Subclinical signs of podocyte injury associated with Circulating Anodic Antigen (CAA) in Schistosoma mansoni-infected patients in Brazil.

BACKGROUND: The long-term effects of schistosomiasis on the glomerulus may contribute to the development of chronic kidney disease. This study aimed to investigate baseline Schistosoma mansoni-Circulating Anodic Antigen (CAA) levels and their association with kidney biomarkers related to podocyte injury and inflammation in long-term follow-up after praziquantel (PZQ) treatment. METHODS: Schistosoma infection was diagnosed by detecting CAA in urine using a quantitative assay based on lateral flow using luminescent up-converting phosphor reporter particles. A cutoff threshold of 0.1 pg/mL CAA was used to diagnose Schistosoma infection (baseline) in a low-prevalence area in Ceará, Northeast, Brazil. Two groups were included: CAA-positive and CAA-negative individuals, both of which received a single dose of PZQ at baseline. Urinary samples from 55 individuals were evaluated before (baseline) and at 1, 2, and 3 years after PZQ treatment. At all time points, kidney biomarkers were quantified in urine and adjusted for urinary creatinine levels. RESULTS: CAA-positive patients had increased baseline albuminuria and proteinuria and showed greater associations between kidney biomarkers. CAA levels correlated only with Vascular Endothelial Growth Factor (VEGF) (podocyte injury) levels. Increasing trends were observed for malondialdehyde (oxidative stress), monocyte chemoattractant protein-1 (inflammation marker), and VEGF. In the follow-up analysis, no relevant differences were observed in kidney biomarkers between the groups and different periods. CONCLUSIONS: S. mansoni-infected individuals presented subclinical signs of glomerular damage that may reflect podocyte injury. However, no causal effect on long-term renal function was observed after PZQ treatment.

Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes.

BACKGROUND: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. METHODS: APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. RESULTS: APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. CONCLUSIONS: Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.

Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes.

BACKGROUND: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. METHODS: Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. RESULTS: Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. CONCLUSIONS: APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

A glomerulus-on-a-chip to recapitulate the human glomerular filtration barrier.

In this work we model the glomerular filtration barrier, the structure responsible for filtering the blood and preventing the loss of proteins, using human podocytes and glomerular endothelial cells seeded into microfluidic chips. In long-term cultures, cells maintain their morphology, form capillary-like structures and express slit diaphragm proteins. This system recapitulates functions and structure of the glomerulus, including permselectivity. When exposed to sera from patients with anti-podocyte autoantibodies, the chips show albuminuria proportional to patients' proteinuria, phenomenon not observed with sera from healthy controls or individuals with primary podocyte defects. We also show its applicability for renal disease modeling and drug testing. A total of 2000 independent chips were analyzed, supporting high reproducibility and validation of the system for high-throughput screening of therapeutic compounds. The study of the patho-physiology of the glomerulus and identification of therapeutic targets are also feasible using this chip.

Urinary podocyte microparticles are associated with disease activity and renal injury in systemic lupus erythematosus.

BACKGROUND: New non-invasive biomarkers are demanded to identify renal damage in various autoimmune-associated kidney diseases. Glomerular podocyte damage mediated by systemic lupus erythematosus (SLE) plays an important role in the pathogenesis and progression of lupus nephritis (LN). This study evaluated whether the podocyte-derived microparticles (MPs) were novel biomarkers of clinical and histological features in SLE patients with LN. METHODS: A cross-sectional study, including 34 SLE patients and 16 healthy controls, was designed. Urinary annexin V+ podocalyxin+ MPs of all participants were quantified by flow cytometry. The correlation of podocyte-derived MPs with clinical and histological parameters of SLE patients was analysed. RESULTS: The number of annexin V+ podocalyxin+ MPs from urine samples were markly increased in patients with SLE. Furthermore, the level of urinary podocyte-derived MPs was positively correlated with the SLE Disease Activity Index (SLEDAI) score, anti-dsDNA antibody titre, erythrocyte sedimentation rate, and proteinuria. Conversely, it was negatively correlated with the level of complement C3 and serum albumin. The number of urinary podocyte-derived MPs was significantly increased in SLE patients with high activity indices. Receiver operating characteristic (ROC) curves were calculated to assess the power for podocyte-derived MP levels in differentiating between SLE patients with and without LN. Podocyte-derived MP levels were able to differentiate between SLE patients with mild disease activity, as well as those with moderate and above disease activity. SLE patients showed increased podocyte-derived MP excretion into the urine. CONCLUSIONS: These findings suggest that the change in urinary podocyte-derived MP levels could be useful for evaluating and monitoring SLE disease activity.

