MAD2B contributes to parietal epithelial cell activation and crescentic glomerulonephritis via Skp2.

Mitotic spindle assembly checkpoint protein 2 (MAD2B), a well-known anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase-ζ, is critical for mitotic control and DNA repair. Previously, we detected a strong increase of MAD2B in the glomeruli from patients with crescentic glomerulonephritis and anti-glomerular basement membrane (anti-GBM) rats, which predominantly originated from activated parietal epithelial cells (PECs). Consistently, in vitro MAD2B was increased in TNF-α-treated PECs, along with cell activation and proliferation, as well as extracellular matrix accumulation, which could be reversed by MAD2B genetic depletion. Furthermore, we found that expression of S phase kinase-associated protein 2 (Skp2), an APC/CCDH1 substrate, was increased in the glomeruli of anti-GBM rats, and TNF-α-stimulated PECs and could be suppressed by MAD2B depletion. Additionally, genetic deletion of Skp2 inhibited TNF-α-induced PEC activation and dysfunction. Finally, TNF-α blockade or glucocorticoid therapy administered to anti-GBM rats could ameliorate MAD2B and Skp2 accumulation as well as weaken PEC activation. Collectively, our data suggest that MAD2B has a pivotal role in the pathogenesis of glomerular PEC activation and crescent formation through induction of Skp2 expression.

(Pro)renin receptor promotes crescent formation via the ERK1/2 and Wnt/ß-catenin pathways in glomerulonephritis.

(Pro)renin receptor [(P)RR] has multiple functions, but its regulation and role in the pathogenesis in glomerulonephritis (GN) are poorly defined. The aims of the present study were to determine the effects of direct renin inhibition (DRI) and demonstrate the role of (P)RR on the progression of crescentic GN. The anti-glomerular basement membrane nephritis rat model developed progressive proteinuria (83.64 ± 10.49 mg/day) and glomerular crescent formation (percent glomerular crescent: 62.1 ± 2.3%) accompanied by increased macrophage infiltration and glomerular expression of monocyte chemoattractant protein (MCP)-1, (P)RR, phospho-extracellular signal-regulated kinase (ERK)1/2, Wnt4, and active ß-catenin. Treatment with DRI ameliorated proteinuria (20.33 ± 5.88 mg/day) and markedly reduced glomerular crescent formation (20.9 ± 2.6%), induction of macrophage infiltration, (P)RR, phospho-ERK1/2, Wnt4, and active ß-catenin. Furthermore, primary cultured parietal epithelial cells stimulated by recombinant prorenin showed significant increases in cell proliferation. Notably, while the ERK1/2 inhibitor PD98059 or (P)RR-specific siRNA treatment abolished the elevation in cell proliferation, DRI treatment did not abrogate this elevation. Moreover, cultured mesangial cells showed an increase in prorenin-induced MCP-1 expression. Interestingly, (P)RR or Wnt4-specific siRNA treatment or the ß-catenin antagonist XAV939 inhibited the elevation of MCP-1 expression, whereas DRI did not. These results suggest that (P)RR regulates glomerular crescent formation via the ERK1/2 signaling and Wnt/ß-catenin pathways during the course of anti-glomerular basement membrane nephritis and that DRI mitigates the progression of crescentic GN through the reduction of (P)RR expression but not inhibition of prorenin binding to (P)RR.

Heparanase in Kidney Disease.

The primary filtration of blood occurs in the glomerulus in the kidney. Destruction of any of the layers of the glomerular filtration barrier might result in proteinuric disease. The glomerular endothelial cells and especially its covering layer, the glycocalyx, play a pivotal role in development of albuminuria. One of the main sulfated glycosaminoglycans in the glomerular endothelial glycocalyx is heparan sulfate. The endoglycosidase heparanase degrades heparan sulfate, thereby affecting glomerular barrier function, immune reactivity and inflammation. Increased expression of glomerular heparanase correlates with loss of glomerular heparan sulfate in many glomerular diseases. Most importantly, heparanase knockout in mice prevented the development of albuminuria after induction of experimental diabetic nephropathy and experimental glomerulonephritis. Therefore, heparanase could serve as a pharmacological target for glomerular diseases. Several factors that regulate heparanase expression and activity have been identified and compounds aiming to inhibit heparanase activity are currently explored.

Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

Lycium barbarum polysaccharides attenuate rat anti-Thy-1 glomerulonephritis through mediating pyruvate dehydrogenase.

Glomerulonephritis is the major cause of chronic kidney disease characterized by mesangial cell proliferation and extracellular matrix deposition. The aim of this study was to investigate the effects of Lycium barbarum polysaccharides (LBPs) on anti-Thy 1 nephritis rats and explore the protective mechanism of LBPs. After the model of glomerulonephritis created by injecting anti-thymocyte serum (ATS), rats were treated with enalapril or LBPs for 8 weeks. The therapeutic effect was evaluated by detection of renal-related biochemical parameters, histological observation and markers of renal fibrosis. Moreover, RNA-seq analysis and experiments in vitro were employed to explore the signaling pathway involved in LBPs treatment. The results found that LBPs treatment significantly suppressed ATS-caused increment at levels of blood urea nitrogen, creatinine, proteinuria, PAI-1 protein expression, glomerular mesangial cell proliferation and extracellular matrix hyperplasia, along with reduction of creatinine clearance. RNA sequencing showed pyruvate metabolism acting as a potential signaling pathway, which was evidenced by the inhibitory effect on up-regulation of pyruvate dehydrogenase and PAI-1 levels via treatment with LBPs in vitro. LBPs are the promising agents for the management of glomerulonephritis through pyruvate metabolism signaling pathway.

Impact of MPO-ANCA-mediated oxidative imbalance on renal vasculitis.

Glomerulonephritis is a severe complication of microscopic polyangiitis (MPA), a small-vessel vasculitis associated with anti-myeloperoxidase antibodies (MPO-ANCA). We previously showed the pathogenic effects of MPO-ANCA that activate MPO to trigger an oxidative burst mainly through HOCl production, contributing to endothelial injury and lung fibrosis. The aim of this study was to investigate the relationship between MPO-induced oxidative stress, anti-oxidant defenses and renal histological lesions in MPA patients. We therefore analyzed histological data from a prospective cohort of ANCA-associated glomerulonephritis. Serum-mediated HOCl production, advanced oxidation protein products (AOPP), and thiol concentration in sera were determined. From 38 patients included, histological classification noted 50% focal glomerulonephritis, 15.8% crescentic-glomerulonephritis, and 34.2% mixed-glomerulonephritis. MPA patients' sera displayed higher HOCl production by MPO ( P < 0.001), higher AOPP ( P < 0.001) and thiol ( P < 0.01) levels, compared with healthy subjects. The presence of cellular crescents was associated with higher serum-mediated HOCl production ( P = 0.049) and lower thiol levels ( P = 0.022) at disease onset. Higher thiol concentrations were associated with focal glomerulonephritis ( P = 0.042), less interstitial fibrosis ( P = 0.039) and hyalinosis ( P = 0.066). In remission, HOCl production was decreased ( P < 0.01), and thiol concentration remained high ( P = 0.39). Our findings suggest that HOCl production by activated MPO could contribute to the very early stage of glomerulonephritis, whereas thiol may exert a protective effect against the development of renal vasculitis and glomerulosclerosis. This study highlights the importance of oxidative defenses to counteract the process of MPO-ANCA associated glomerulonephritis.

An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis.

