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.

Albumin induces CD44 expression in glomerular parietal epithelial cells by activating extracellular signal-regulated kinase 1/2 pathway.

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a prosclerotic and migratory PEC phenotype in glomerulosclerosis. However, the regulatory mechanisms underlying CD44 expression by activated PECs remain largely unknown. This study was performed to examine the mediators responsible for CD44 induction in glomerular PECs in association with diabetes. CD44 expression and localization were evaluated in the glomeruli of Zucker diabetic rat kidneys and primary cultured PECs upon albumin stimulation. Real-time polymerase chain reaction confirmed an albuminuria-associated upregulation of the CD44 gene in the glomeruli of diabetic rats. Immunostaining analysis of diabetic kidneys further revealed an increase in CD44 in hypertrophic PECs, which often contain albumin-positive vesicles. Losartan treatment significantly attenuated albuminuria and lowered CD44 protein levels in the diabetic kidneys. In primary cultured rat PECs, rat serum albumin (0.25-1 mg/ml) caused a dose-dependent upregulation of CD44, claudin-1, and megalin protein expression, which was accompanied by an activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. Albumin-induced CD44 and claudin-1 expression were greatly suppressed in the presence of the ERK1/2 inhibitor, U0126. In addition, knockdown of megalin by small interfering RNA interference in PECs resulted in a significant reduction of albumin-induced CD44 and claudin-1 proteins. Taken together, our results demonstrate that albumin induces CD44 expression by PECs via the activation of the ERK signaling pathway, which is partially mediated by endocytic receptor megalin.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli.

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Cinacalcet-mediated activation of the CaMKKß-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy.

Apoptosis and autophagy are harmoniously regulated biological processes for maintaining tissue homeostasis. AMP-activated protein kinase (AMPK) functions as a metabolic sensor to coordinate cellular survival and function in various organs, including the kidney. We investigated the renoprotective effects of cinacalcet in high-glucose treated human glomerular endothelial cells (HGECs), murine podocytes and C57BLKS/J-db/db mice. In cultured HGECs and podocytes, cinacalcet decreased oxidative stress and apoptosis and increased autophagy that were attributed to the increment of intracellular Ca2+ concentration and the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinaseß (CaMKKß)-Liver kinase B1 (LKB1)-AMPK and their downstream signals including the phosphorylation of endothelial nitric oxide synthase (eNOS) and increases in superoxide dismutases and B cell leukemia/lymphoma 2/BCL-2-associated X protein expression. Interestingly, intracellular chelator BAPTA-AM reversed cinacalcet-induced CaMKKß elevation and LKB1 phosphorylation. Cinacalcet reduced albuminuria without influencing either blood glucose or Ca2+ concentration and ameliorated diabetes-induced renal damage, which were related to the increased expression of calcium-sensing receptor and the phosphorylation of CaMKKß-LKB1. Subsequent activation of AMPK was followed by the activation of peroxisome proliferator-activated receptor γ coactivator-1α and phospho-Ser1177eNOS-nitric oxide, resulting in a decrease in apoptosis and oxidative stress as well as an increase in autophagy.Our results suggest that cinacalcet increases intracellular Ca2+ followed by an activation of CaMKKß-LKB1-AMPK signaling in GECs and podocytes in the kidney, which provides a novel therapeutic means for type 2 diabetic nephropathy by modulation of apoptosis and autophagy.

Ste20-like kinase, SLK, a novel mediator of podocyte integrity.