CRISPR-Cas9-Mediated Correction of the G189R-PAX2 Mutation in Induced Pluripotent Stem Cells from a Patient with Focal Segmental Glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is defined by focal (involving few glomeruli) and segmental sclerosis of the glomerular tuft that manifests with nephrotic syndrome. Mutations in genes involved in the maintenance of structure and function of podocytes have been found in a minority of these patients. A family with adult-onset autosomal dominant FSGS was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2. Here, we efficiently corrected this point mutation in patient-derived induced pluripotent stem cells (iPSCs) by means of CRISPR-Cas9-based homology-directed repair. The iPSC lines were differentiated into podocytes, which were tested for their motility. Editing the PAX2 p.G189R mutation restored podocyte motility, which was altered in podocytes derived from patient iPSCs.

PMab-213: A Monoclonal Antibody for Immunohistochemical Analysis Against Pig Podoplanin.

Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking. In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometric screening. One of the mAbs, PMab-213 (IgG2b, kappa), could specifically detect CHO/pPDPN cells through flow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was determined to be 2.1 × 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.

PMab-210: A Monoclonal Antibody Against Pig Podoplanin.

Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa), was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.

Podocyturia in paediatric patients with Fabry disease. / Podocituria en pacientes pediátricos con enfermedad de Fabry.

INTRODUCTION: Fabry disease (FD) is a hereditary disorder caused by a deficiency of α-galactosidase A enzyme activity. The transmission of the disorder is linked to the X chromosome. OBJECTIVES: The objectives of the study were: 1. To quantify the presence of podocytes in paediatric patients with FD and compare them with the value of the measured podocyturia in healthy controls. 2. To determine whether a greater podocyturia is related to the onset of pathological albuminuria in patients with FD. 3. To determine the risk factors associated with pathological albuminuria. METHODS: We performed an analytical, observational study of Fabry and control subjects, which were separated into 2groups in accordance with the absence of the disease (control group) or the presence of the disease (Fabry group). RESULTS: We studied 31 patients, 11 with FD and 20 controls, with a mean age of 11.6 years. The difference between the mean time elapsed from the diagnosis of FD to the measurement of podocyturia (40 months) and the onset of pathological albuminuria (34 months) was not significant (p=0.09). Podocytes were identified by staining for the presence of synaptopodin and the mean quantitative differences between both podocyturias were statistically significant (p=0.001). Albuminuria was physiological in 4 of the patients with FD and the relative risk to develop pathological albuminuria according to podocyturia was 1.1 in the control group and 3.9 in the Fabry group, with a coefficient of correlation between podocyturia and albuminuria in the Fabry group of 0.8354. Finally, the 2 risk factors associated with the development of pathological albuminuria were podocyturia (OR: 14) and being aged over 10 years (OR: 18). We found no significant risk with regard to glomerular filtrate renal (GFR) (OR: 0.5) or gender (OR: 1.3). The mean GFR remained within normal values. CONCLUSION: The detection of podocyturia in paediatric patients with FD could be used as an early marker of renal damage, preceding and proportional to the occurrence of pathological albuminuria.

Expression of DENDRIN in several glomerular diseases and correlation to pathological parameters and renal failure - preliminary study.