Non-selective inhibitors of spleen tyrosine kinase (SYK) efficiently suppress disease in T cell-dependent models of crescentic glomerulonephritis. However, the therapeutic potential of selective SYK inhibitors in this disease has not been established. In addition, we lack knowledge regarding SYK expression in non-myeloid cells in glomerulonephritis. We addressed these two issues in a rat model of nephrotoxic serum nephritis (NTN) using a SYK inhibitor, GS-492429. Disease was induced in Sprague-Dawley rats (Study 1) or Wistar-Kyoto (WKY) rats (Study 2) by immunization with sheep IgG and administration of sheep anti-rat nephrotoxic serum. Animals were untreated or received GS-492429 (30 mg/kg/bid) or vehicle treatment from 2 h before nephrotoxic serum injection until being killed 3 or 24 h later (Study 1) or 14 days later (Study 2). Two-colour confocal microscopy found that SYK expression in NTN kidney was restricted to myeloid cells and platelets, with no evidence of SYK expression by T cells, mesangial cells, podocytes or tubular epithelial cells. In Study 1, GS-492429 treatment significantly reduced glomerular neutrophil and macrophage infiltration, with protection from glomerular thrombosis and proteinuria. In Study 2, GS-492429 treatment reduced glomerular crescent formation by 70% on day 14 NTN in conjunction with reduced glomerular thrombosis, glomerulosclerosis and tubular damage. This was accompanied by a marked reduction in markers of inflammation (CCL2, TNF-α, NOS2, MMP-12). Importantly, the protective effects of GS-492429 were independent of T cell infiltration and activation and independent of JAK/STAT3 signalling. In conclusion, this study demonstrates that a SYK inhibitor can suppress the development of crescentic glomerulonephritis through effects upon myeloid cells and platelets.

Sirt1 activation prevents anti-Thy 1.1 mesangial proliferative glomerulonephritis in the rat through the Nrf2/ARE pathway.

Mesangial proliferative glomerulonephritis (MsPGN) is characterized by glomerular mesangial cells proliferation and extracellular matrix deposition in mesangial area, which develop into glomerulosclerosis. Both silent information regulator 2-related protein 1 (Sirt1) and nuclear factor erythroid 2-related factor 2/anti-oxidant response element (Nrf2/ARE) pathway had remarkable renoprotective effects. However, whether Sirt1 and Nrf2/ARE pathway can regulate the pathological process of MsPGN remains unknown. Here, we found that Sirt1 activation by SRT1720 decreased mesangial hypercellularity and mesangial matrix areas, reduced renal Col4 and α-SMA expressions, lowered 24 h proteinuria, and eventually reduced FN and TGF-ß1 expressions in rats received anti-Thy 1.1 IgG. Further study showed that SRT1720 markedly enhanced the activity of Nrf2/ARE pathway including promoting the nuclear content and ARE-binding ability of Nrf2, elevating the protein levels of HO-1 and SOD1, two target genes of Nrf2, which eventually increased total SOD activity and decreased malondialdehyde level in the kidney tissues of experimental anti-Thy 1.1 MsPGN rats. Taken together, Sirt1 prevented the pathological process of experimental anti-Thy 1.1 MsPGN through promoting the activation of Nrf2/ARE pathway, which warrants further elucidation. Sirt1 might be a potential therapeutic target for treating MsPGN.

Inactivation of MAP3K7 in FOXD1-expressing cells results in loss of mesangial PDGFRΒ and juvenile kidney scarring.