SLK is essential for embryonic development and may play a key role in wound healing, tumor growth, and metastasis. Expression and activation of SLK are increased in kidney development and during recovery from ischemic acute kidney injury. Overexpression of SLK in glomerular epithelial cells/podocytes in vivo induces injury and proteinuria. Conversely, reduced SLK expression leads to abnormalities in cell adhesion, spreading, and motility. Tight regulation of SLK expression thus may be critical for normal renal structure and function. We produced podocyte-specific SLK-knockout mice to address the functional role of SLK in podocytes. Mice with podocyte-specific deletion of SLK showed reduced glomerular SLK expression and activity compared with control. Podocyte-specific deletion of SLK resulted in albuminuria at 4-5 mo of age in male mice and 8-9 mo in female mice, which persisted for up to 13 mo. At 11-12 mo, knockout mice showed ultrastructural changes, including focal foot process effacement and microvillous transformation of podocyte plasma membranes. Mean foot process width was approximately twofold greater in knockout mice compared with control. Podocyte number was reduced by 35% in knockout mice compared with control, and expression of nephrin, synaptopodin, and podocalyxin was reduced in knockout mice by 20-30%. In summary, podocyte-specific deletion of SLK leads to albuminuria, loss of podocytes, and morphological evidence of podocyte injury. Thus, SLK is essential to the maintenance of podocyte integrity as mice age.

Heme oxygenase-1 is a potent inhibitor of placental ischemia-mediated endothelin-1 production in cultured human glomerular endothelial cells.

Preeclampsia is a pregnancy-specific disorder of maternal hypertension and reduced renal hemodynamics linked to reduced endothelial function. Placental ischemia is thought to be the culprit of this disease, as it causes the release of factors like tumor necrosis factor (TNF)-α that induce vascular endothelin-1 (ET-1) production. Interestingly, placental ischemia-induced hypertension in rats [reduced uterine perfusion pressure (RUPP) model] is abolished by ETA receptor blockade, suggesting a critical role for ET-1. Although it has been found that systemic induction of heme oxygenase (HO)-1 is associated with reduced ET-1 production and attenuated hypertension, it is unclear whether HO-1 directly modulates the increased ET-1 response to placental factors. We tested the hypothesis that HO-1 or its metabolites inhibit ET-1 production in human glomerular endothelial cells induced by serum of RUPP rats or TNF-α. Serum (5%) from RUPP hypertensive (mean arterial blood pressure 119 ± 9 mmHg) vs. normotensive pregnant (NP, 101 ± 6 mmHg, P < 0.001) rats increased ET-1 production (RUPP 168.8 ± 18.1 pg/ml, NP 80.3 ± 22.7 pg/ml, P < 0.001, n = 12/group). HO-1 induction [25 µM cobalt photoporphyrin (CoPP)] abolished RUPP serum-induced ET-1 production (1.6 ± 0.8 pg/ml, P < 0.001), whereas bilirubin (10 µM) significantly attenuated ET-1 release (125.3 ± 5.2 pg/ml, P = 0.005). Furthermore, TNF-α-induced ET-1 production (TNF-α 31.0 ± 8.4 vs. untreated 7.5 ± 0.4 pg/ml, P < 0.001) was reduced by CoPP (1.5 ± 0.8 pg/ml, P < 0.001) and bilirubin (10.5 ± 4.3 pg/ml, P < 0.001). These results suggest that circulating factors released during placental ischemia target the maternal glomerular endothelium to increase ET-1, and that pharmacological induction of HO-1 or bilirubin could be a treatment strategy to block this prohypertensive pathway in preeclampsia.

Matrix metalloproteinase-12 deficiency attenuates experimental crescentic anti-glomerular basement membrane glomerulonephritis.