BACKGROUND: In glomerular injury dendrin translocates from the slit diaphragm to the podocyte nucleus, inducing apoptosis. We analyzed dendrin expression in IgA glomerulonephritis and Henoch Schönlein purpura (IgAN/HSP) versus in podocytopathies minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and compared it to pathohistological findings and renal function at the time of biopsy and the last follow-up. METHODS: Twenty males and 13 females with median of age 35 years (min-max: 3-76) who underwent percutaneous renal biopsy and had diagnosis of glomerular disease (GD) were included in this retrospective study. Fifteen patients had IgAN/HSP and eighteen podocytopathy. Control group consisted of ten patients who underwent nephrectomy due to renal cancer. Dendrin expression pattern (membranous, dual, nuclear or negative), number of dendrin positive nuclei and proportion of dendrin negative glomeruli were analyzed. RESULTS: In GD and the control group significant differences in number of dendrin positive nuclei and proportion of dendrin negative glomeruli were found (P = 0.004 and P = 0.003, respectively). Number of dendrin positive nuclei was higher in podocytopathies than in IgAN/HSP, 3.90 versus 1.67 (P = 0.028). Proportion of dendrin negative glomeruli correlated to higher rates of interstitial fibrosis (P = 0.038), tubular atrophy (P = 0.011) and globally sclerotic glomeruli (P = 0.008). Dual and nuclear dendrin expression pattern were connected with lower rate of interstitial fibrosis and tubular atrophy than negative dendrin expression pattern (P = 0.024 and P = 0.017, respectively). Proportion of dendrin negative glomeruli correlated with lower creatinine clearance (CC) at the time of biopsy and the last follow-up (P = 0.010 and P < 0.001, respectively). Dendrin expression pattern correlated to CC at the last follow-up (P = 0.009), being lower in patients with negative than nuclear or dual dendrin expression (P = 0.034 and P = 0.004, respectively). CONCLUSION: In this pilot study the number of dendrin positive nuclei was higher in podocytopathies than in inflammatory GD. Negative dendrin expression pattern correlated to chronic tubulointerstitial changes and lower CC, which needs to be confirmed in a larger series.

RANK Deficiency Ameliorates Podocyte Injury by Suppressing Calcium/Calcineurin/ NFATc1 Signaling.

BACKGROUND/AIMS: Podocyte injury and loss contribute to proteinuria, glomerulosclerosis and eventually kidney failure. Receptor activator of NF-κB (RANK) belongs to the TNF receptor superfamily, which plays a key role in the pathogenesis of podocyte injury. However, the mechanism underlying the effect of RANK in podocyte injury remains unclear. Here, we sought to explore the possible molecular mechanisms involved in podocyte injury caused by RANK. METHODS: Immortalized mouse podocytes were treated with siRNA targeting RANK for 48 h or ionomycin for 24 h before harvest. Western blot, quantitative RT-PCR and immunofluorescence staining were used to evaluate the expression and function of RANK, nuclear factor of activated T cells c1 (NFATc1), transient receptor potential cation channel, subfamily C, member 6 (TRPC6) and calcineurin in podocytes. The Calcineurin Cellular Activity Assay kit was used to detect the phosphatase activity of calcineurin in cultured podocytes. A Ca2+ influx assay was performed to analyze alterations in Ca2+ entry under different conditions. Co-immunoprecipitation assays were used to observe the relationship between RANK and TRPC6. RESULTS: RANK mRNA and protein expression were markedly increased in injured podocytes (ionomycin stimulation). Further study found that translocation of NFATc1 to the nucleus was significantly reduced after knocking down RANK by siRNA. Meanwhile, we also demonstrated that loss of RANK suppressed the phosphatase activity of calcineurin and attenuated the ionomycin-induced increase in Ca2+ influx. In addition, we showed that RANK knockdown in cultured podocytes decreased TRPC6 protein expression. Co-immunoprecipitation experiments suggested that RANK binds to TRPC6 and that ionomycin enhanced the binding of RANK to TRPC6. CONCLUSION: Our findings demonstrated that RANK deficiency ameliorates podocyte injury by suppressing calcium/calcineurin/NFATc1 signaling, which may present a promising target for therapeutic intervention.

Value of immunohistochemical expression of podocalyxin in active lupus nephritis.

