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Macrophage accumulation and activation play an essential role in kidney fibrosis; however, the underlying mechanisms remain to be explored. By analyzing the kidney tissues from patients and animal models with kidney fibrosis, we found a large induction of PP2Acα in macrophages. We then generated a mouse model with inducible macrophage ablation of PP2Acα. The knockouts developed less renal fibrosis, macrophage accumulation, or tubular cell death after unilateral ureter obstruction or ischemic reperfusion injury compared to control littermates. In cultured macrophages, PP2Acα deficiency resulted in decreased cell motility by inhibiting Rap1 activity. Moreover, co-culture of PP2Acα−/− macrophages with tubular cells resulted in less tubular cell death attributed to downregulated Stat6-mediated tumor necrosis factor α (TNFα) production in macrophages. Together, this study demonstrates that PP2Acα promotes macrophage accumulation and activation, hence accelerates tubular cell death and kidney fibrosis through regulating Rap1 activation and TNFα production.

Ras homolog enriched in brain (Rheb1), a small GTPase, plays a crucial role in regulating cell growth, differentiation, and survival. However, the role and mechanisms for Rheb1 in tubular cell survival and acute kidney injury (AKI) remain unexplored. Here we found that Rheb1 signaling was activated in kidney tubule of AKI patients and cisplatin-treated mice. A mouse model of tubule-specific deletion of Rheb1 (Tubule-Rheb1−/−) was generated. Compared to control littermates, Tubule-Rheb1−/− mice were phenotypically normal within 2 months after birth but developed more severe kidney dysfunction, tubular cell death including apoptosis, necroptosis and ferroptosis, mitochondrial defect and less PGC-1α expression after cisplatin injection. In primary cultured tubular cells, Rheb1 ablation exacerbated cisplatin-induced cell death and mitochondrial defect. Furthermore, haploinsufficiency for Tsc1 in tubular cells led to Rheb1 activation and mitigated cisplatin-induced cell death, mitochondrial defect and AKI. Together, this study uncovers that Rheb1 may protect against cisplatin-induced tubular cell death and AKI through maintaining mitochondrial homeostasis.

The role and mechanisms for upregulating complement factor B (CFB) expression in podocyte dysfunction in diabetic kidney disease (DKD) are not fully understood. Here, analyzing Gene Expression Omnibus {"type":"entrez-geo","attrs":{"text":"GSE30528","term_id":"30528"}}GSE30528 data, we identified genes enriched in mTORC1 signaling, CFB, and complement alternative pathways in podocytes from patients with DKD. In mouse models, podocyte mTOR complex 1 (mTORC1) signaling activation was induced, while blockade of mTORC1 signaling reduced CFB upregulation, alternative complement pathway activation, and podocyte injury in the glomeruli. Knocking down CFB remarkably alleviated alternative complement pathway activation and DKD in diabetic mice. In cultured podocytes, high glucose treatment activated mTORC1 signaling, stimulated STAT1 phosphorylation, and upregulated CFB expression, while blockade of mTORC1 or STAT1 signaling abolished high glucose–upregulated CFB expression. Additionally, high glucose levels downregulated protein phosphatase 2Acα (PP2Acα) expression, while PP2Acα deficiency enhanced high glucose–induced mTORC1/STAT1 activation, CFB induction, and podocyte injury. Taken together, these findings uncover a mechanism by which CFB mediates podocyte injury in DKD.

Energy metabolism is crucial in maintaining cellular homeostasis and adapting to stimuli for tubular cells. However, the underlying mechanisms remain largely unknown. Here, we report that PP2Acα was upregulated in damaged tubular cells from patients and animal models with acute or chronic kidney injury. Using in vitro and in vivo model, we demonstrated that PP2Acα induction in damaged tubular cells suppresses fatty acid oxidation and promotes glycolysis, leading to cell death and fibrosis. Mechanistically, we revealed that PP2Acα dephosphorylates ACC through interaction with B56δ, leading to the regulation of fatty acid oxidation. Furthermore, PP2Acα also dephosphorylates p-Glut1 (Thr478) and suppresses Trim21-mediated Glut1 ubiquitination and degradation, leading to the promotion of glucose intake and glycolysis. Thus, this study adds new insight into the tubular cell metabolic alterations in kidney diseases. PP2Acα may be a promising therapeutic target for kidney injury.