Transforming growth factor-ß (TGFß) plays a central role in renal scarring, controlling extracellular matrix deposition by interstitial cells and mesangial cells. TGFß signals through Smad and mitogen-activated protein kinase (MAPK) pathways. To understand the role of MAPK in interstitial and mesangial cells, we genetically inactivated TGFß-activated kinase-1 ( Map3k7) using Foxd1+/cre. Embryonic kidney development was unperturbed in mutants, but spontaneous scarring of the kidney ensued during the first postnatal week, with retention of embryonic nephrogenic rests and accumulation of collagen IV in the mesangium. MAPK signaling in the mesangium of mutant mice was skewed, with depressed p38 but elevated c-Jun NH2-terminal kinase (JNK) activation at postnatal day 3. Despite normal expression of platelet-derived growth factor receptor-ß (PDGFRß) in the mesangium of mutants at birth, expression was lost concomitantly with the increase in JNK activation, and studies in isolated mesangial cells revealed that JNK negatively regulates Pdgfrß. In summary, we show that MAP3K7 balances MAPK signaling in mesangial cells, suppressing postnatal JNK activation. We propose that the balance of MAPK signaling is essential for appropriate postnatal regulation of mesangial PDGFRß expression.

Ubiquitin C-Terminal Hydrolase L1 is required for regulated protein degradation through the ubiquitin proteasome system in kidney.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.

Parathyroid hormone-related protein modulates inflammation in mouse mesangial cells and blunts apoptosis by enhancing COX-2 expression.

Injury of mesangial cells (MC) is a prominent feature of glomerulonephritis. Activated MC secrete inflammatory mediators that induce cell apoptosis. Parathyroid hormone-related peptide (PTHrP) is a locally active cytokine that enhances cell survival and is upregulated by proinflammatory factors in many cell types. The aim of this study was to analyze the regulation of PTHrP expression by inflammatory cytokines and to evaluate whether PTHrP itself acts as a proinflammatory and/or survival factor on male murine MC in primary culture. Our results showed that IL-1ß (10 ng/ml) and TNF-α (10 ng/ml) rapidly and transiently upregulated PTHrP expression in MC. The effects of IL-1ß were both transcriptional and posttranscriptional, with stabilization of the PTHrP mRNA by human antigen R (HuR). Proteome profiler arrays showed that PTHrP itself enhanced cytokines within 2 h in cell lysates, mainly IL-17, IL-16, IL-1α, and IL-6. PTHrP also stimulated sustained expression (2-4 h) of chemokines, mainly regulated upon activation normal T cell expressed and secreted (RANTES)/C-C motif chemokine 5 (CCL5) and macrophage inflammatory protein-2 (MIP-2)/C-X-C motif chemokine 2 (CXCL2), thymus and activation-regulated chemokine (TARC)/CCL17, and interferon-inducible T cell α-chemoattractant (I-TAC)/CXCL11. Moreover, PTHrP markedly enhanced cyclooxygenase-2 (COX-2) expression and elicited its autoinduction through the activation of the NF-κB pathway. PTHrP induced MC survival via the COX-2 products, and PTHrP overexpression in MC blunted the apoptotic effects of IL-1ß and TNF-α. Altogether, these findings suggest that PTHrP functions as a booster of glomerular inflammatory processes and may be a negative feedback loop preserving MC survival.

Reduction in podocyte SIRT1 accelerates kidney injury in aging mice.

Both the incidence and prevalence of chronic kidney disease are increasing in the elderly population. Although aging is known to induce kidney injury, the underlying molecular mechanisms remain unclear. Sirtuin 1 (Sirt1), a longevity gene, is known to protect kidney cell injury from various cellular stresses. In previous studies, we showed that the podocyte-specific loss of Sirt1 aggravates diabetic kidney injury. However, the role of Sirt1 in aging-induced podocyte injury is not known. Therefore, in this study we sought to determine the effects of podocyte-specific reduction of Sirt1 in age-induced kidney injury. We employed the inducible podocyte-specific Sirt1 knockdown mice that express shRNA against Sirt1 (Pod-Sirt1RNAi) and control mice that express shRNA for luciferase (Pod-LuciRNAi). We found that reduction of podocyte Sirt1 led to aggravated aging-induced glomerulosclerosis and albuminuria. In addition, urinary level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, was markedly increased in aged Pod-Sirt1RNAi mice compared with aged Pod-LuciRNAi mice. Although podocyte-specific markers decreased in aged mice compared with the young controls, the decrease was further exacerbated in aged Pod-Sirt1RNAi compared with Pod-LuciRNAi mice. Interestingly, expression of cellular senescence markers was significantly higher in the glomeruli of Pod-Sirt1RNAi mice than Pod-LuciRNAi mice, suggesting that cellular senescence may contribute to podocyte loss in aging kidneys. Finally, we confirmed that Pod-Sirt1RNAi glomeruli were associated with reduced activation of the transcription factors peroxisome proliferator-activated receptor (PPAR)-α coactivador-1 (PGC1α)/PPARγ, forkhead box O (FOXO)3, FOXO4, and p65 NF-κB, through SIRT1-mediated deacetylation. Together, our data suggest that SIRT1 may be a potential therapeutic target to treat patients with aging-related kidney disease.

