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Background: Clonal hematopoiesis of indeterminate potential (CHIP) is a common inflammatory condition of aging that causes myriad end-organ damage. We have recently shown associations for CHIP with acute kidney injury and with kidney function decline in the general population, with stronger associations for CHIP driven by mutations in genes other than DNMT3A (non- DNMT3A CHIP). Longitudinal kidney function endpoints in individuals with pre-existing chronic kidney disease (CKD) and CHIP have been examined in two previous studies, which reported conflicting findings and were limited by small sample sizes., Methods: In this study, we examined the prospective associations between CHIP and CKD progression events in four cohorts of CKD patients (total N = 5,772). The primary outcome was a composite of 50% kidney function decline or kidney failure. The slope of eGFR decline was examined as a secondary outcome. Mendelian randomization techniques were then used to investigate potential causal effects of CHIP on eGFR decline. Finally, kidney function was assessed in adenine-fed CKD model mice having received a bone marrow transplant recapitulating Tet2 -CHIP compared to controls transplanted wild-type bone marrow., Results: Across all cohorts, the average age was 66.4 years, the average baseline eGFR was 42.6 ml/min/1.73m 2 , and 24% had CHIP. Upon meta-analysis, non- DNMT3A CHIP was associated with a 59% higher relative risk of incident CKD progression (HR 1.59, 95% CI: 1.02-2.47). This association was more pronounced among individuals with diabetes (HR 1.29, 95% CI: 1.03-1.62) and with baseline eGFR ≥ 30 ml/min/1.73m (HR 1.80, 95% CI: 1.11-2.90). Additionally, the annualized slope of eGFR decline was steeper among non- DNMT3A CHIP carriers, relative to non-carriers (β -0.61 ± 0.31 ml/min/1.73m 2 , p = 0.04). Mendelian randomization analyses suggested a causal role for CHIP in eGFR decline among individuals with diabetes. In a dietary adenine mouse model of CKD, Tet2 -CHIP was associated with lower GFR as well as greater kidney inflammation, tubular injury, and tubulointerstitial fibrosis., Conclusion: Non- DNMT3A CHIP is a potentially targetable novel risk factor for CKD progression.

Age is a predominant risk factor for acute kidney injury (AKI), yet the biological mechanisms underlying this risk are largely unknown. Clonal hematopoiesis of indeterminate potential (CHIP) confers increased risk for several chronic diseases associated with aging. Here we sought to test whether CHIP increases the risk of AKI. In three population-based epidemiology cohorts, we found that CHIP was associated with a greater risk of incident AKI, which was more pronounced in patients with AKI requiring dialysis and in individuals with somatic mutations in genes other than DNMT3A, including mutations in TET2 and JAK2. Mendelian randomization analyses supported a causal role for CHIP in promoting AKI. Non-DNMT3A-CHIP was also associated with a nonresolving pattern of injury in patients with AKI. To gain mechanistic insight, we evaluated the role of Tet2-CHIP and Jak2 V617F -CHIP in two mouse models of AKI. In both models, CHIP was associated with more severe AKI, greater renal proinflammatory macrophage infiltration and greater post-AKI kidney fibrosis. In summary, this work establishes CHIP as a genetic mechanism conferring impaired kidney function recovery after AKI via an aberrant inflammatory response mediated by renal macrophages., (© 2024. The Author(s).)

Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor activated by thrombin. In the platelet, response to thrombin PAR4 contributes to the predominant procoagulant microparticle formation, increased fibrin deposition, and initiation of platelet-stimulated inflammation. In addition, PAR4 is expressed in other cell types, including endothelial cells. Under inflammatory conditions, PAR4 is overexpressed via epigenetic demethylation of the PAR4 gene, F2RL3 . PAR4 knockout (KO) studies have determined a role for PAR4 in ischemia-reperfusion injury in the brain, and PAR4 KO mice display normal cardiac function but present less myocyte death and cardiac dysfunction in response to acute myocardial infarction. Although PAR4 has been reported to be expressed within the kidney, the contribution of PAR4 to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 KO mice are protected against kidney injury in two mouse models. First, PAR4 KO mice are protected against induction of markers of both fibrosis and inflammation in two different models of kidney injury: 1 ) 7 days following unilateral ureter obstruction (UUO) and 2 ) an AKI-CKD model of ischemia-reperfusion followed by 8 days of contralateral nephrectomy. We further show that PAR4 expression in the kidney is low in the control mouse kidney but induced over time following UUO. PAR4 KO mice are protected against blood urea nitrogen (BUN) and glomerular filtration rate (GFR) kidney function pathologies in the AKI-CKD model. Following the AKI-CKD model, PAR4 is expressed in the collecting duct colocalizing with Dolichos biflorus agglutinin (DBA), but not in the proximal tubule with Lotus tetragonolobus lectin (LTL). Collectively, the results reported in this study implicate PAR4 as contributing to the pathology in mouse models of acute and chronic kidney injury. NEW & NOTEWORTHY The contribution of the thrombin receptor protease-activated receptor 4 (PAR4) to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 expression is upregulated after kidney injury and PAR4 knockout (KO) mice are protected against fibrosis following kidney injury in two mouse models. First, PAR4 KO mice are protected against unilateral ureter obstruction. Second, PAR4 KO mice are protected against an AKI-CKD model of ischemia-reperfusion followed by contralateral nephrectomy.

