Renal fibrosis, particularly tubulointerstitial fibrosis, is the common final outcome of almost all progressive chronic kidney diseases. Renal fibrosis is also a reliable predictor of prognosis and a major determinant of renal insufficiency. Irrespective of the initial causes, renal fibrogenesis is a dynamic and converging process that consists of four overlapping phases: priming, activation, execution and progression. Nonresolving inflammation after a sustained injury sets up the fibrogenic stage (priming) and triggers the activation and expansion of matrix-producing cells from multiple sources through diverse mechanisms, including activation of interstitial fibroblasts and pericytes, phenotypic conversion of tubular epithelial and endothelial cells and recruitment of circulating fibrocytes. Upon activation, matrix-producing cells assemble a multicomponent, integrin-associated protein complex that integrates input from various fibrogenic signals and orchestrates the production of matrix components and their extracellular assembly. Multiple cellular and molecular events, such as tubular atrophy, microvascular rarefaction and tissue hypoxia, promote scar formation and ensure a vicious progression to end-stage kidney failure. This Review outlines our current understanding of the cellular and molecular mechanisms of renal fibrosis, which could offer novel insights into the development of new therapeutic strategies.

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Cellular and molecular mechanisms of renal fibrosis

TGF-β1 has been long considered as a key mediator in renal fibrosis and induces renal scarring largely by activating its downstream Smad signaling pathway. Interestingly, while mice overexpressing active TGF-β1 develop progressive renal injury, latent TGF-β1 plays a protective role in renal fibrosis and inflammation. Under disease conditions, Smad2 and Smad3 are highly activated, while Smad7 is degraded through the ubiquitin proteasome degradation mechanism. In addition to TGF-β1, many pathogenic mediators such as angiotensin II and advanced glycation end products can also activate the Smad pathway via both TGF-β-dependent and independent mechanisms. Smads interact with other signaling pathways, such as the MAPK and NF-κB pathways, to positively or negatively regulate renal inflammation and fibrosis. Studies from gene knockout mice demonstrate that TGF-β1 acts by stimulating its downstream Smads to diversely regulate kidney injury. In the context of renal fibrosis and inflammation, Smad3 is pathogenic, while Smad2 and Smad7 are protective. Smad4 exerts its diverse roles by transcriptionally enhancing Smad3-mediated renal fibrosis while inhibiting NF-κB-driven renal inflammation via a Smad7-dependent mechanism. Furthermore, we also demonstrated that TGF-β1 acts by stimulating Smad3 to positively or negatively regulate microRNAs to exert its fibrotic role in kidney disease. In conclusion, TGF-β/Smad signaling is a major pathway leading to kidney disease. Smad3 is a key mediator in renal fibrosis and inflammation, whereas Smad2 and Smad7 are renoprotective. Smad4 exerts its diverse role in promoting renal fibrosis while inhibiting inflammation. Thus, targeting the downstream TGF-β/Smad3 signaling pathway by gene transfer of either Smad7 or Smad3-dependent microRNAs may represent a specific and effective therapeutic strategy for kidney disease.

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Diverse roles of TGF-β/Smads in renal fibrosis and inflammation.

https://www.kidney-international.org/article/S0085-2538(15)54927-2/pdf

Apoptosis and acute kidney injury.

Chronic kidney disease (CKD) is a devastating condition that is reaching epidemic levels owing to the increasing prevalence of diabetes mellitus, hypertension and obesity, as well as ageing of the population. Regardless of the underlying aetiology, CKD is slowly progressive and leads to irreversible nephron loss, end-stage renal disease and/or premature death. Factors that contribute to CKD progression include parenchymal cell loss, chronic inflammation, fibrosis and reduced regenerative capacity of the kidney. Current therapies have limited effectiveness and only delay disease progression, underscoring the need to develop novel therapeutic approaches to either stop or reverse progression. Preclinical studies have identified several approaches that reduce fibrosis in experimental models, including targeting cytokines, transcription factors, developmental and signalling pathways and epigenetic modulators, particularly microRNAs. Some of these nephroprotective strategies are now being tested in clinical trials. Lessons learned from the failure of clinical studies of transforming growth factor β1 (TGFβ1) blockade underscore the need for alternative approaches to CKD therapy, as strategies that target a single pathogenic process may result in unexpected negative effects on simultaneously occurring processes. Additional promising avenues include preventing tubular cell injury and anti-fibrotic therapies that target activated myofibroblasts, the main collagen-producing cells. Here, the authors review drivers of fibrogenesis, including epithelial cell injury, inflammation, regeneration pathways and factors that promote the AKI-to-CKD transition. They discuss direct targeting of fibrotic pathways and therapeutic approaches that have reportedly decreased kidney fibrosis in preclinical and/or clinical studies.