Risk Factors for Renal Survival in Chinese Patients with Myeloperoxidase-ANCA-Associated GN.

BACKGROUND AND OBJECTIVES: Our study explored the association of histopathologic classification of ANCA-associated GN with renal survival in Chinese patients with myeloperoxidase-ANCA-associated GN. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Two hundred fifteen patients with biopsy-proven myeloperoxidase-ANCA-associated GN were included from January of 1996 to December of 2014. The biopsies included focal (n=27), mixed (n=82), crescentic (n=47), and sclerotic (n=59) classes. The long-term renal outcome and risk factors of myeloperoxidase-ANCA-associated GN for different histopathologic classes were retrospectively analyzed. RESULTS: During a median follow-up time of 22 (9-51) months, 88 (40.9%) patients reached ESRD. The 5-year renal survival (overall 58.7%) was highest in the focal class (100.0%) and lowest in the sclerotic class (20.7%), with no difference between the mixed (58.9%) and crescentic (67.4%) classes. Patients in the mixed (hazard ratio, 0.34; 95% confidence interval, 0.20 to 0.57; P<0.001) and crescentic (hazard ratio, 0.31; 95% confidence interval, 0.16 to 0.59; P<0.001) classes were at lower risk for ESRD compared with patients in the sclerotic class, as were patients who received glucocorticoids plus mycophenolate mofetil (hazard ratio, 0.32; 95% confidence interval, 0.18 to 0.60; P<0.001) compared with those receiving glucocorticoids alone. In addition, patients with a serum creatinine level ≥4 mg/dl (hazard ratio, 2.93; 95% confidence interval, 1.77 to 4.85; P<0.001) or hypoalbuminemia (hazard ratio, 2.11; 95% confidence interval, 1.32 to 3.34; P=0.002) were at higher risk for ESRD. A serum creatinine level ≥4 mg/dl and a percentage of global sclerotic glomeruli ≥60% were the two independent risk factors for ESRD in the sclerotic class. CONCLUSIONS: The histopathologic classification of ANCA-associated GN in combination with serum creatinine and serum albumin levels and treatment regimen is associated with renal outcome in myeloperoxidase-ANCA-associated GN. The evaluation of serum creatinine level and percentage of global sclerotic glomeruli provides additional information on the risk of renal survival in the sclerotic class of myeloperoxidase-ANCA-associated GN.

Deglycosylation of myeloperoxidase uncovers its novel antigenicity.