The AE and damage characteristics of three types of pore-structured rock under the same working conditions are studied by means of uniaxial cyclic loading and unloading tests. The results suggest that with repeated loading and unloading, AE ringing increases as a "jump", and the denser the structure, the earlier the "jump" occurs. The AE cumulative energy shows a "step" upward trend, but there is a significant difference in the "step" spacing. By comparing the energy distribution of rocks with different pore structures, it can be seen that the smaller the porosity and the smaller the pore size, the greater the energy input and storage, and the earlier the internal failure. Compared with the other two energy-based damage calculation methods, the damage calculation method defined in this paper is closer to the true internal damage level of the rock loading cycle. The NSE value of the modified damage variable calculation method was significantly improved and it was shown that the dissipated energy before pore compaction is the main energy causing damage, after pore compaction the combined effects of dissipated energy and plastic deformation energy result in rock damage., (© 2024. The Author(s).)

Loxoprofen sodium is a chiral drug with two chiral centers. In our previous work, we found that the elimination of its four isomers showed stereospecificity in rats, while how the stereospecific behavior occurred in vivo was unclear. To clarify this issue, each single isomer of loxoprofen sodium was prepared by a chiral semi-preparative high-performance liquid chromatography (HPLC) and then administered to rats. By analysis of each isomer in rat plasma utilizing an analytical chiral HPLC, it was discovered that the chiral inversion occurred only to its (2R)-isomers, one from (1'S,2R)- to (1'S,2S)-isomer and the other from (1'R,2R)- to (1'R,2S)-isomer. The reduction of α-substituted cyclopentanone occurred only to its (1'R)-isomers, with (1'R,2R)-isomer reduced to (2'S,1'R,2R)-trans-alcohol and (1'R,2S)- to (2'S,1'R,2S)-trans-alcohol. Interestingly, both the inversion and the reduction reaction occurred to its (1'R,2R)-isomer due to the special stereo-structure with both (2R)- and (1'R)-configuration, and conversely, neither of them occurred to its (1'S,2S)-isomer, which caused the significantly different elimination rate in vivo. These new findings were meaningful for evaluation of the safety and efficacy of chiral drugs., (© 2023 Wiley-VCH GmbH.)

Fibrosis is the progressive accumulation of excess extracellular matrix and can cause organ failure. Fibrosis can affect nearly every organ including kidney and there is no specific treatment currently. Although Epidermal Growth Factor Receptor (EGFR) signaling pathway has been implicated in development of kidney fibrosis, underlying mechanisms by which EGFR itself mediates kidney fibrosis have not been elucidated. We find that EGFR expression increases in interstitial myofibroblasts in human and mouse fibrotic kidneys. Selective EGFR deletion in the fibroblast/pericyte population inhibits interstitial fibrosis in response to unilateral ureteral obstruction, ischemia or nephrotoxins. In vivo and in vitro studies and single-nucleus RNA sequencing analysis demonstrate that EGFR activation does not induce myofibroblast transformation but is necessary for the initial pericyte/fibroblast migration and proliferation prior to subsequent myofibroblast transformation by TGF-ß or other profibrotic factors. These findings may also provide insight into development of fibrosis in other organs and in other conditions., (© 2023. The Author(s).)

