Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G 2 /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 G 2 /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 G 2 /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 G 1 /S phase, and increased COL1 expression in the G 2 /M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G 2 /M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G 2 /M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G 2 /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 G 2 /M arrest.

BackgroundAbnormal 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.MethodsWe 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.ResultsA 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.ConclusionsA 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.

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-␤ signaling from the cytosol to the nucleus and HSP72 assists in protein folding and facilitates nuclear translocation, we investigated whether HSP72 inhibits TGF-␤-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-⌬PBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (HSP72-⌬NLS). Overexpression of wild-type HSP72 or HSP72-⌬NLS inhibited TGF-␤1-induced EMT, but HSP72-⌬PBD did not, suggesting a critical role for the PBD in this inhibition. HSP72 overexpression inhibited TGF-␤1-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-␤1-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. 21: 598 -609, 201021: 598 -609, . doi: 10.1681 Transforming growth factor ␤ (TGF-␤) and related factors regulate a wide variety of biologic activities. 1 Furthermore, abnormalities in TGF-␤ signaling lead to various human diseases. 2 Signals from TGF-␤ are mainly transduced by the Smad family of transcription factors. Upon TGF-␤ stimulation, Smad2 and Smad3 are phosphorylated at their carboxy-tails by the activated TGF-␤ type I receptor kinase, 3 forming a stable complex with Smad4 in cytoplasm and then accumulating in the nucleus to regulate transcription of target genes. In contrast, Smad7 inhibits the TGF-␤ receptor type I-dependent Smad2/3 activation. It has been demonstrated that Smad3 phosphorylation plays a predominant role in guiding the complex through the nuclear pore. 4 cytoplasm. 5 Blockade of TGF-␤ signaling events, specifically the phosphorylation of Smad2 and Smad3 and/or nuclear translocation, prevent epithelial-to-mesenchymal transition (EMT) and tissue fibrosis. 6,7 Heat shock protein 72 (HSP72) exerts cytoprotective effects by assisting in protein folding, assembly/disassembly and translocation of client proteins across membranes, protein degradation, and signal transduction. 8,9 However, some cytoprotective effects do not require the HSP72 chaperone function. 10 HSP72 contains distinct domains: a C-terminal peptide-binding domain (PBD) responsible...

BackgroundAKI 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.MethodsWe 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.ResultsSurprisingly, 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.ConclusionsDeletion 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.

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 cJun 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 LPSassociated 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 LPSassociated 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.