Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Regulation of NLRP3 Inflammasome Activation and Inflammatory Exosome Release in Podocytes by Acid Sphingomyelinase During Obesity.

The activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been reported to importantly contribute to glomerular inflammation and injury under different pathological conditions such as obesity. However, the mechanism mediating NLRP3 inflammasome activation in podocytes and subsequent glomerular injury remains poorly understood. Given that the ceramide signaling pathway has been reported to be implicated in obesity-related glomerulopathy (ORG), the present study was designed to test whether the ceramide-producing enzyme, acid sphingomyelinase (ASM), determines NLRP3 inflammasome activation and inflammatory exosome release in podocytes leading to glomerular inflammation and injury during ORG. In Smpd1trg/Podocre mice, podocyte-specific overexpression of Smpd1 gene which encodes ASM significantly exaggerated high-fat diet (HFD)-induced NLRP3 inflammasome activation in podocytes and immune cell infiltration in glomeruli compared to WT/WT mice. Smpd1 gene deletion, however, blocked these pathological changes induced by HFD in Smpd1-/- mice. Accompanied with NLRP3 inflammasome activation and glomerular inflammation, urinary excretion of exosomes containing podocyte marker and NLRP3 inflammasome products (IL-1ß and IL-18) in Smpd1trg/Podocre mice on the HFD was much higher than that in WT/WT mice. In contrast, Smpd1-/- mice on the HDF had significantly lower urinary exosome excretion than WT/WT mice. Correspondingly, HFD-induced podocyte injury, glomerular sclerosis, and proteinuria were more severe in Smpd1trg/Podocre mice, but milder in Smpd1-/- mice compared to WT/WT mice. Using podocytes isolated from these mice, we demonstrated that visfatin, a prototype pro-inflammatory adipokine, induced NLRP3 inflammasome activation and enrichment of multivesicular bodies (MVBs) containing IL-1ß in podocytes, which was much stronger in podocytes from Smpd1trg/Podocre mice, but weaker in those from Smpd1-/- mice than WT/WT podocytes. By quantitative analysis of exosomes, it was found that upon visfatin stimulation, podocytes from Smpd1trg/Podocre mice released much more exosomes containing NLRP3 inflammasome products, but podocytes from Smpd1-/- mice released much less exosomes compared to WT/WT podocytes. Super-resolution microscopy demonstrated that visfatin inhibited lysosome-MVB interaction in podocytes, indicating impaired MVB degradation by lysosome. The inhibition of lysosome-MVB interaction by visfatin was amplified by Smpd1 gene overexpression but attenuated by Smpd1 gene deletion. Taken together, our results suggest that ASM in podocytes is a crucial regulator of NLRP3 inflammasome activation and inflammatory exosome release that instigate glomerular inflammation and injury during obesity.

Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel.

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.

Cerebral Salt-Wasting Syndrome in a Patient With Active Pulmonary Tuberculosis.

A 37-year-old female with a medical history of recently diagnosed active pulmonary tuberculosis and a new intracranial lesion presented with altered mental status, nausea, and vomiting for two days. An initial physical examination revealed that the patient was euvolemic. Laboratory findings revealed a serum sodium concentration of 105 mEq/L. During her admission, she was initially managed with lactated ringer solution in the emergency department, followed by 3% normal saline in the intensive care unit, and, eventually, on oral sodium chloride and fluid restriction on discharge. Once she was stabilized, she had episodes of dizziness, and concerns were raised about the salt-wasting syndrome.

Podocyte Sphingolipid Signaling in Nephrotic Syndrome.

Podocytes play a vital role in the pathogenesis of nephrotic syndrome (NS), which is clinically characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, and peripheral edema. The pathogenesis of NS has evolved through several hypotheses ranging from immune dysregulation theory and increased glomerular permeability theory to the current concept of podocytopathy. Podocytopathy is characterized by dysfunction or depletion of podocytes, which may be caused by unknown permeability factor, genetic disorders, drugs, infections, systemic disorders, and hyperfiltration. Over the last two decades, numerous studies have been done to explore the molecular mechanisms of podocyte injuries or NS and to develop the novel therapeutic strategies targeting podocytopathy for treatment of NS. Recent studies have shown that normal sphingolipid metabolism is essential for structural and functional integrity of podocytes. As a basic component of the plasma membrane, sphingolipids not only support the assembly of signaling molecules and interaction of receptors and effectors, but also mediate various cellular activities, such as apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation. This review briefly summarizes current evidence demonstrating the regulation of sphingolipid metabolism in podocytes and the canonical or noncanonical roles of podocyte sphingolipid signaling in the pathogenesis of NS and associated therapeutic strategies.