Inadequate potassium (K + ) consumption correlates with increased mortality and poor cardiovascular outcomes. Potassium effects on blood pressure have been described previously; however, whether or not low K + independently affects kidney disease progression remains unclear. Here, we demonstrate that dietary K + deficiency causes direct kidney injury. Effects depend on reduced blood K + and are kidney specific. In response to reduced K + , the channel Kir4.2 mediates altered proximal tubule (PT) basolateral K + flux, causing intracellular acidosis and activation of the enzyme glutaminase and the ammoniagenesis pathway. Deletion of either Kir4.2 or glutaminase protects from low-K + injury. Reduced K + also mediates injury and fibrosis in a model of aldosteronism. These results demonstrate that the PT epithelium, like the distal nephron, is K + sensitive, with reduced blood K + causing direct PT injury. Kir4.2 and glutaminase are essential mediators of this injury process, and we identify their potential for future targeting in the treatment of chronic kidney disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Obesity and obesity-related health complications are increasing in prevalence. Adipose tissue from obese subjects has low-grade, chronic inflammation, leading to insulin resistance. Adipose tissue macrophages (ATMs) are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. In response to a high fat diet (HFD), ATM numbers initially increase by proliferation of resident macrophages, but subsequent increases also result from infiltration in response to chemotactic signals from inflamed adipose tissue. To elucidate the underlying mechanisms regulating the increases in ATMs and their proinflammatory phenotype, we investigated the role of activation of ATM epidermal growth factor receptor (EGFR). A high fat diet increased expression of EGFR and its ligand amphiregulin in ATMs. Selective deletion of EGFR in ATMs inhibited both resident ATM proliferation and monocyte infiltration into adipose tissue and decreased obesity and development of insulin resistance. Therefore, ATM EGFR activation plays an important role in adipose tissue dysfunction., (© 2022. The Author(s).)

A rapid reversed-phase high performance liquid chromatographic (HPLC) methodology for chiral separation of three profen compounds has been developed and then applied to enantiomeric impurity testing of their corresponding enantiopure drugs. The assay is specific, allowing quantitation of the enantiomeric impurities at levels of 0.0078%, 0.0105%, and 0.0416% relative to S-ibuprofen, S-naproxen, and S-ketoprofen, respectively. In order to gain a better insight into the chiral recognition mechanisms of chiral profens on an FLM Chiral NQ(2)-RH column, molecular docking studies were carried out using AutoDock 4.0 software. It was found that hydrogen bonding, hydrophobic interactions, and π-π stacking were all involved in stereoselective interactions, and the calculated binding energy (BE) obtained reflected the binding strength of each enantiomer interacting with a chiral selector. The higher the BE value, the harder it was to elute the corresponding enantiomer, which also accorded with the enantiomer elution order observed in the actual enantiomeric separation. Additionally, thermodynamic analysis revealed that the enantioseparation process at 15-40°C was driven mainly by entropic contributions. The methodology was further validated according to the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q2 (R1) and proved to be sensitive, linear, precise, and accurate for determining R-profen impurities in three commercially available single-enantiomer S-profen drugs. As expected, in the case of products acquired in actual pharmacies, the levels of all of the monitored impurities were found to be lower than the allowable impurity limits., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Obesity-associated complications are causing increasing morbidity and mortality worldwide. Expansion of adipose tissue in obesity leads to a state of low-grade chronic inflammation and dysregulated metabolism, resulting in insulin resistance and metabolic syndrome. Adipose tissue macrophages (ATMs) accumulate in obesity and are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. Macrophages are rich sources of cyclooxygenase (COX), the rate limiting enzyme for prostaglandin E2 (PGE2) production. When mice were fed a high-fat diet (HFD), ATMs increased expression of COX-2. Selective myeloid cell COX-2 deletion resulted in increased monocyte recruitment and proliferation of ATMs, leading to increased proinflammatory ATMs with decreased phagocytic ability. There were increased weight gain and adiposity, decreased peripheral insulin sensitivity and glucose utilization, increased adipose tissue inflammation and fibrosis, and abnormal adipose tissue angiogenesis. HFD pair-feeding led to similar increases in body weight, but mice with selective myeloid cell COX-2 still exhibited decreased peripheral insulin sensitivity and glucose utilization. Selective myeloid deletion of the macrophage PGE2 receptor subtype, EP4, produced a similar phenotype, and a selective EP4 agonist ameliorated the metabolic abnormalities seen with ATM COX-2 deletion. Therefore, these studies demonstrated that an ATM COX-2/PGE2/EP4 axis plays an important role in inhibiting adipose tissue dysfunction.