Podocalyxin is an electronegative sialoglycoprotein that prevents the podocyte foot process from collapsing. The aim of this study was to detect an association between the glomerular immunohistochemical (IHC) expression of podocalyxin and the degree of podocyte effacement detected by electron microscopy, and to evaluate the role of podocalyxin IHC expression as a novel marker for disease activity in lupus nephritis (LN). METHODS: Thirty-two renal biopsies of active lupus nephritis patients were studied. Clinical assessment by the systemic lupus activity measure (SLAM-R) score and laboratory data were included [serum creatinine, 24-h urinary protein, antinuclear antibodies (ANA), anti-double-strand DNA antibodies (anti-dsDNA), C3 and C4]. Light (L/M) and electron microscopic (E/M) examination was conducted. Podocyte loss was evaluated by immunohistochemistry with monoclonal anti-podocalyxin antibodies by means of a semiquantitative score that was graded from 0 to 4+ according to the percentage of glomerular involvement. RESULTS: 22 cases (68.8%) with LN class IV, 6 (18.8%) with class III and 4 (12.5%) with class V. The mean age was (25.41±10.13) years. There was a significant negative correlation between IHC podocalyxin score and LN class, and NIH activity parameters such as leukocyte infiltration, endocapillary proliferation, fibrinoid necrosis and cellular crescent and disease activity index but not chronicity index. There was a highly significant negative correlation between IHC podocalyxin and podocyte effacement by E/M (rs=-0.903, P=0.000), and E/M immune deposits (r=-0.53, P=0.001), and a significant association with degree of proteinuria, ANA and SLAM score (P<0.05). CONCLUSIONS: Podocyte loss indicated by podocalyxin immunohistochemical expression reflects the degree of activity and severity of LN and the degree of podocyte effacement by E/M.

Automated segmentation and quantification of actin stress fibres undergoing experimentally induced changes.

The actin cytoskeleton is a main component of cells and it is crucially involved in many physiological processes, e.g. cell motility. Changes in the actin organization can be effected by diseases or vice versa. Due to the nonuniform pattern, it is difficult to quantify reasonable features of the actin cytoskeleton for a significantly high cell number. Here, we present an approach capable to fully segment and analyse the actin cytoskeleton of 2D fluorescence microscopic images with a special focus on stress fibres. The extracted feature data include length, width, orientation and intensity distributions of all traced stress fibres. Our approach combines morphological image processing techniques and a trace algorithm in an iterative manner, classifying the segmentation result with respect to the width of the stress fibres and in nonfibre-like actin. This approach enables us to capture experimentally induced processes like the condensation or the collapse of the actin cytoskeleton. We successfully applied the algorithm to F-actin images of cells that were treated with the actin polymerization inhibitor latrunculin A. Furthermore, we verified the robustness of our algorithm by a sensitivity analysis of the parameters, and we benchmarked our algorithm against established methods. In summary, we present a new approach to segment actin stress fibres over time to monitor condensation or collapse processes.

Accumulation of worn-out GBM material substantially contributes to mesangial matrix expansion in diabetic nephropathy.

Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN), generally considered to emerge from different sites of overproduction: GBM components from podocytes and mesangial matrix from mesangial cells. Reevaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that mesangial cells also synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence, and in situ hybridization studies clearly show that the thickening of the GBM is due not only to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes: overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes.

Ang-(1-7) inhibited mitochondrial fission in high-glucose-induced podocytes by upregulation of miR-30a and downregulation of Drp1 and p53.

BACKGROUND: The aim of this study was to investigate the possible effects of angiotensin-(1-7) [Ang-(1-7)] on podocytes and the mitochondrial signaling pathway in a high-glucose (HG) environment. METHODS: We established a model of HG-induced podocytes by incubating podocytes in RPMI 1640 containing 33mM glucose. The cells were divided into the following groups: (1) normal glucose group as control incubated in Roswell Park Memorial Institute (RPMI) 1640 containing 5mM glucose; (2) Ang-(1-7), 10nM, incubated in RPMI 1640 containing 5mM glucose; (3) the HG group incubated in RPMI 1640 containing 33mM glucose; and (4) Ang-(1-7), 10nM, incubated in HG group incubated in RPMI 1640 containing 33mM glucose. After a period of 24 hours, mitochondrial fission and podocyte fusion were observed by electron microscope. Additionally, p53 and Drp1 were tested by Western blot, the position of Drp1 was detected by immunofluorescence, and miR-30a was analyzed by quantitative real-time polymerase chain reaction. RESULTS: Ang-(1-7) inhibited mitochondrial fission in HG-treated podocytes. However, Ang-(1-7) also significantly reduced the expression of Drp1 and p53, and improved the expression of miR-30a in HG-induced podocytes. CONCLUSION: Ang-(1-7) inhibited mitochondrial fission in HG-induced podocytes by upregulation of miR-30a and downregulation of Drp1 and p53.