Myeloperoxidase (MPO) is a common target antigen of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and is recognized in one-third of patients with anti-glomerular basement membrane (GBM) disease. Our previous study identified over 60% of patients with anti-GBM disease recognizing linear peptides of MPO heavy chain. Here we tested whether aberrant glycosylation alters MPO antigenicity through exposure of neo-epitopes on MPO molecules. Atypical glycosylated MPO molecules, including all possible glycosylation types, were prepared by exoglycosidase and endoglycosidase treatments. Antibodies were detected from the sera of 40 patients with anti-GBM disease without the coexistence of MPO-ANCA. Circulating antibodies against aberrant glycosylated MPO existed in 21 of these patients. Non-glycan MPO and MPO with only N-acetylglucosamine had high frequencies of recognition (16 and 15 patients, respectively). Antibodies binding to aberrant glycosylated MPO could not be inhibited by intact MPO or GBM antigen. When applied to ethanol-fixed neutrophils from normal individuals, these antibodies yielded a typical cytoplasmic staining pattern (c-ANCA). Antigen specificity was detected in 90% of the antibodies using five peptides containing one of the five N-glycosylation sites each, mostly on N323, N355, and N391. The antibodies were restricted to IgG1 subclass, could activate complement, and induce neutrophil degranulation in vitro. Thus, aberrant glycosylated MPO exposed neo-epitopes and was recognized by half of the patients with anti-GBM disease. Their antibodies possessed pathogenic characteristics and may be associated with kidney injury.

CD8+ T Cells Effect Glomerular Injury in Experimental Anti-Myeloperoxidase GN.

Observations in patients with ANCA-associated vasculitis suggest that CD8+ T cells participate in disease, but there is no experimental functional evidence of pathologic involvement for these cells. Myeloperoxidase (MPO) is a well defined autoantigen in ANCA-associated vasculitis. Studies in experimental models of anti-MPO GN suggest that, after ANCA-induced neutrophil localization, deposited MPO within glomeruli is recognized by autoreactive T cells that contribute to injury. We tested the hypothesis that CD8+ T cells mediate disease in experimental ANCA-associated vasculitis. CD8+ T cell depletion in the effector phase of disease attenuated injury in murine anti-MPO GN. This protection associated with decreased levels of intrarenal IFN-γ, TNF, and inflammatory chemokines and fewer glomerular macrophages. Moreover, we identified a pathogenic CD8+ T cell MPO epitope (MPO431-439) and found that cotransfer of MPO431-439-specific CD8+ T cell clones exacerbated disease mediated by MPO-specific CD4+ cells in Rag1-/- mice. Transfer of MPO431-439-specific CD8+ cells without CD4+ cells mediated glomerular injury when MPO was planted in glomeruli. These results show a pathogenic role for MPO-specific CD8+ T cells, provide evidence that CD8+ cells are a therapeutic target in ANCA-associated vasculitis, and suggest that a molecular hotspot within the MPO molecule contains important CD8+, CD4+, and B cell epitopes.

Cyclosporine A induced histological changes of Cathepsin L and CD2AP expression in renal glomeruli and tubules.

BACKGROUND: Cyclosporine A (CsA) is used globally as an immunosuppressant for the treatment of immune-mediated nephrotic syndrome (NS). However, its long-term use causes nephrotoxicity characterized by tubulointerstitial injury and glomerulosclerosis. The present study aimed to investigate the associations between histomorphological findings and immunohistological expression of Cathepsin L (CatL) and CD2-associated protein (CD2AP) in patients with NS mediated with CsA. METHODS: A total of 18 patients with child-onset NS were divided into two groups after treatment with CsA for 2 years (group A; n = 10) and more than 4 years (group B; n = 8), respectively. Analyses of relationships between tubulointerstitial disorders and expression of CatL and CD2AP proteins were performed using immunohistochemistry of paired renal specimens. RESULTS: Glomeruli with arteriole hyalinization were significantly increased in both groups depending on dosage periods, although degrees of tubule and interstitial injury did not differ between groups. CD2AP expression was significantly greater in podocytes (P = 0.046) and was significantly less in proximal tubule cells (P = 0.014) in patients of group B compared with those of group A. Moreover, CD2AP expression was significantly increased in lateral tubule cells in both groups (group A, P = 0.02; group B, P = 0.001), and CatL expression in glomeruli and tubule cells did not change with the duration of CsA treatment in either patient group. CONCLUSIONS: CD2AP expression in renal tubules may histologically associate with tissue hypoxia and reflected recovery from CsA-mediated renal injury in patients, even with mild histological features of tubulointerstitial disorder.