Abnormal podocyte TRPML1 channel activity and exosome release in mice with podocyte-specific Asah1 gene deletion.

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1fl/fl/PodoCre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1fl/fl/PodoCre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1fl/fl/PodoCre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1fl/fl/PodoCre mice compared to WT/WT mice. In the podocytes from Asah1fl/fl/PodoCre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited in the podocytes of Asah1fl/fl/PodoCre mice. By GCaMP3 Ca2+ imaging, it was found that lysosomal Ca2+ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1fl/fl/PodoCre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1fl/fl/PodoCre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1fl/fl/PodoCre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.

Podocytopathy and Nephrotic Syndrome in Mice with Podocyte-Specific Deletion of the Asah1 Gene: Role of Ceramide Accumulation in Glomeruli.

Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of the α subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatography-tandem mass spectrometry was demonstrated in isolated glomeruli of Asah1fl/fl/PodoCre mice compared with their littermates. By crossbreeding Asah1fl/fl/PodoCre mice with Smpd1-/- mice, we also produced a double knockout strain, Smpd1-/-/Asah1fl/fl/PodoCre, that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1fl/fl/PodoCre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for the maintenance of the structural and functional integrity of podocytes.

Impaired Delivery of Cholesterol Effluxed From Macrophages to Hepatocytes by Serum From CKD Patients May Underlie Increased Cardiovascular Disease Risk.

INTRODUCTION: Although chronic kidney disease (CKD) is associated with increased risk for coronary artery disease (CAD), the underlying mechanisms are not completely defined. In the present study, we tested the hypothesis that flux of cholesterol from macrophage foam cells to liver is impaired in subjects with CKD. METHODS: Consecutive healthy patients, patients with at least 1 CAD risk factor, patients with established CAD, and patients with CKD stages G3 to G5 (n ≥ 15/group) were recruited prospectively. The ability of total patient serum without any modifications to (i) facilitate efflux of cholesterol from human THP1-macrophage foam cells under physiological conditions (cholesterol efflux capacity [CEC]) and (ii) to deliver this effluxed cholesterol to primary hepatocytes with physiological expression of high-density lipoprotein (HDL) receptor SR-BI (capacity to deliver cholesterol to hepatocytes [CDCH]) was evaluated. RESULTS: Although healthy patients, patients with at least 1 CAD risk factor, and patients with established CAD all showed similar CEC, patients with CKD showed significantly higher CEC. CDCH was significantly lower in all groups compared with the healthy patients; however, when corrected for higher CEC, CDCH in patients with CKD was significantly lower than in patients with CAD. CDCH correlated with age, body mass index, metabolic parameters, inflammatory markers, and kidney function markers (estimated glomerular filtration rate [eGFR], serum creatinine, and serum cystatin C). CONCLUSIONS: These results suggest that aberrations in delivery of cholesterol effluxed from macrophage foam cells to liver for final elimination or the last step of reverse cholesterol transport, may underlie the increased risk of CAD in patients with CKD.

Ultra-short duration direct acting antiviral prophylaxis to prevent virus transmission from hepatitis C viremic donors to hepatitis C negative kidney transplant recipients.

We conducted an adaptive design single-center pilot trial between October 2017 and November 2018 to determine the safety and efficacy of ultra-short-term perioperative pangenotypic direct acting antiviral (DAA) prophylaxis for deceased hepatitis C virus (HCV)-nucleic acid test (NAT) positive donors to HCV negative kidney recipients (D+/R-). In Group 1, 10 patients received one dose of SOF/VEL (sofusbuvir/velpatasvir) pretransplant and one dose on posttransplant Day 1. In Group 2A (N = 15) and the posttrial validation (Group 2B; N = 25) phase, patients received two additional SOF/VEL doses (total 4) on Days 2 and 3 posttransplant. Development of posttransplant HCV transmission triggered 12-week DAA therapy. For available donor samples (N = 27), median donor viral load was 1.37E + 06 IU/mL (genotype [GT]1a: 70%; GT2: 7%; GT3: 23%). Overall viral transmission rate was 12% (6/50; Group 1:30% [3/10]; Group 2A:13% [2/15]; Group 2B:4% [1/25]). For the 6 viremic patients, 5 (83%) achieved sustained virologic response (3 with first-line DAA therapy; and two after retreatment with second-line DAA). At a median follow-up of 8 months posttransplant, overall patient and allograft survivals were 98%, respectively. The 4-day strategy reduced viral transmission to 7.5% (3/40; 95% confidence interval [CI]: 1.8%-20.5%) and could result in avoidance of prolonged posttransplant DAA therapy for most D+/R - transplants.

Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression.