In silico Structural characterization of podocin and assessment of nephrotic syndrome-associated podocin mutants.

Nephrotic syndrome (NS) is manifested by hyperproteinuria, hypoalbuminemia, and edema. NPHS2 that encodes podocin was found to have most mutations among the genes that are involved in the pathophysiology of NS. Podocin, an integral membrane protein belonging to stomatin family, is expressed exclusively in podocytes and is localized to slit-diaphragm (SD). Mutations in podocin are known to be associated with steroid-resistant NS and rapid progression to end-stage renal disease, thus signifying its role in maintaining SD integrity and podocyte function. The structural insights of podocin are not known, and the precise mechanism by which podocin contributes to the architecture of SD is yet to be elucidated. In this study, we deduced a model for human podocin, discussed the details of transmembrane localization and intrinsically unstructured regions, and provide an understanding of how podocin interacts with other SD components. Intraprotein interactions were assessed in wild-type podocin and in some of its mutants that are associated with idiopathic NS. Mutations in podocin alter the innate intraprotein interactions affecting the native structure of podocin and its ability to form critical complex with subpodocyte proteins. © 2016 IUBMB Life, 68(7):578-588, 2016.

Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys.

Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R.

Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice.

Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes.

WT1 immunoenzyme staining using SurePath(™) processed urine cytology helps to detect kidney disease.

OBJECTIVES: Damage and detachment of podocytes and loss into the urine have been implicated in the progression of kidney diseases. The purpose of this study was to investigate the potential role of urine cytology based on SurePath(™) combined with immunoenzyme staining using Wilms' tumour 1 (WT1) antibody as a podocyte marker in the discrimination of normality and non-renal urinary tract disease from kidney disease. METHODS: Sixty-six patients with kidney disease, 45 patients with lower urinary tract disease and 30 healthy volunteers were examined. Urine cytology slides were prepared using the SurePath method and immunoenzyme stained with WT1 antibody, and the number of WT1-positive cells was counted. RESULTS: In kidney disease, WT1-positive cells were found in 33 (50%) of 66 samples. No WT1-positive cells were found in 45 patients with lower urinary tract disease or in 30 healthy volunteers. The positive rates for WT1 varied with disease type, but not significantly: immunoglobulin A (IgA) nephropathy, (14/23); membranous glomerulonephritis, (4/10); Henoch-Schönlein purpura nephritis, (3/5); diabetic glomerulopathy, (5/5); minor glomerular abnormality/minimal change nephrotic syndrome (0/4). CONCLUSIONS: The results suggest that WT1 immunoenzyme staining of urine cytology can be used to detect some types of kidney disease.

B7-1 Is Not Induced in Podocytes of Human and Experimental Diabetic Nephropathy.

The incidence of progressive kidney disease associated with diabetes continues to rise worldwide. Current standard therapy with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers achieves only partial renoprotection, increasing the need for novel therapeutic approaches. Previous studies described B7-1 induction in podocytes of patients with proteinuria, including those with FSGS and type 2 diabetic nephropathy (DN). These findings sparked great excitement in the renal community, implying that abatacept, a costimulatory inhibitor that targets B7-1, could be a novel therapy for diabetic renal disease. Given previous concerns over the value of B7-1 immunostaining and the efficacy of abatacept in patients with recurrent FSGS after renal transplantation, we investigated B7-1 expression in human and experimental DN before embarking on clinical studies of the use of B7-1 targeting strategies to treat proteinuria in DN. Immunohistochemical analysis of kidney specimens using different antibodies revealed that B7-1 is not induced in podocytes of patients with DN, independent of disease stage, or BTBR ob/obmice, a model of type 2 diabetes. These results do not support the use of abatacept as a therapeutic strategy for targeting podocyte B7-1 for the prevention or treatment of DN.