Aristolochic acid (AA) is the causative nephrotoxic alkaloid in AA nephropathy, which results in a tubulointerstitial fibrosis. AA causes direct proximal tubule damage as well as an influx of macrophages, although the role of macrophages in pathogenesis is poorly understood. Here, we demonstrate that AA directly stimulates migration, inflammation, and ROS production in macrophages ex vivo. Cells lacking interferon regulatory factor 4 (IRF4), a known regulator of macrophage migration and phenotype, had a reduced migratory response, though effects on ROS production and inflammation were preserved or increased relative to WT cells. Macrophage-specific IRF4-knockout mice were protected from both acute and chronic kidney effects of AA administration based on functional and histological analysis. Renal macrophages from kidneys of AA-treated macrophage-specific IRF4-knockout mice demonstrated increased apoptosis and ROS production compared with WT controls, indicating that AA directly polarizes macrophages to a promigratory and proinflammatory phenotype. However, knockout mice had reduced renal macrophage abundance following AA administration. While macrophages lacking IRF4 can adopt a proinflammatory phenotype upon AA exposure, their inability to migrate to the kidney and increased rates of apoptosis upon infiltration provide protection from AA in vivo. These results provide evidence of direct AA effects on macrophages in AA nephropathy and add to the growing body of evidence that supports a key role of IRF4 in modulating macrophage function in kidney injury.

Following acute injury to the kidney, macrophages play an important role in recovery of functional and structural integrity, but organ fibrosis and progressive functional decline occur with incomplete recovery. Pro-resolving macrophages are characterized by increased cyclooxygenase 2 (COX-2) expression and this expression was selectively increased in kidney macrophages following injury and myeloid-specific COX-2 deletion inhibited recovery. Deletion of the myeloid prostaglandin E2 (PGE2) receptor, E-type prostanoid receptor 4 (EP4), mimicked effects seen with myeloid COX-2-/- deletion. PGE2-mediated EP4 activation induced expression of the transcription factor MafB in kidney macrophages, which upregulated anti-inflammatory genes and suppressed pro-inflammatory genes. Myeloid Mafb deletion recapitulated the effects seen with either myeloid COX-2 or EP4 deletion following acute kidney injury, with delayed recovery, persistent presence of pro-inflammatory kidney macrophages, and increased kidney fibrosis. Thus, our studies identified a previously unknown mechanism by which prostaglandins modulate macrophage phenotype following acute organ injury and provide new insight into mechanisms underlying detrimental kidney effects of non-steroidal anti-inflammatory drugs that inhibit cyclooxygenase activity., (Published by Elsevier Inc.)

In the present study, an accurate, rapid, and simple chiral HPLC-UV method with amylose tris(3-chloro-5-methylphenylcarbamate) as stationary phase was developed and applied for enantiomeric determination of six nonsteroidal anti-inflammatory drugs (NSAIDs) in the commercial pharmaceutical formulations, including (R,S)-ibuprofen, S-ibuprofen, (R,S)-ketoprofen, S-ketoprofen, S-naproxen, and (R,S)-loxoprofen sodium. Experiments on the influence of mobile phase composition, proportion of organic modifier, percentage of acid additives, and column temperature on enantioseparation were conducted to obtain the best separation condition. It was indicated that one mobile phase simply composed of acetonitrile-water (0.1% formic acid, v/v) at the proportion of 50:50 (v/v) with a flow rate of 0.6 ml/min at 22°C could simultaneously provide the excellent enantiomeric resolutions for all selected NSAIDs, which made the enantioseparation process more applicable and operable. The newly developed method was then applied for determination of NSAID enantiomers in pharmaceutical formulations containing racemic mixtures or single stereoisomers. Calibration curve of each enantiomer at the concentration of 5.0-100 ug/ml showed good linearity with the correlation coefficient above 0.9996. Satisfactory recovery (96.54-101.54%), good intra-day precision (RSD 0.52-1.46%), and inter-day precision (RSD 0.13-1.09%) were also obtained. The newly developed method was then applied for determination of NSAID enantiomers in pharmaceutical formulations containing racemic mixtures or single stereoisomers. Quantitative results of the commercial capsules and tablets demonstrated that the difference between the declared and measured values did not exceed 1.52%., (© 2021 Wiley Periodicals LLC.)