Kidney fibroblasts play a crucial role in dictating tubular cell fate and the outcome of acute kidney injury (AKI). The underlying mechanisms remain to be determined. Here, we found that mTOR signaling was activated in fibroblasts from mouse kidneys with ischemia/reperfusion injury (IRI). Ablation of fibroblast Rheb or Rictor promoted, while ablation of fibroblast Tsc1 protected against tubular cell death and IRI in mice. In tubular cells cultured with conditioned media (CM) from Rheb(−/−) or Rictor(−/−) fibroblasts, less hepatocyte growth factor (HGF) receptor c-met signaling activation or staurosporine-induced cell apoptosis was observed. While CM from Tsc1(−/−) fibroblasts promoted tubular cell c-met signaling activation and inhibited staurosporine-induced cell apoptosis. In kidney fibroblasts, blocking mTOR signaling downregulated the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and HGF. Downregulating fibroblast HGF expression or blocking tubular cell c-met signaling facilitated tubular cell apoptosis. Notably, renal PPARγ and HGF expression was less in mice with fibroblast Rheb or Rictor ablation, but more in mice with fibroblast Tsc1 ablation than their littermate controls, respectively. Together, these data suggest that mTOR signaling activation in kidney fibroblasts protects against tubular cell death and dictates the outcome of AKI through stimulating PPARγ and HGF expression.

Our previous study demonstrated that the mammalian target of rapamycin complex 2 (mTORC2) signaling alleviates renal inflammation and protects against cisplatin-induced AKI. However, the underlying mechanisms for mTORC2 in regulating renal inflammation in AKI remain to be determined. In this study, we found that lipopolysaccharide (LPS) could activate mTORC2 signaling in NRK-52E cells, and blockage of mTORC2 signaling led to Yap/Taz degradation, which in turn activated NF-κB signaling and induced inflammatory cytokines secretion. Overexpression of constitutively active Taz (Taz-S89A) could attenuate the inflammation-amplified role of mTORC2 blockage. In mouse models, tubule-specific deletion of Rictor had higher blood urea nitrogen level, severe morphological injury as well as more inflammatory cells accumulation compared with those in their littermate controls. Overall, these results demonstrate that mTORC2 signaling protects against renal inflammation and dictates the outcome of AKI by modulating Yap/Taz degradation.

Protein Phosphatase 2A (PP2A), a main serine/threonine phosphatase, plays a profibrotic role in the development of different organs. However, the role and mechanisms of PP2Acα in fibroblast activation and kidney fibrosis are not fully known. Here we found that PP2Acα expression was upregulated in kidney tissue of chronic kidney disease (CKD) patients and unilateral ureter obstructive (UUO) mice. Ablation of fibroblast PP2Acα alleviates fibroblast activation and kidney fibrosis in mouse kidneys with UUO nephropathy compared with the control littermates. In primary cultured fibroblasts, PP2Acα deletion restrains TGFβ1-induced fibroblast activation, which is accompanied by increased phosphorylation of the extracellular regulated kinase (ERK). Blocking ERK pathway activation by PD98059 could promote fibroblast activation, indicating that PP2Acα promotes TGFβ1-induced fibroblast activation via suppressing ERK pathway. Consistently, in vivo, the activation of ERK pathway was upregulated by PP2Acα ablation in kidney fibroblasts. Together, these data uncover that PP2Acα may promote fibroblast activation and kidney fibrosis via suppressing ERK pathway, suggesting that targeting PP2Acα may provide a therapeutic effect for CKD.