BACKGROUND: The associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function. METHODS: Renal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls. RESULTS: The Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia. CONCLUSIONS: Butyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.

Curcumin-mediated regulation of intestinal barrier function: The mechanism underlying its beneficial effects.

Curcumin has anti-inflammatory, anti-oxidant and anti-proliferative properties established largely by in vitro studies. Accordingly, oral administration of curcumin beneficially modulates many diseases including diabetes, fatty-liver disease, atherosclerosis, arthritis, cancer and neurological disorders such as depression, Alzheimer's or Parkinson's disease. However, limited bioavailability and inability to detect curcumin in circulation or target tissues has hindered the validation of a causal role. We established curcumin-mediated decrease in the release of gut bacteria-derived lipopolysaccharide (LPS) into circulation by maintaining the integrity of the intestinal barrier function as the mechanism underlying the attenuation of metabolic diseases (diabetes, atherosclerosis, kidney disease) by curcumin supplementation precluding the need for curcumin absorption. In view of the causative role of circulating LPS and resulting chronic inflammation in the development of diseases listed above, this review summarizes the mechanism by which curcumin affects the several layers of the intestinal barrier and, despite negligible absorption, can beneficially modulate these diseases.

Use of Diuretics in the Treatment of Heart Failure in Older Adults.

Diuretics are the most commonly prescribed class of drugs in patients with heart failure, and in the short term they remain the most effective treatment for relief from fluid congestion. This article reviews the mode of action of the various diuretic classes and the physiologic adaptations that follow and sets up the basis for their use in the treatment of volume-retaining states, particularly as applies to the elderly. In addition, the article reviews the common side effects related to diuretics.

Sphingolipids in obesity and related complications.

Sphingolipids are biologically active lipids ubiquitously produced in all vertebrate cells. Asides from structural components of cell membrane, sphingolipids also function as intracellular and extracellular mediators that regulate many important physiological cellular processes including cell survival, proliferation, apoptosis, differentiation, migration and immune processes. Recent studies have also indicated that disruption of sphingolipid metabolism is strongly associated with different diseases that exhibit diverse neurological and metabolic consequences. Here, we briefly summarize current evidence for understanding of sphingolipid pathways in obesity and associated complications. The regulation of sphingolipids and their enzymes may have a great impact in the development of novel therapeutic modalities for a variety of metabolic diseases.

Characterization and Activation of NLRP3 Inflammasomes in the Renal Medulla in Mice.

BACKGROUND/AIMS: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. METHODS AND RESULTS: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1ß production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. CONCLUSION: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response.

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.

Pre-transplant phospholipase A2 receptor autoantibody concentration is associated with clinically significant recurrence of membranous nephropathy post-kidney transplantation.

Previous studies that have assessed the association of pre-transplant antiphospholipase A2 receptor autoantibody (PLA2R-Ab) concentration with a recurrence of membranous nephropathy (rMN) post-kidney transplant have yielded variable results. We tested 16 consecutive transplant patients with a history of iMN for pre-transplant PLA2R-Ab. Enzyme-linked immunosorbent assay titers (Euroimmun, NJ, USA) >14 RU/mL were considered positive. A receiver operating characteristic (ROC) analysis was performed after combining data from Quintana et al. (n = 21; Transplantation February 2015) to determine a PLA2R-Ab concentration which could predict rMN. Six of 16 (37%) patients had biopsy-proven rMN at a median of 3.2 yr post-transplant. Of these, five of six (83%) had a positive PLA2R-Ab pre-transplant with a median of 82 RU/mL (range = 31-1500). The only patient who had rMN with negative PLA2R-Ab was later diagnosed with B-cell lymphoma. One hundred percent (n = 10) of patients with no evidence of rMN (median follow-up = five yr) had negative pre-transplant PLA2R-Ab. In a combined ROC analysis (n = 37), a pre-transplant PLA2R-Ab > 29 RU/mL predicted rMN with a sensitivity of 85% and a specificity of 92%. Pre-transplant PLA2R-Ab could be a useful tool for the prediction of rMN. Patients with rMN in the absence of PLA2R-Ab should be screened for occult malignancy and/or alternate antigens.

A model of acute kidney injury in mice with cirrhosis and infection.