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single-cell-based RNA sequencing (RNA-seq) of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) has not previously been compared with that of T1DN. We analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26 fsTRAP ; eNOS -/- ; db/db mice and compared it with that of streptozotocin-induced T1DN in podocin-Cre; Rosa26 fsTRAP ; eNOS -/- mice using translating ribosome affinity purification (TRAP) and RNA-seq. More than 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN than in T1DN. TGF-β signaling pathway genes were upregulated, while MAPK pathway genes were downregulated only in T2DN, while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased, while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number of diabetes-induced genes not previously linked to podocyte injury were confirmed in both mouse and human DN. On the basis of differences and similarities in the podocyte translatome in T2DN and T1DN, investigators can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury., (© 2021 by the American Diabetes Association.)

Background: AKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear., Methods: We used mice with myeloid or macrophage cell-specific deletion of Irf4 (MΦ Irf4 -/- ) to evaluate Irf4's role in renal macrophage polarization and development of fibrosis after severe AKI., Results: Surprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow-derived monocytes (BMDMs) from MΦ Irf4 -/- mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4 -/- BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4 -/- mice or in wild-type mice with inhibition of AKT activity., Conclusions: Deletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype., (Copyright © 2021 by the American Society of Nephrology.)

Renal epidermal growth factor receptor (EGFR) signaling is activated in models of diabetic nephropathy (DN), and inhibition of the EGFR signaling pathway protects against the development of DN. We have now determined that in cultured podocytes, high glucose led to increases in activation of EGFR signaling but decreases in autophagy activity as indicated by decreased beclin-1 and inhibition of LC3B autophagosome formation as well as increased rubicon (an autophagy inhibitor) and SQSTM1 (autophagy substrate). Either genetic (small interfering [si]EGFR) or pharmacologic (AG1478) inhibition of EGFR signaling attenuated the decreased autophagy activity. In addition, rubicon siRNA knockdown prevented high glucose-induced inhibition of autophagy in podocytes. We further examined whether selective EGFR deletion in podocytes affected the progression of DN in type 2 diabetes. Selective podocyte EGFR deletion had no effect on body weight or fasting blood sugars in either db/db mice or nos3 -/- ; db/db mice, a model of accelerated type 2 DN. However selective podocyte EGFR deletion led to relative podocyte preservation and marked reduction in albuminuria and glomerulosclerosis, renal proinflammatory cytokine/chemokine expression, and decreased profibrotic and fibrotic components in nos3 -/- ; db/db mice. Podocyte EGFR deletion led to decreased podocyte expression of rubicon, in association with increased podocyte autophagy activity. Therefore, activation of EGFR signaling in podocytes contributes to progression of DN at least in part by increasing rubicon expression, leading to subsequent autophagy inhibition and podocyte injury., (© 2020 by the American Diabetes Association.)

Purpose: Vitamin K deficiency, expressed by a high level of desphospho-uncarboxylated matrix GLA protein (dp-ucMGP), is highly prevalent in dialysis patients. However, the predictive ability of the vitamin K status remains unclear in continuous ambulatory peritoneal dialysis (CAPD) patients., Methods: 158 prevalent CAPD patients with a median level of dp-ucMGP of 1093 (752, 1485) pmol/L were enrolled. Patient outcomes including all-cause mortality and cardiovascular events (CVEs) were recorded during follow-up. Survival curves were performed using Kaplan-Meier method, and the influences of dp-ucMGP on outcomes were analyzed by Cox regression models., Results: A total of 59 deaths and 82 new episodes of CVEs occurred during median follow-up of 31.4 ± 13.1 months (range: 3.8-48.0 months). Kaplan-Meier analysis revealed patients with higher dp-ucMGP levels (≥ 1093 pmol/L) had an increased risk for both mortality (P = 0.005) and CVEs (P < 0.001). Multivariable Cox regression confirmed that higher dp-ucMGP levels increase the mortality risk [hazard ratio (HR), 1.763; 95% CI 1.045-3.291] and CVEs (HR, 1.846; 95% CI 1.074-3.172). For every 100 pmol/L increase in serum dp-ucMGP, the adjusted HRs for mortality and CVEs were 1.054 (95% CI 1.008-1.106) and 1.034 (95% CI 1.012-1.089), respectively., Conclusions: Vitamin K deficiency, as expressed by high dp-ucMGP levels, showed independently associations with mortality and CVEs in CAPD patients.

