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.

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.

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.

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.

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.

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.

Nod-like receptor protein 3 (NLRP3) inflammasome activation and podocyte injury via thioredoxin-interacting protein (TXNIP) during hyperhomocysteinemia.

NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1ß production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1ß production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.

Activation of inflammasomes in podocyte injury of mice on the high fat diet: Effects of ASC gene deletion and silencing.

Inflammasome, an intracellular inflammatory machinery, has been reported to be involved in a variety of chronic degenerative diseases such as atherosclerosis, autoinflammatory diseases and Alzheimer's disease. The present study hypothesized that the formation and activation of inflammasomes associated with apoptosis associated speck-like protein (ASC) are an important initiating mechanism resulting in obesity-associated podocyte injury and consequent glomerular sclerosis. To test this hypothesis, Asc gene knockout (Asc(-/-)), wild type (Asc(+/+)) and intrarenal Asc shRNA-transfected wild type (Asc shRNA) mice were fed a high fat diet (HFD) or normal diet (ND) for 12 weeks to produce obesity and associated glomerular injury. Western blot and RT-PCR analyses demonstrated that renal tissue Asc expression was lacking in Asc(-/-) mice or substantially reduced in Asc shRNA transfected mice compared to Asc(+/+) mice. Confocal microscopic and co-immunoprecipitation analysis showed that the HFD enhanced the formation of inflammasome associated with Asc in podocytes as shown by colocalization of Asc with Nod-like receptor protein 3 (Nalp3). This inflammasome complex aggregation was not observed in Asc(-/-) and local Asc shRNA-transfected mice. The caspase-1 activity, IL-1ß production and glomerular damage index (GDI) were also significantly attenuated in Asc(-/-) and Asc shRNA-transfected mice fed the HFD. This decreased GDI in Asc(-/-) and Asc shRNA transfected mice on the HFD was accompanied by attenuated proteinuria, albuminuria, foot process effacement of podocytes and loss of podocyte slit diaphragm molecules. In conclusion, activation and formation of inflammasomes in podocytes are importantly implicated in the development of obesity-associated glomerular injury.

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.

Post-paracentesis circulatory derangements are related to monocyte activation.

BACKGROUND & AIMS: Post-paracentesis circulatory dysfunction is associated with development of hepatorenal syndrome and increased mortality. The impact of large volume paracentesis (LVP) on the 24-h blood pressure (BP) profile is unknown, and the relationship to Na+-retentive and pro-inflammatory cytokines also remains unknown. The aims of this study were to (i) define the effects of LVP with albumin administration on 24-h BP profiles, and (ii) relate changes in BP over time to changes in Na+-retentive hormones, clinical factors and inflammatory cytokines. METHODS: Ten patients undergoing LVP had 24-h ambulatory BP monitoring performed pre- and post-paracentesis. Markers of the innate immune system, bacterial translocation and Na+-retentive hormones were drawn pre- and post-LVP. RESULTS: Mean arterial pressure (MAP) dropped in nine of the 10 patients in the 24 h following a paracentesis compared to 24 h preceding the procedure (mean drop of 5.5 mmHg, P<0.005). A mixed effects model was used to define time-covariate interactions in predicting changes in BP profile. Monocyte chemotactic protein-1 (MCP1) was associated with Δsystolic BP (ß=-0.011, P<0.05), Δdiastolic BP (ß=-0.012, P<0.05) and ΔMAP (ß=-0.012, P<0.05). Plasma renin activity was also significantly associated with Δsystolic BP (ß=-0.21, P<0.05). Renal function was also significantly reduced following LVP. CONCLUSIONS: Systolic, diastolic and MAP decreased over 24 h after LVP compared to the 24 h pre-LVP. This drop is related to increases in MCP-1 after LVP. Increased MCP-1, a marker of monocyte activation, was strongly related to changes in BP.

Curcumin and chronic kidney disease (CKD): major mode of action through stimulating endogenous intestinal alkaline phosphatase.

Curcumin, an active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa), has significant anti-inflammatory properties. Chronic kidney disease (CKD), an inflammatory disease, can lead to end stage renal disease resulting in dialysis and transplant. Furthermore, it is frequently associated with other inflammatory disease such as diabetes and cardiovascular disorders. This review will focus on the clinically relevant inflammatory molecules that play a role in CKD and associated diseases. Various enzymes, transcription factors, growth factors modulate production and action of inflammatory molecules; curcumin can blunt the generation and action of these inflammatory molecules and ameliorate CKD as well as associated inflammatory disorders. Recent studies have shown that increased intestinal permeability results in the leakage of pro-inflammatory molecules (cytokines and lipopolysaccharides) from gut into the circulation in diseases such as CKD, diabetes and atherosclerosis. This change in intestinal permeability is due to decreased expression of tight junction proteins and intestinal alkaline phosphatase (IAP). Curcumin increases the expression of IAP and tight junction proteins and corrects gut permeability. This action reduces the levels of circulatory inflammatory biomolecules. This effect of curcumin on intestine can explain why, despite poor bioavailability, curcumin has potential anti-inflammatory effects in vivo and beneficial effects on CKD.