BACKGROUND & AIMS: Infectious acute kidney injury (AKI) is a life threatening complication of cirrhosis with limited therapeutic options. The aim of this study was to develop a model of infectious AKI in cirrhotic mice. METHODS: Cirrhosis was established by intragastric administration of carbon tetrachloride (CCl4 ). Systemic haemodynamics was assessed invasively while cardiac function was assessed by echocardiography. AKI was induced using varying doses of lipopolysaccharide (LPS) titrated to produce 50% lethality. Renal function was assessed from serum creatinine and urine output (UOP). Renal injury was evaluated by urinalysis (proteinuria and casts) and renal histology. These mice were compared to: (i) normal mice, (ii) normal mice + LPS, and (iii) mice treated with CCl4 alone. RESULTS: Cirrhosis with increased cardiac output, decreased systemic vascular resistance, activation of renin-angiotensin-aldosterone axis developed after 12 weeks of CCl4 administration. LPS injection produced a dose-dependent increase in mortality (33% at 2 mg/kg vs. 80% at 6 mg/kg) without urine (casts or proteinuria) or histological evidence of tubular injury. 2 mg/kg LPS injection produced a rise in creatinine (0.79 ± 0.27 mg/dl in CCl4 +LPS compared to 0.45 ± 0.14 in CCl4 alone, P < 0.05) and a decrease in UOP (0.86 ± 0.4 ml/16 h in CCl4 + LPS compared to 1.70 ± 0.7 ml/16 h in CCl4 mice, P < 0.05). UOP remained low in mice that died while it recovered over 48-72 h in those that recovered. Control mice treated with 2 mg/kg LPS did not experience AKI. CONCLUSIONS: Cirrhotic CCl4 treated mice develop functional AKI and mimic most of the features of infectious AKI following LPS injection.

High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide.

A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.

Enhanced epithelial-to-mesenchymal transition associated with lysosome dysfunction in podocytes: role of p62/Sequestosome 1 as a signaling hub.

BACKGROUND: Autophagy is of importance in the regulation of cell differentiation and senescence in podocytes. It is possible that derangement of autophagy under different pathological conditions activates or enhances Epithelial-to-Mesenchymal Transition (EMT) in podocytes, resulting in glomerular sclerosis. To test this hypothesis, the present study produced lysosome dysfunction by inhibition of the vacuolar H(+)-ATPase (V-ATPase) to test whether deficiency of autophagic flux leads to enhancement of EMT in podocytes. METHODS AND RESULTS: By Western blot and confocal analysis, lysosome inhibition using a V-ATPase inhibitor or its siRNA was found to markedly decreases the epithelial markers (P-cadherin and ZO-1) and increases the mesenchymal markers (FSP-1 and α-SMA). This enhancement was accompanied by deficient autophagic flux, as demonstrated by marked increases in LC3B-II and p62/Sequestosome 1. However, inhibition of autophagosome formation using spaudin-1 significantly attenuated both enhancement of EMT and deficiency of autophagic flux. To explore the mechanisms by which deficient autophagic flux enhances EMT, we tested the role of accumulated p62 as a signal hub in this process. Neither the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear kappa-light-chain-enhancer pathways of p62 contributed to enhanced EMT. However, inhibition of cyclin-dependent kinase 1 (CDK1) activity reduced the phosphorylation of p62 and enhanced EMT in podocytes similar to lysosome dysfunction. CONCLUSION: The lack of phosphorylated p62 leads to a faster exit from cell mitosis, enhanced EMT associated with lysosome dysfunction may be attributed to accumulation of p62 and associated reduction of p62 phosphorylation.

Differences in coagulation in clotting of vascular access in hemodialysis patients.

Arteriovenous graft (AVG) thrombosis is a frequent cause of graft failure. We evaluated coagulation protein concentrations, platelet function, and viscoelasticity factors in 20 hemodialysis (HD) patients with AVGs. The goal was to determine whether significant differences in protein concentrations, platelet function, and viscoelasticity factors exist among dialysis patients requiring frequent AVG declot procedures vs. those who do not. Twenty HD patients were enrolled: 10 frequent clotters (>3 declots in the previous year) and 10 were nonclotters. Patients on antiplatelets or chronic anticoagulation were excluded. Laboratories were drawn pretreatment and heparinase was added to counteract any potential heparin effect. Coagulation protein concentrations including tissue factor (TF), thrombin/antithrombin III complex (TAT), and prothrombin fragment 1 + 2 (F(1+2)) were assayed. The time to clot onset was measured by force onset time (FOT). Platelet contractile force (PCF) measured the force produced by platelets during clot retraction, whereas clot rigidity was measured as clot elastic modulus (CEM). FOT, CEM, and PCF were measured by Hemodyne. Both groups had upregulation of the TF pathway, as TF, TAT, and F(1+2) levels were similarly increased over baseline levels. Hemodialysis patients with frequent AVG clotting had higher levels of both PCF and CEM compared with nonclotters. Additionally, the frequent clotters had a lower FOT relative to nonclotters, although both were considered in the normal range. Our study suggests that HD patients with recurrent AVG thrombotic events form clots with higher tensile strength compared with HD patients without recurrent graft thrombosis.