AIM: Matrix metalloproteinase-12 (MMP-12; macrophage elastase) is an enzyme that can cleave various extracellular matrix proteins and is required for macrophage infiltration and pulmonary fibrosis in experimental emphysema. We have shown previously that MMP-12 is highly up-regulated in experimental anti-glomerular basement membrane (GBM) disease. The aim of this study was to determine whether MMP-12 is required for glomerular macrophage infiltration and crescent formation in anti-GBM glomerulonephritis. METHODS: Accelerated anti-GBM disease was induced in groups of MMP-12 gene deficient mice (MMP-12-/-) and wild-type C57BL/6J controls, which were killed 12 days after injection of anti-GBM serum. RESULTS: Wild-type and MMP-12-/- mice developed glomerular damage and glomerular tuft adhesions to Bowman's capsule. Both groups developed severe proteinuria. Wild-type mice also developed significant loss of renal function and crescents in 22% of glomeruli, which were associated with macrophage infiltration and Bowman's capsule rupture. In contrast, MMP-12-/- mice were partially protected from renal function decline, crescent formation and Bowman's capsule rupture. This was associated with reduced macrophage infiltration in both glomeruli and the interstitium, and with reduced expression of CCL2, TNF-α and iNOS mRNA in MMP-12-/- kidneys. In addition, KIM-1 mRNA levels were reduced in MMP-12-/- mice indicating less tubular damage. CONCLUSION: These data demonstrate that endogenous MMP-12 facilitates macrophage accumulation and activation in anti-GBM glomerulonephritis which is required for glomerular crescent formation, Bowman's capsule rupture, tubular damage and renal function decline.

The persistent inhibitory properties of saxagliptin on renal dipeptidyl peptidase-4: Studies with HK-2 cells in vitro and normal rats in vivo.

Saxagliptin, a potent and selective DPP-4 inhibitor, exhibits a slow dissociation from DPP-4. We investigated the sustained effects of saxagliptin on renal DPP-4 activity in a washout study using renal tubular (HK-2) cells, and in a pharmacodynamic study using normal rats. In HK-2 cells, the inhibitory potency of saxagliptin on DPP-4 activity persisted after washout, while that of sitagliptin was clearly reduced. In normal rats, a single treatment of saxagliptin or sitagliptin inhibited the plasma DPP-4 activity to similar levels. The inhibitory action of saxagliptin on the renal DPP-4 activity was retained, even when its inhibitory effect on the plasma DPP-4 activity disappeared. However, the inhibitory action of sitagliptin on the renal DPP-4 activity was abolished in correlation with the inhibition of the plasma DPP-4 activity. In situ staining showed that saxagliptin suppressed the DPP-4 activity in both glomerular and tubular cells and its inhibitory effects were significantly higher than those of sitagliptin. Saxagliptin exerted a sustained inhibitory effect on the renal DPP-4 activity in vitro and in vivo. The long binding action of saxagliptin in renal tubular cells might involve the sustained inhibition of renal DPP-4.

Reciprocal regulation of miR-214 and PTEN by high glucose regulates renal glomerular mesangial and proximal tubular epithelial cell hypertrophy and matrix expansion.

Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications, including renal hypertrophy and matrix protein accumulation. Reduced expression of phosphatase and tensin homolog (PTEN) by hyperglycemia contributes to these processes. We considered involvement of miR in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'-UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both of these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates glycogen synthase kinase-3ß, PRAS40, and tuberin. In contrast, quenching of miR-214 inhibited high-glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by small interfering RNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mammalian target of rapamycin complex 1 (mTORC1) activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high-glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high-glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.

Glomerular Epithelial Cells-Targeted Heme Oxygenase-1 Over Expression in the Rat: Attenuation of Proteinuria in Secondary But Not Primary Injury.