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.

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.

Pretransplant transcriptome profiles identify among kidneys with delayed graft function those with poorer quality and outcome.

Robust biomarkers are needed to identify donor kidneys with poor quality associated with inferior early and longer-term outcome. The occurrence of delayed graft function (DGF) is most often used as a clinical outcome marker to capture poor kidney quality. Gene expression profiles of 92 preimplantation biopsies were evaluated in relation to DGF and estimated glomerular filtration rate (eGFR) to identify preoperative gene transcript changes associated with short-term function. Patients were stratified into those who required dialysis during the first week (DGF group) versus those without (noDGF group) and subclassified according to 1-month eGFR of >45 mL/min (eGFR(hi)) versus eGFR of ≤45 mL/min (eGFR(lo)). The groups and subgroups were compared in relation to clinical donor and recipient variables and transcriptome-associated biological pathways. A validation set was used to confirm target genes. Donor and recipient characteristics were similar between the DGF versus noDGF groups. A total of 206 probe sets were significant between groups (P < 0.01), but the gene functional analyses failed to identify any significantly affected pathways. However, the subclassification of the DGF and noDGF groups identified 283 probe sets to be significant among groups and associated with biological pathways. Kidneys that developed postoperative DGF and sustained an impaired 1-month function (DGF(lo) group) showed a transcriptome profile of significant immune activation already preimplant. In addition, these kidneys maintained a poorer transplant function throughout the first-year posttransplant. In conclusion, DGF is a poor marker for organ quality and transplant outcome. In contrast, preimplant gene expression profiles identify "poor quality" grafts and may eventually improve organ allocation.

A rapid and simple chemiluminescence method for screening levels of inosine and hypoxanthine in non-traumatic chest pain patients.

A rapid and simple chemiluminescence method was developed for detection of inosine and hypoxanthine in human plasma. The method utilized a microplate luminometer with direct injectors to automatically dispense reagents during sample analysis. Enzymatic conversions of inosine to hypoxanthine, followed by hypoxanthine to xanthine to uric acid, generated superoxide anion radicals as a useful metabolic by-product. The free radicals react with Pholasin(®) , a sensitive photoprotein used for chemiluminescence detection, to produce measurable blue-green light. The use of Pholasin(®) and a chemiluminescence signal enhancer, Adjuvant-K™, eliminated the need for plasma clean-up steps prior to analysis. The method used 20 µL of heparinized plasma, with complete analysis of total hypoxanthine levels (inosine is metabolized to hypoxanthine using purine nucleoside phosphorylase) in approximately 3.7 min. The rapid chemiluminescence method demonstrated the capability of differentiating total hypoxanthine levels between healthy individuals, and patients presenting with non-traumatic chest pain and potential acute cardiac ischemia. The results support the potential use of chemiluminescence methodology as a diagnostic tool to rapidly screen for elevated levels of inosine and hypoxanthine in human plasma, potential biomarkers of acute cardiac ischemia.

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.

A pilot study of genetic polymorphisms and hemodialysis vascular access thrombosis.

Vascular access thrombosis (VAT) remains a significant problem worldwide. This study determined the association between VAT and 7 candidate gene polymorphisms (factor V Leiden 1691G>A, factor II 20210G>A, methylenetetrahydrofolate reductase 677C>T, angiotensin converting enzyme 287 base pair (bp) insertion/deletion, transforming growth factor-beta1 869T>C and 915G>C, NOS3 -786T>C and intron 4 27 bp tandem repeat, and endotoxin receptor CD14 -159C>T). This was a retrospective case-control pilot study conducted in 101 hemodialysis patients at a large tertiary-care, University health-science center. Sixty cases that experienced frequent VAT and 41 controls that had not experienced VAT in at least 3 years were evaluated for demographics and genotyping. These data were summarized, and univariable and multivariable regression models were constructed. Univariate VAT predictors included the NOS3 420 bp allele (P=0.03) and the presence of a central venous dialysis catheter (P<0.01). Aspirin use was protective against VAT (P=0.02). In the multivariate analysis, the dialysis access type remained a significant predictor of thrombosis (P<0.01), while aspirin use retained its protective status (P=0.01). Statin use was associated with the cases (P=0.02); however, the NOS3 420 bp allele failed to improve the model. These data confirm that central venous dialysis catheter access is associated with thrombosis, while aspirin use appears protective. The NOS3 420 bp allele may have an association with thrombosis; however, further epidemiologic data evaluating large dialysis registries are needed to confirm our observation.