Increased high mobility group box 1 (HMGB1) in dialysis effluence is associated with the presence of peritoneal dialysis-related peritonitis in patients and peritoneal dysfunction in acute peritonitis mice model, but it remains unclear whether HMGB1 is involved in peritoneal mesothelial cell injury and functions via molecular posttranslational modifications by acetylation in this process. Here we first showed correlation between HMGB1 acetylation level in dialysis effluence of patients and occurrence of Gram-negative peritonitis. The increased level of acetylated HMGB1 was similarly observed under the lipopolysaccharides (LPS) treatment in both human peritoneal mesothelial cell line (HMrSV5) and mice visceral peritoneum tissue. Overexpression of wild-type, but not hypoacetylation mutant of HMGB1, enhanced LPS-induced apoptosis in HMrSV5 cells, which was accompanied by elevated protein levels of BAX and cleaved-caspase 3 compared to the control. Pretreatment of HMrSV5 cell with JNK inhibitor attenuated LPS-induced HMGB1 acetylation. Consistently, primary peritoneal mesothelial cells from Jnk1 -/- mice showed a lower protein contents of acetylated HMGB1, fewer apoptosis, and decreased protein expression of BAX and cleaved-caspase3 after LPS exposure, as compared to those from wild-type mice. In conclusion, our data demonstrated HMGB1 promotes LPS-induced peritoneal mesothelial cells apoptosis, which is associated with JNK1-mediated upregulation of HMGB1 acetylation.

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G2/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G2/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G2/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G1/S phase, and increased COL1 expression in the G2/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G2/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G2/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G2/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G2/M arrest.

Heat shock protein 72 (HSP72) has been shown to attenuate unilateral ureteral obstruction-induced kidney fibrosis. It remains unknown whether HSP72 has direct effects on fibroblast proliferation in the renal fibrotic evolution. Herein, we first confirmed that increased HSP72 expression occurred in fibrotic human kidneys. Using three different animal models of kidney fibrosis, pharmacological down-regulation or genetic deletion of endogenous HSP72 expression exacerbated STAT3 phosphorylation, fibroblast proliferation, and tubulointerstitial fibrosis. In contrast, treatment with geranylgeranyl acetone, a specific inducer of HSP72, reduced phosphorylated STAT3 and protected animals from kidney fibrosis. In cultured renal interstitial fibroblasts, overexpression of HSP72 blocked transforming growth factor (TGF)-β1-induced cell activation and proliferation, as evidenced by inhibiting expression of α-smooth muscle actin, fibronectin, and collagen I/III, as well as by reducing cell numbers and DNA synthesis. Mechanical studies showed that overexpressed HSP72 attenuated TGF-β-induced phosphorylation and nuclear translocation of STAT3 and its downstream protein expression. However, siRNA knockdown of HSP72 increased TGF-β-induced STAT3 activity and fibroblast proliferation. Ectopic expression of a constitutively active STAT3 conferred resistance to HSP72 inhibition of fibroblast proliferation. Thus, HSP72 blocks fibroblast activation and proliferation in renal fibrosis via targeting the STAT3 pathway and may serve as a novel therapeutic agent for chronic kidney disease regardless of the etiology., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Although high body mass index (BMI) appears to confer a survival advantage in hemodialysis patients, the association of BMI with mortality in continuous ambulatory peritoneal dialysis (CAPD) patients is uncertain. We enrolled incident CAPD patients and BMI was categorized according to World Health Organization classification for Asian population. BMI at baseline and one year after the initiation of peritoneal dialysis (PD) treatment was assessed to calculate the BMI change (∆BMI). Patients were split into four categories according quartiles of ∆BMI. Kaplan-Meier method and Cox regression proportional hazard analysis were performed to assess the association of BMI on outcomes. A total of 1263 CAPD patients were included, with a mean age of 47.8 ± 15.0 years, a mean BMI of 21.58 ± 3.13 kg/m². During a median follow-up of 25.3 months, obesity was associated with increased risk for cardiovascular diseases (CVD) death (adjusted hazard ratio (AHR) 2.01; 95% CI 1.14, 3.54), but not all-cause mortality. Additionally, patients with more BMI decline (>0.80%) during the first year after CAPD initiation had an elevated risk for both all-cause (AHR: 2.21, 95% CI 1.23-3.95) and CVD mortality (AHR 2.31, 95% CI 1.11, 4.84), which was independent of baseline BMI values.