BACKGROUND/AIMS: Induction of heme oxygenase 1 (HO-1) in glomerular epithelial cells (GEC) in response to injury is poor and this may be a disadvantage. We, therefore, explored whether HO-1 overexpression in GEC can reduce proteinuria induced by puromycin aminonucleoside (PAN) or in anti-glomerular basement membrane (GBM) antibody (Ab)-mediated glomerulonephritis (GN). METHODS: HO-1 overexpression in GEC (GECHO-1) of Sprague-Dawley rats was achieved by targeting a FLAG-human (h) HO-1 using transposon-mediated transgenesis. Direct GEC injury was induced by a single injection of PAN. GN was induced by administration of an anti-rat GBM Ab and macrophage infiltration in glomeruli was assessed by immunohistochemistry and western blot analysis, which was also used to assess glomerular nephrin expression. RESULTS: In GECHO-1 rats, FLAG-hHO-1 transprotein was co-immunolocalized with nephrin. Baseline glomerular HO-1 protein levels were higher in GECHO-1 compared to wild type (WT) rats. Administration of either PAN or anti-GBM Ab to WT rats increased glomerular HO-1 levels. Nephrin expression markedly decreased in glomeruli of WT or GECHO-1 rats treated with PAN. In anti-GBM Ab-treated WT rats, nephrin expression also decreased. In contrast, it was preserved in anti-GBM Ab-treated GECHO-1 rats. In these, macrophage infiltration in glomeruli and the ratio of urine albumin to urine creatinine (Ualb/Ucreat) were markedly reduced. There was no difference in Ualb/Ucreat between WT and GECHO-1 rats treated with PAN. CONCLUSION: Depending on the type of injury, HO-1 overexpression in GEC may or may not reduce proteinuria. Reduced macrophage infiltration and preservation of nephrin expression are putative mechanisms underlying the protective effect of HO-1 overexpression following immune 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.

Antioxidant diet and sex interact to regulate NOS isoform expression and glomerular mesangium proliferation in Zucker diabetic rat kidney.

Oxidative stress contributes substantially to the pathophysiology of diabetic nephropathy (DN). Consumption of an antioxidant-fortified (AO) diet from an early age prevents or delays later development of DN in the Zucker rat female with type 2 diabetes. We hypothesize this is due to effects on mesangial matrix and renal nitric oxide synthase (NOS) distribution and to sex-specific differences in NOS responses in the diabetic kidney. Total glomerular tuft area (GTA) and PAS-positive tuft area (PTA), endothelial (e), neuronal (n) and inducible (i) NOS were quantified in males and females on AO or regular (REG) diet at 6 and 20 weeks of age. eNOS was observed in glomeruli and tubules. nNOS predominantly localized to tubular epithelium in both cortex and medulla. iNOS was expressed in proximal and distal tubules and collecting ducts. Sex, diabetes duration and AO diet affected the distribution of the three isoforms. GTA and PTA increased with duration of hyperglycemia and showed a negative correlation with renal levels of all NOS isoforms. AO diet in both genders was associated with less PAS-positive staining and less mesangial expansion than the REG diet, an early increase in cortical iNOS in males, and sex-specific changes in cortical eNOS at 20 weeks. These effects of AO diet may contribute to sex-specific preservation of renal function in females.

Matrix metalloproteinase 12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity.

Obesity-induced kidney injury contributes to albuminuria, which is characterized by a progressive decline in renal function leading to glomerulosclerosis and renal fibrosis. Matrix metalloproteinases (MMPs) modulate inflammation and fibrosis by degrading a variety of extracellular matrix and regulating the activities of effector proteins. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm, atherosclerosis, and emphysema, but the underlying mechanisms remain unclear. The present study examined the function of MMP-12 in glomerular fibrogenesis and inflammation using apo E(-/-) or apo E(-/-)MMP-12(-/-) mice and maintained on a high-fat-diet (HFD) for 3, 6, or 9 months. MMP-12 deletion reduced glomerular matrix accumulation, and downregulated the expression of NADPH oxidase 4 and the subunit-p67(phox), indicating the inhibition of renal oxidative stress. In addition, the expression of the inflammation-associated molecule MCP-1 and macrophage marker-CD11b was decreased in glomeruli of apo E(-/-)MMP-12(-/-) mice fed HFD. MMP-12 produced by macrophages infiltrating into glomeruli contributed to the degradation of collagen type IV and fibronectin. Crescent formation due to renal oxidative stress in Bowman's space was a major factor in the development of fibrogenesis and inflammation. These results suggest that regulating MMP-12 activity could be a therapeutic strategy for the treatment of crescentic glomerulonephritis and fibrogenesis.

Heparan sulfate 6-O-endosulfatases, Sulf1 and Sulf2, regulate glomerular integrity by modulating growth factor signaling.