Our previously published study demonstrated that mammalian target of rapamycin complex 2 (mTORC2) signaling mediates TGFβ1-induced fibroblast activation. However, the underlying mechanisms for mTORC2 in stimulating fibroblast activation remain poorly understood. Here, we found that TGFβ1 could stimulate mTORC2 and Yap/Taz activation in NRK-49F cells. Blocking either mTORC2 or Yap/Taz signaling diminished TGFβ1-induced fibroblast activation. In addition, blockade of mTORC2 could down-regulate the expression of Yap/Taz, connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). Overexpression of constitutively active Taz (Taz-S89A) could restore fibroblast activation suppressed by PP242, an mTOR kinase inhibitor in NRK-49F cells. In mouse kidneys with unilateral ureter obstructive (UUO) nephropathy, both mTORC2 and Yap/Taz were activated in the interstitial myofibroblasts. Ablation of Rictor in fibroblasts/pericytes or blockade of mTOR signaling with PP242 attenuated Yap/Taz activation and UUO nephropathy in mice. Together, this study uncovers that targeting mTORC2 retards fibroblast activation and kidney fibrosis through suppressing Yap/Taz activation.

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome–lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.

The Wnt/β-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/β-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4- or TGFβ1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro Conversely, inhibition of Wnt/β-catenin signaling prevented these IL-4- or TGFβ1-induced processes. In a mouse model, induced deletion of β-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/β-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

M2 macrophage polarization is known to underlie kidney fibrosis. We previously reported that most of the members of the Wnt family of signaling proteins are induced in fibrotic kidneys. Dysregulation of the signaling protein Wnt5a is associated with fibrosis, but little is known about the role of Wnt5a in regulating M2 macrophage activation that results in kidney fibrosis. Here, using murine Raw 264.7 cells and bone marrow–derived macrophages, we found that Wnt5a enhanced transforming growth factor β1 (TGFβ1)-induced macrophage M2 polarization as well as expression of the transcriptional regulators Yes-associated protein (Yap)/transcriptional coactivator with PDZ-binding motif (Taz). Verteporfin blockade of Yap/Taz inhibited both Wnt5a- and TGFβ1-induced macrophage M2 polarization. In mouse models of kidney fibrosis, shRNA-mediated knockdown of Wnt5a expression diminished kidney fibrosis, macrophage Yap/Taz expression, and M2 polarization. Moreover, genetic ablation of Taz in macrophages attenuated kidney fibrosis and macrophage M2 polarization in mice. Collectively, these results indicate that Wnt5a promotes kidney fibrosis by stimulating Yap/Taz-mediated macrophage M2 polarization.

The CD38, possessing ADP-ribosyl cyclase (ADPR-cyclase) and cyclic ADP-ribose hydrolase (cADPR-hydrolase), is able to regulate a variety of cellular activities. However, the role and mechanisms for CD38 in macrophage activation and sepsis-induced acute kidney injury (AKI) remain to be determined. Here we report that in cultured macrophages, Lipopolysaccharide (LPS) could upregulate CD38 expression in time and dose dependent manner. Knocking down or blockade of CD38 in macrophages could inhibit LPS-induced macrophage M1 polarization accompanied by diminished NF-κB signaling activation. In mouse model with LPS-induced acute kidney injury, blocking CD38 with quercetin could significantly relieve kidney dysfunction, kidney pathological changes as well as inflammatory cell accumulation. Similar to those in the cultured cells, quercetin could inhibit macrophage M1 polarization and NF-κB signaling activation in macrophages from kidneys and spleens in mice after LPS injection. Together, these results demonstrate that CD38 mediates LPS-induced macrophage activation and AKI, which may be treated as a therapeutic target for sepsis-induced AKI in patients.

The Wnt/β-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/β-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4– or TGFβ1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro. Conversely, inhibition of Wnt/β-catenin signaling prevented these IL-4– or TGFβ1-induced processes. In a mouse model, induced deletion of β-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/β-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.