Interplay between transglutaminases and heparan sulphate in progressive renal scarring.

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-ß1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.

Heme Oxygenase 1 Up-Regulates Glomerular Decay Accelerating Factor Expression and Minimizes Complement Deposition and Injury.

Complement-activation controllers, including decay accelerating factor (DAF), are gaining emphasis as they minimize injury in various dysregulated complement-activation disorders, including glomerulopathies. Heme oxygenase (HO)-1 overexpression or induction has been shown to attenuate injury in complement-dependent models of glomerulonephritis. This study investigated whether up-regulation of DAF by heme oxygenase 1 (HO-1) is an underlying mechanism by using Hmox-1-deficient rats (Hmox1+/-; Hmox1-/-) or rats with HO-1 overexpression targeted to glomerular epithelial cells (GECHO-1), which are particularly vulnerable to complement-mediated injury owing to their terminally differentiated nature. Constitutively expressed DAF was decreased in glomeruli of Hmox1-/- rats and augmented in glomeruli of GECHO-1 rats. In GECHO-1 rats with anti-glomerular basement membrane antibody mediated, complement-dependent injury, complement component C3 fragment b (C3b) deposition was reduced, whereas proteinuria was diminished. In glomeruli of wild-type rats, the natural Hmox substrate, hemin, induced glomerular DAF. This effect was attenuated in glomeruli of Hmox1-/- rats and augmented in glomeruli of GECHO-1 rats. Hemin analogues differing in either metal or porphyrin ring functionalities, acting as competitive Hmox-substrate inhibitors, also increased glomerular DAF and reduced C3b deposition after spontaneous complement activation. In the presence of a DAF-blocking antibody, the reduction in C3b deposition was reversed. These observations establish HO-1 as a physiologic regulator of glomerular DAF and identify hemin analogues as inducers of functional glomerular DAF able to minimize C3b deposition.

Instigation of NLRP3 inflammasome activation and glomerular injury in mice on the high fat diet: role of acid sphingomyelinase gene.

Ceramide has been reported to initiate inflammasome formation and activation in obesity and different pathological conditions. The present study was performed to explore the role of acid sphingomyelinase (Asm) in the development of high fat diet (HFD)-induced inflammasome and activation and consequent glomerular injury. Asm knockout (Asm(-/-)) and wild type (Asm(+/+)) mice with or without Asm short hairpin RNA (shRNA) transfection were fed a HFD or normal chow for 12 weeks to produce obesity and associated glomerular injury. HFD significantly enhanced the Asm activity, ceramide production, colocalization of Nlrp3 (Nod-like receptor protein 3) with ASC (apoptosis-associated speck-like protein) or Caspase-1, NADPH-dependent superoxide (O2(•-)) production in glomeruli of Asm(+/+) mice than in control diet-fed mice. However, such HFD-induced increases in Asm activity, ceramide production, colocalization of Nlrp3 with ASC or Caspase-1, superoxide (O(2•-)) production was attenuated in Asm(-/-) or Asm shRNA-transfected wild-type mice. In consistency with decreased inflammasome formation, the caspase-1 activity and IL-1ß production was significantly attenuated in Asm(-/-) or Asm shRNA-transfected wild-type mice fed a HFD. Morphological examinations showed that HFD-induced profound injury in glomeruli of Asm(+/+) mice which was markedly attenuated in Asm(-/-) mice. The decreased glomerular damage index in Asm(-/-) mice was accompanied by attenuated proteinuria. Fluorescent immunohistochemical examinations using podocin as a podocyte marker showed that inflammasome formation induced by the HFD were mostly located in podocytes as demonstrated by co-localization of podocin with Nlrp3. In conclusion, these observations disclose a pivotal role of Asm in the HFD-induced inflammasome formation and consequent glomerular inflammation and injury.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production.

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.