Glomerular integrity and functions are maintained by growth factor signaling. Heparan sulfate, the major component of glomerular extracellular matrixes, modulates growth factor signaling, but its roles in glomerular homeostasis are unknown. We investigated the roles of heparan sulfate 6-O-endosulfatases, sulfatase (Sulf)1 and Sulf2, in glomerular homeostasis. Both Sulf1 and Sulf2 were expressed in the glomeruli of wild-type (WT) mice. Sulf1 and Sulf2 double-knockout (DKO) mice showed glomerular hypercellularity, matrix accumulation, mesangiolysis, and glomerular basement membrane irregularity. Platelet-derived growth factor (PDGF)-B and PDGF receptor-ß were upregulated in Sulf1 and Sulf2 DKO mice compared with WT mice. Glomeruli from Sulf1 and Sulf2 DKO mice in vitro stimulated by either PDGF-B, VEGF, or transforming growth factor-ß similarly showed reduction of phospho-Akt, phospho-Erk1/2, and phospho-Smad2/3, respectively. Since glomerular lesions in Sulf1 and Sulf2 DKO mice were reminiscent of diabetic nephropathy, we examined the effects of Sulf1 and Sulf2 gene disruption in streptozotocin-induced diabetes. Diabetic WT mice showed an upregulation of glomerular Sulf1 and Sulf2 mRNA by in situ hybridization. Diabetic DKO mice showed significant increases in albuminuria and serum creatinine and an acceleration of glomerular pathology without glomerular hypertrophy; those were associated with a reduction of glomerular phospho-Akt. In conclusion, Sulf1 and Sulf2 play indispensable roles to maintain glomerular integrity and protective roles in diabetic nephropathy, probably by growth factor modulation.

HDAC expression and activity is upregulated in diseased lupus-prone mice.

Prior studies have shown that pan-HDAC inhibition can decrease disease in lupus mice; however, the mechanisms(s) remain to be elucidated. MRL/MpJ-Fas(lpr) (MRL/lpr) mice develop a lupus-like disease characterized by anti-dsDNA production, lymphoproliferation, and immune complex-mediated glomerulonephritis. Early- and late-disease (12 and 20weeks-of-age respectively) female MRL/lpr mice were compared to age-matched, healthy C57BL/6 mice for HDAC expression and activity in bone marrow (BM) B cells, splenic B and T cells, and glomerular cells. We found that HDAC6 was significantly overexpressed in B cells, splenic T cells and glomerular cells, whereas HDAC9 expression was significantly increased in splenic T cells, BM B cells and glomerular cells. Due to the overexpression of HDAC6, we tested whether treatment with a selective HDAC6 inhibitor (ACY-738) or a pan-HDAC inhibitor (TsA) would decrease HDAC activity. ACY-738 significantly reduced cytoplasmic HDAC activity whereas TsA significantly decreased both nuclear and cytoplasmic HDAC activity. In vitro studies in mesangial cells showed that ACY-738 increased α-tubulin and Hsp90 acetylation resulting in decreased nuclear activation of NF-κB. Treatment of pre-B cells with ACY-738 decreased the Bcl-2:Bax ratio leading to a pro-apoptotic environment. These results suggest that increased HDAC6 expression and activity contribute to SLE pathogenesis, and isoform-selective HDAC inhibitors may prove beneficial in the treatment of SLE by acetylating key signaling and transcription factors in inflammation and cell activation.

Antibodies to m-type phospholipase A2 receptor in children with idiopathic membranous nephropathy.