https://www.nature.com/articles/s41581-019-0248-y.pdf?proof=t

Targeting the progression of chronic kidney disease

Purpose of reviewTubulointerstitial injury in the kidney is complex, involving a number of independent and overlapping cellular and molecular pathways, with renal interstitial fibrosis and tubular atrophy (IFTA) as the final common pathway. Furthermore, there are multiple ways to assess IFTA.Recent

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Renal interstitial fibrosis: mechanisms and evaluation.

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.

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2016.1190071

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis.

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-β signaling from the cytosol to the nucleus and HSP72 assists in protein folding and facilitates nuclear translocation, we investigated whether HSP72 inhibits TGF-β-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-ΔPBD), which is responsible for substrate binding and refolding, or the nuclear localization signal (HSP72-ΔNLS). Overexpression of wild-type HSP72 or HSP72-ΔNLS inhibited TGF-β1-induced EMT, but HSP72-ΔPBD did not, suggesting a critical role for the PBD in this inhibition. HSP72 overexpression inhibited TGF-β1-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-β1-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.

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HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

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.

Heat shock protein 72 enhances autophagy as a protective mechanism in lipopolysaccharide-induced peritonitis in rats.

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.

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Serum Potassium Levels and Its Variability in Incident Peritoneal Dialysis Patients: Associations with Mortality

Esophageal squamous cell carcinoma (ESCC) is a common invasive malignancy worldwide with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been reported to be involved in cancer development. However, lncRNAs that are functional in ESCC and the underlying molecular mechanisms remain largely unknown.Transcriptomic analysis was performed to identify dysregulated lncRNAs in ESCC tissue samples. The high expression of LINC00680 in ESCC was validated by RT-qPCR, and the oncogenic functions of LINC00680 was investigated by cell proliferation, colony formation, migration and invasion assays in ESCC cells in vitro and xenografts derived from ESCC cells in mice. RNA-seq, competitive endogenous RNA (ceRNA) network analysis, and luciferase reporter assays were carried out to identify LINC00680 target genes and the microRNAs (miRNAs) bound to LINC00680. Antisense oligonucleotides (ASOs) were used for in vivo treatment.Transcriptome profiling revealed that a large number of lncRNAs was dysregulated in ESCC tissues. Notably, LINC00680 was highly expressed, and upregulation of LINC00680 was associated with large tumor size, advanced tumor stage, and poor prognosis. Functionally, knockdown of LINC00680 restrained ESCC cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, LINC00680 was found to act as a ceRNA by sponging miR-423-5p to regulate PAK6 (p21-activated kinase 6) expression in ESCC cells. The cell viability and motility inhibition induced by LINC00680 knockdown was significantly reversed upon PAK6 restoration and miR-423-5p inhibition. Furthermore, ASO targeting LINC00680 substantially suppressed ESCC both in vitro and in vivo.An oncogenic lncRNA, LINC00680, was identified in ESCC, which functions as a ceRNA by sponging miR-423-5p to promote PAK6 expression and ESCC. LINC00680/miR-423-5p/PAK6 axis may serve as promising diagnostic and prognostic biomarkers and therapeutic targets for ESCC. 

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Shirong Cao

Papers

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis.

HSP72 Inhibits Smad3 Activation and Nuclear Translocation in Renal Epithelial-to-Mesenchymal Transition

Heat shock protein 72 enhances autophagy as a protective mechanism in lipopolysaccharide-induced peritonitis in rats.

Serum Potassium Levels and Its Variability in Incident Peritoneal Dialysis Patients: Associations with Mortality

Long non-coding RNA LINC00680 functions as a ceRNA to promote esophageal squamous cell carcinoma progression through the miR-423-5p/PAK6 axis