclusion: In this study, it was found that 22% were older patients with TMJD. A higher level of anxiety was shown in both patients’ groups, regardless of shorter pain experience in the older patients.     

Significance of Urinary Full-Length Megalin in Patients with IgA Nephropathy

Background and Objectives

Megalin is highly expressed at the apical membranes of proximal tubular epithelial cells. A urinary full-length megalin (C-megalin) assay is linked to the severity of diabetic nephropathy in type 2 diabetes. This study examined the relationship between levels of urinary C-megalin and histological findings in adult patients with IgA nephropathy (IgAN).

Design, Setting, Participants, & Measurements

Urine samples voided in the morning on the day of renal biopsy were obtained from 73 patients with IgAN (29 men and 44 women; mean age, 33 years) and 5 patients with membranous nephropathy (MN). Renal pathologic variables were analyzed using the Oxford classification of IgAN, the Shigematsu classification and the Clinical Guidelines of IgAN in Japan. The levels of urinary C-megalin were measured by sandwich ELISA.

Results

Histological analysis based on the Oxford classification revealed that the levels of urinary C-megalin were correlated with mesangial hypercellularity in IgAN patients (OR = 1.76, 95% CI: 1.04–3.27, P<0.05). There was a significant correlation between the levels of urinary C-megalin and the severity of chronic extracapillary abnormalities according to the Shigematsu classification in IgAN patients (β = 0.33, P = 0.008). The levels of urinary C-megalin were significantly higher in all risk levels of IgAN patients requiring dialysis using the Clinical Guidelines of IgAN in Japan than in the control group. The levels of urinary C-megalin were significantly higher in the high risk and very high risk grades than in the low risk grade (P<0.05). The levels of urinary C-megalin were significantly higher in MN patients compared to the control group.

Conclusions

The levels of urinary C-megalin are associated with histological abnormalities in adult IgAN patients. There is a possibility that urinary C-megalin is an independent predictor of disease progression of IgAN. In addition, our results suggest that urinary C-megalin is a marker of glomerular abnormalities in various glomerular diseases as well as IgAN.     

Presence of lysosomal enzymes in the normal glomerular basement membrane matrix

Summary The question posed in the present study was: are there hydrolytic enzymes, including proteases, present in the extracellular matrix of the glomerular basement membrane? If these enzymes are present they may play a role in the catabolism of the glomerular basement membrane (GBM) and removal of macromolecular debris resulting from ultrafiltration. Enzymes, acid phosphatase - the marker for lysosomal enzymes - β-galactosidase, β-glucuronidase and acid protease (using albumin as substrate) were biochemically assayed in purified basement membrane preparations. It was found that all enzymes were present in significant amounts in the basement membrane. Compared to other enzymes, acid protease activity was present in much higher amounts. The pH optima of these enzymes were variable but all had significant activity at neutral pH. A method was developed to localize the marker enzyme, acid phosphatase, ultrastructurally in the basement membrane in order to substantiate the biochemical findings. Activity was shown by the presence of dense deposits of lead phosphate. Staining for acid phosphatase could also be shown on isolated, purified basement membrane. The demonstration of acid hydrolases in the GBM matrix argues for their role in (i) the extracellular turnover of basement membrane macromolecules, and (ii) clearance of debris of ultrafiltration which tend to clog the membrane pores.     

Genetic Variation in miR-146a Is Not Associated with Susceptibility to IgA Nephropathy in Adults from a Chinese Han Population

Background

MicroRNA 146a (miR-146a) is a 19 to 23 nucleotide long, small non-coding RNA with gene regulatory functions that has influence on the pathogenesis of many diseases. A single nucleotide polymorphism (rs2910164 C>G) in pre-miR-146a is correlated with the expression of miR-146a. The aim of this study was to perform an association analysis of rs2910164 with IgA nephropathy in adult patients from a Chinese Han population.

Methods

A total of 145 patients with renal biopsy-proved IgA nephropathy (IgAN) and 179 healthy controls were recruited to the current study. rs2910164 was genotyped by the polymerase chain reaction (PCR) and high-resolution melting methods (HRM). Clinical characteristics and pathology grading of patients with IgAN were recorded at the time of kidney biopsy.

Result

There were significant differences among the population of patients grouped by different age of onset in a co-dominant model (CG vs. CC vs. GG) (p = 0.033) and a recessive model (CG+CC vs. GG) (p = 0.001). However, no significant difference was observed in the distribution of genotypes between cases and controls (p = 0.144). There was also no significant difference between rs2910164 and patient quantitative traits (all p > 0.003) or different pathology grading (Lee’s grading system and tubular atrophy/interstitial fibrosis in the Oxford classification) (all p > 0.05).

Conclusions

There was no association of rs2910164 with susceptibility to IgAN in adults from a Chinese Han population. However, rs2910164 was correlated with the age of onset of IgAN in adult patients.     

LG3 fragment of endorepellin is a possible biomarker of severity in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by deposition of IgA in the glomerular mesangium. The diagnosis of IgAN still requires a kidney biopsy that cannot easily be repeated in the same patient during follow‐up. Therefore, identification of noninvasive urinary biomarkers would be very useful for monitoring patients with IgAN. We first used bidimensional electrophoresis (2DE) coupled to MALDI‐TOF‐TOF and Western blot to identify some urinary biomarkers associated with IgAN. Urine of IgAN patients showed an increase of albumin fragments, α‐1‐antitrypsin and α‐1‐β‐glycoprotein, along with a decrease of a single spot that was identified as the laminin G‐like 3 (LG3) fragment of endorepellin. The urinary proteomes of 43 IgAN patients were compared to those of 30 healthy individuals by ELISA. Quantification of LG3 confirmed a significant decrease in the urine of IgAN patients compared to healthy controls, except in ten patients in whom LG3 was increased. These ten patients had a more severe disease with lower glomerular filtration rate values. We found a significant inverse correlation between LG3 levels and glomerular filtration rate in the 43 patients with IgAN, which was not observed in 65 patients with other glomerular diseases including membranous nephropathy (23), lupus nephropathy (13), focal segmental glomerulosclerosis (15), diabetic nephropathy (14), and six patients with nonglomerular diseases. Therefore, we suggest that the LG3 fragment of endorepellin could be associated with IgAN severity and might be related to pathogenesis of IgAN.