Idiopathic membranous nephropathy (IMN), the commonest cause of adult nephrotic syndrome (NS), accounts for only a minority of paediatric NS. Antibodies to m-type phospholipase A2 receptor (PLA2R) are seen in two-thirds of adult IMN cases. PLA2R staining in glomerular deposits is observed in 74% and 45% of adult and paediatric IMN cases, respectively. However, there are no reports of anti-PLA2R in paediatric IMN. We evaluated anti-PLA2R levels and PLA2R in gloemrular deposits in paediatric IMN seen at our center. Five cases were enrolled, all the cases stained for PLA2R in glomeruli and three (60%) had antibodies to PLA2R antigen. There was a parellel reduction in proteinuria and anti-PLA2R titer. The present report suggests that PLA2R has a contributory role in the pathogenesis of paediatric IMN.

Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

Aldo-keto reductase 1b7, a novel marker for renin cells, is regulated by cyclic AMP signaling.

We previously identified aldo-keto reductase 1b7 (AKR1B7) as a marker for juxtaglomerular renin cells in the adult mouse kidney. However, the distribution of renin cells varies dynamically, and it was unknown whether AKR1B7 maintains coexpression with renin in response to different developmental, physiological, and pathological situations, and furthermore, whether similar factor(s) simultaneously regulate both proteins. We show here that throughout kidney development, AKR1B7 expression-together with renin-is progressively restricted in the kidney arteries toward the glomerulus. Subsequently, when formerly renin-expressing cells reacquire renin expression, AKR1B7 is reexpressed as well. This pattern of coexpression persists in extreme pathological situations, such as deletion of the genes for aldosterone synthase or Dicer. However, the two proteins do not colocalize within the same organelles: renin is found in the secretory granules, whereas AKR1B7 localizes to the endoplasmic reticulum. Interestingly, upon deletion of the renin gene, AKR1B7 expression is maintained in a pattern mimicking the embryonic expression of renin, while ablation of renin cells resulted in complete abolition of AKR1B7 expression. Finally, we demonstrate that AKR1B7 transcription is controlled by cAMP. Cultured cells of the renin lineage reacquire the ability to express both renin and AKR1B7 upon elevation of intracellular cAMP. In vivo, deleting elements of the cAMP-response pathway (CBP/P300) results in a stark decrease in AKR1B7- and renin-positive cells. In summary, AKR1B7 is expressed within the renin cell throughout development and perturbations to homeostasis, and AKR1B7 is regulated by cAMP levels within the renin cell.

Critical role of renal dipeptidyl peptidase-4 in ameliorating kidney injury induced by saxagliptin in Dahl salt-sensitive hypertensive rats.

Saxagliptin, a potent dipeptidyl peptidase-4 (DPP-4) inhibitor, is currently used to treat type 2 diabetes mellitus, and it has been reported to exhibit a slower rate of dissociation from DPP-4 compared with another DPP-4 inhibitor, sitagliptin. In this study, we compared the effects of saxagliptin and sitagliptin on hypertension-related renal injury and the plasma and renal DPP-4 activity levels in Dahl salt-sensitive hypertensive (Dahl-S) rats. The high-salt diet (8% NaCl) significantly increased the blood pressure and quantity of urinary albumin excretion and induced renal glomerular injury in the Dahl-S rats. Treatment with saxagliptin (14mg/kg/day via drinking water) for 4 weeks significantly suppressed the increase in urinary albumin excretion and tended to ameliorate glomerular injury without altering the blood glucose levels and systolic blood pressure. On the other hand, the administration of sitagliptin (140mg/kg/day via drinking water) did not affect urinary albumin excretion and glomerular injury in the Dahl-S rats. Meanwhile, the high-salt diet increased the renal DPP-4 activity but did not affect the plasma DPP-4 activity in the Dahl-S rats. Both saxagliptin and sitagliptin suppressed the plasma DPP-4 activity by 95% or more. Although the renal DPP-4 activity was also inhibited by both drugs, the inhibitory effect of saxagliptin was more potent than that of sitagliptin. These results indicate that saxagliptin has a potent renoprotective effect in the Dahl-S rats, independent of its glucose-lowering actions. The inhibition of the renal DPP-4 activity induced by saxagliptin may contribute to ameliorating renal injury in hypertension-related renal injury.

Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.