High prevalence of vitamin D deficiency in African American kidney transplant recipients.

Kidney transplant patients are at high risk for developing Vitamin D(3) deficiency. The prevalence rates of 25(OH) Vitamin D(3) deficiency and its association with parathyroid hormone (PTH) levels in African American kidney transplant recipients have not been examined. We measured 25(OH) Vitamin D(3) and intact PTH concentrations in 38 African American transplant patients at our center in October 2006. We collected various laboratory data including serum creatinine, calcium, phosphate, alkaline phosphatase, and glomerular filtration rate. Vitamin D(3) deficiency was present in 57.8% of the patients and 94.7% had insufficiency. Ten of 22 (45%) patients with chronic kidney disease stage 3 had intact PTH more than or equal to 70 pg/mL. On multivariate analysis, 25(OH) Vitamin D(3) level was negatively correlated with intact PTH (P<0.01) and alkaline phosphatase level was positively associated with intact PTH levels (P<0.002). Vitamin D(3) deficiency and insufficiency is present in most of the African American kidney transplant patients.

An in vivo-in vitro study of cefepime and cefazolin dialytic clearance during high-flux hemodialysis.

STUDY OBJECTIVES: To assess the influence of in vitro and in vivo hemodialysis with a new high-flux dialyzer on the clearance of cefazolin and cefepime; to assess the correlation of in vivo dialytic clearance of these antibiotics with blood flow rate; and to assess the correlation between in vitro and in vivo dialytic clearances of these antibiotics. DESIGN: Prospective, open-label, dialysis clearance study. SETTING: A tertiary-care, university health science center. PATIENTS: Five adults who received high-flux hemodialysis 3 times/week. Intervention. For the in vivo experiment, patients received a single intravenous infusion of cefazolin 1 g and cefepime 1 g before dialysis and then underwent a modified hemodialysis session. For the in vitro experiment, a buffered simulated plasma water (SPW) solution containing cefazolin and cefepime was used. Hemodialysis for both experiments was performed with use of a new high-flux polysulfone dialyzer. MEASUREMENTS AND MAIN RESULTS: Cefazolin and cefepime dialytic clearances were determined at blood and/or SPW flow rates of 100, 200, 300, and 400 ml/minute after a 15-minute equilibration period. The degree of correlation of in vitro and in vivo clearances with blood flow rate was determined. Cefepime dialytic clearance increased proportionally with blood flow rate (p<0.01), reaching a maximum mean +/- SD value of 178.9 +/- 24.3 ml/minute at a blood flow rate of 400 ml/minute. Cefazolin dialytic clearance ranged from a mean +/- SD of 42.3 +/- 7.7 to 52.7 +/- 16 ml/minute; no significant correlation was noted between blood flow rate and dialytic clearance. In vitro cefazolin and cefepime dialytic clearances increased proportionally with SPW flow rate (p<0.05). After adjusting the in vitro cefazolin and cefepime dialytic clearances based on their degrees of protein binding, the correlation between the in vitro and in vivo cefepime dialytic clearances was significant (r(2)=0.91, p=0.04), but no significant correlation was noted between the in vitro and in vivo cefazolin clearances (r(2)=0.61, p=0.22). CONCLUSION: The in vivo hemodialysis clearances of cefepime and cefazolin with the new high-flux polysulfone dialyzer used in this study are markedly higher than values reported with conventional dialyzers but similar to values observed with other high-flux hemodialyzers. The in vivo dialytic clearance of cefazolin was significantly lower than the in vitro values, most likely due to cefazolin's high degree of protein binding. These results highlight the limitation of directly applying in vitro data to clinical situations.

Use of diuretics in the treatment of heart failure in the elderly.

Diuretics are tools of considerable therapeutic importance. First, they effectively reduce blood pressure, while at the same time decreasing the morbidity and mortality associated with hypertension. Diuretics are currently recommended as first-line therapy for the treatment of hypertension. In addition, they remain an important component of heart failure therapy, in that they improve the symptoms of congestion, which typify the more advanced stages of heart failure. This article reviews the mode of action of the various diuretic classes and the physiologic adaptations that follow; sets up the basis for their use in the treatment of volume-retaining states, particularly as applies to the elderly; and reviews diuretic-related side effects that are normally encountered.