Background: The prognostic value of pulmonary hypertension at the start of peritoneal dialysis (PD) in patient survival is unclear., Methods: We conducted a retrospective study of incident patients who initiated PD therapy from January 2007 to December 2011, and followed up through June 2013. Pulmonary hypertension was defined as an estimated systolic pulmonary artery pressure (PAP) of ≥ 35 mm Hg using echocardiography. Clinical parameters and laboratory findings were compared between patients with and without pulmonary hypertension and a logistic regression model was elaborated. Patient outcomes (all-cause and cardiovascular mortality) were recorded during follow-up. Survival curves were constructed by the Kaplan-Meier method, and the influences of pulmonary hypertension on outcomes were analyzed by Cox regression models., Results: Pulmonary hypertension was prevalent in 99 (16.0%) of the 618 patients studied. The independent risk factors for pulmonary hypertension were female (odds ratio [OR] = 2.12; 95% confidence interval [CI]: 1.29 - 3.46), left atrial diameter (OR = 1.15; 95% CI: 1.10 - 1.20), left ventricular ejection fraction (OR = 0.97; 95% CI: 0.95 - 0.99), and serum sodium (OR = 0.94; 95% CI: 0.89 - 0.99). Over a median follow-up of 29.4 months, 93 patients (15.0%) died, 59.1% of them due to cardiovascular disease. Kaplan-Meier survival analysis showed that patients with pulmonary hypertension had worse overall rates of survival and cardiovascular death-free survival than those without pulmonary hypertension. After multivariate adjustment, pulmonary hypertension was independently associated with increased risk for both all-cause and cardiovascular mortality, with hazard ratios (HRs) of 2.10 (95% CI: 1.35 - 3.27) and 2.60 (95% CI: 1.48 - 4.56), respectively., Conclusions: The prevalence of pulmonary hypertension at the start of PD was common and associated with increased risk of both all-cause and cardiovascular mortality in incident PD patients., (Copyright © 2015 International Society for Peritoneal Dialysis.)

Background: Abnormal serum potassium is associated with an increased risk of mortality in dialysis patients. However, the impacts of serum potassium levels on short- and long-term mortality and association of potassium variability with death in peritoneal dialysis (PD) patients are uncertain., Methods: We examined mortality-predictability of serum potassium at baseline and its variability in PD patients treated in our center January 2006 through December 2010 with follow-up through December 2012. The hazard ratios (HRs) were used to assess the relationship between baseline potassium levels and short-term (≤1 year) as well as long-term (>1 year) survival. Variability of serum potassium was defined as the coefficient of variation of serum potassium (CVSP) during the first year of PD., Results: A total of 886 incident PD patients were enrolled, with 248 patients (27.9%) presented hypokalemia (serum potassium <3.5 mEq/L). During a median follow-up of 31 months (range: 0.5-81.0 months), adjusted all-cause mortality hazard ratio (HR) and 95% confidence interval (CI) for baseline serum potassium of <3.0, 3.0 to <3.5, 3.5 to <4.0, 4.5 to <5.0, and ≥5.0 mEq/L, compared with 4.0 to <4.5 (reference), were 1.79 (1.02-3.14), 1.15 (0.72-1.86), 1.31 (0.82-2.08), 1.33 (0.71-2.48), 1.28 (0.53-3.10), respectively. The increased risk of lower potassium with mortality was evident during the first year of follow-up, but vanished thereafter. Adjusted all-cause mortality HR for CVSP increments of 7.5% to <12.0%; 12.0% to <16.7% and ≥16.7%, compared with <7.5% (reference), were 1.35 (0.67-2.71), 2.00 (1.05-3.83) and 2.18 (1.18-4.05), respectively. Similar association was found between serum potassium levels and its variability and cardiovascular mortality., Conclusions: A lower serum potassium level was associated with all-cause and cardiovascular mortality during the first year of follow-up in incident PD patients. In addition, higher variability of serum potassium levels conferred an increased risk of death in this population.

High mobility group box 1 (HMGB1), a DNA-binding nuclear protein, has been implicated as an endogenous danger signal in the pathogenesis of infection diseases. However, the potential role and source of HMGB1 in the peritoneal dialysis (PD) effluence of patients with peritonitis are unknown. First, to evaluate HMDB1 levels in peritoneal dialysis effluence (PDE), a total of 61 PD patients were enrolled in this study, including 42 patients with peritonitis and 19 without peritonitis. Demographic characteristics, symptoms, physical examination findings and laboratory parameters were recorded. HMGB1 levels in PDE were determined by Western blot and ELISA. The concentrations of TNF-α and IL-6 in PDE were quantified by ELISA. By animal model, inhibition of HMGB1 with glycyrrhizin was performed to determine the effects of HMGB1 in LPS-induced mice peritonitis. In vitro, a human peritoneal mesothelial cell line (HMrSV5) was stimulated with lipopolysaccharide (LPS), HMGB1 extracellular content in the culture media and intracellular distribution in various cellular fractions were analyzed by Western blot or immunofluorescence. The results showed that the levels of HMGB1 in PDE were higher in patients with peritonitis than those in controls, and gradually declined during the period of effective antibiotic treatments. Furthermore, the levels of HMGB1 in PDE were positively correlated with white blood cells (WBCs) count, TNF-α and IL-6 levels. However, pretreatment with glycyrrhizin attenuated LPS-induced acute peritoneal inflammation and dysfunction in mice. In cultured HMrSV5 cells, LPS actively induced HMGB1 nuclear-cytoplasmic translocation and release in a time and dose-dependent fashion. Moreover, cytosolic HMGB1 was located in lysosomes and secreted via a lysosome-mediated secretory pathway following LPS stimulation. Our study demonstrates that elevated HMGB1 levels in PDE during PD-related peritonitis, at least partially, from peritoneal mesothelial cells, which may be involved in the process of PD-related peritonitis and play a critical role in acute peritoneal dysfunction.

Peritoneal dialysis-related peritonitis causes the denudation of mesothelial cells and, ultimately, membrane integrity alterations and peritoneal dysfunction. Because heat shock protein 72 (HSP72) confers protection against apoptosis and because autophagy mediates survival in response to cellular stresses, we examined whether autophagy contributes to HSP72-mediated cytoprotection in lipopolysaccharide (LPS)-induced peritonitis. Exposure of cultured peritoneal mesothelial cells to LPS resulted first in autophagy and later, apoptosis. Inhibition of autophagy by 3-methyladenine or Beclin-1 small-interfering RNA sensitized cells to apoptosis and abolished the antiapoptotic effect of HSP72, suggesting that autophagy activation acts as a prosurvival mechanism. Overexpression of HSP72 augmented autophagy through c-Jun N-terminal kinase (JNK) phosphorylation and Beclin-1 up-regulation. Suppression of JNK activity reversed HSP72-mediated Beclin-1 up-regulation and autophagy, indicating that HSP72-mediated autophagy is JNK dependent. In a rat model of LPS-associated peritonitis, autophagy occurred before apoptosis in peritoneum. Up-regulation of HSP72 by geranylgeranylacetone increased autophagy, inhibited apoptosis, and attenuated peritoneal injury, and these effects were blunted by down-regulation of HSP72 with quercetin. Additionally, blocking autophagy by chloroquine promoted apoptosis and aggravated LPS-associated peritoneal dysfunction. Thus, HSP72 protects peritoneum from LPS-induced mesothelial cells injury, at least in part by enhancing JNK activation-dependent autophagy and inhibiting apoptosis. These findings imply that HSP72 induction might be a potential therapy for peritonitis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Although heat shock protein 72 (HSP72) ameliorates renal tubulointerstitial fibrosis by inhibiting epithelial-to-mesenchymal transition (EMT), the underlying mechanism is unknown. Because Smad proteins transduce TGF-beta signaling from the cytosol to the nucleus and HSP72 assists in protein folding and facilitates nuclear translocation, we investigated whether HSP72 inhibits TGF-beta-induced EMT by modulating Smad expression, activation, and nuclear translocation. To evaluate the roles of distinct HSP72 structural domains in these processes, we constructed vectors that expressed wild-type HSP72 or mutants lacking either the peptide-binding domain (HSP72-DeltaPBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (HSP72-DeltaNLS). Overexpression of wild-type HSP72 or HSP72-DeltaNLS inhibited TGF-beta1-induced EMT, but HSP72-DeltaPBD did not, suggesting a critical role for the PBD in this inhibition. HSP72 overexpression inhibited TGF-beta1-induced phosphorylation and nuclear translocation of Smad3 and p-Smad3, but not Smad2; these inhibitory effects required the PBD but not the NLS. Coimmunoprecipitation assays suggested a physical interaction between Smad3 and the PBD. siRNA knockdown of endogenous HSP72 enhanced both TGF-beta1-induced Smad3 phosphorylation and EMT and confirmed the interaction of HSP72 with both Smad3 and p-Smad3. In vivo, induction of HSP72 by geranylgeranylacetone suppressed Smad3 phosphorylation in renal tubular cells after unilateral ureteral obstruction. In conclusion, HSP72 inhibits EMT in renal epithelial cells primarily by exerting domain-specific effects on Smad3 activation and nuclear translocation.