HNF4A P2 isoform alleviates kidney fibrosis by inhibiting dedifferentiation of proximal tubular cells through JAG1/NOTCH signaling.

Tubular cell dedifferentiation and peritubular inflammation are coupled by the transcription regulator Id1 in renal fibrogenesis

Renal cell dedifferentiation, redifferentiation, and proliferation could resort to kidney repair and regeneration both theoretically and practically. The vertebrate kidney has an intrinsic capability to regenerate following acute impairment. Impaired tubular epithelial cells’ rapid alternate and reconstitution of ordinary tubular role are required by the injured kidney’s successful regeneration. Identifying the cells participating in the regeneration process as well as the molecular mechanisms implicated may unveil therapeutic objectives for kidney disease’s therapy. Renal regeneration is connected with the expression of genetic pathways requisite for kidney organogenesis, indicating that the regenerating tubular epithelium may be “reprogrammed” to a less-differentiated, progenitor state. Proximal tubular cell and podocyte dedifferentiation serve as two critical approaches of regenerative medicine in nephrology. For acute kidney injury, proximal tubular cell damage is the main pathophysiological reason. The mechanism and morphological changes of proximal tubular cell dedifferentiation, redifferentiation, migration, and proliferation are articulated in this review. Several sorts of stem cells, like bone marrow-derived cells, adipocyte-derived mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells, are utilized for renal regeneration in a similar way. Endogenous or lineage reprogrammed renal progenitor cells symbolize a magnetic probability for differentiation into multiple renal cell types. Additionally, podocyte dysfunction could bring about other categories of nephron-related disease, such as diabetic nephropathy and HIV-associated nephropathy. Interestingly, podocyte dedifferentiation is observed in the usual pathological process of HIV-associated nephropathy, which could provide an excellent research model for exploring underlying mechanism of podocyte differentiation.

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.

a human kidney contains approximately one million functional units (the nephrons) that consist of a filter (the glomerulus) and a processing portion (the proximal, intermediate, and distal tubule). The glomeruli produce ∼180 liters of primary filtrate every day of which only 1 to 2 liters are

Sustained Activation of EGFR Triggers Renal Fibrogenesis after Acute Kidney Injury

ERK, p38, and Smad Signaling Pathways Differentially Regulate Transforming Growth Factor-β1 Autoinduction in Proximal Tubular Epithelial Cells

While NLRP3 contributes to kidney fibrosis, the function of most NOD-like receptors (NLRs) in chronic kidney disease (CKD) remains unexplored. To identify further NLR members involved in the pathogenesis of CKD, we searched for NLR genes expressed by normal kidneys and differentially expressed in human CKD transcriptomics databases. For NLRP6, lower kidney expression correlated with decreasing glomerular filtration rate. The role and molecular mechanisms of Nlrp6 in kidney fibrosis were explored in wild-type and Nlrp6-deficient mice and cell cultures. Data mining of single-cell transcriptomics databases identified proximal tubular cells as the main site of Nlrp6 expression in normal human kidneys and tubular cell Nlrp6 was lost in CKD. We confirmed kidney Nlrp6 downregulation following murine unilateral ureteral obstruction. Nlrp6-deficient mice had higher kidney p38 MAPK activation and more severe kidney inflammation and fibrosis. Similar results were obtained in adenine-induced kidney fibrosis. Mechanistically, profibrotic cytokines transforming growth factor beta 1 (TGF-β1) and TWEAK decreased Nlrp6 expression in cultured tubular cells, and Nlrp6 downregulation resulted in increased TGF-β1 and CTGF expression through p38 MAPK activation, as well as in downregulation of the antifibrotic factor Klotho, suggesting that loss of Nlrp6 promotes maladaptive tubular cell responses. The pattern of gene expression following Nlrp6 targeting in cultured proximal tubular cells was consistent with maladaptive transitions for proximal tubular cells described in single-cell transcriptomics datasets. In conclusion, endogenous constitutive Nlrp6 dampens sterile kidney inflammation and fibrosis. Loss of Nlrp6 expression by tubular cells may contribute to CKD progression.

Kidney fibrosis is the final common pathway of chronic kidney diseases. Proximal tubular epithelial cells (PTECs) arrested in G2/M phase of the cell cycle play a pivotal role in kidney fibrosis. Phosphofurin acidic cluster sorting protein 2 (PACS-2) is a multifunctional protein involved in various cellular activities including cell cycle regulation, yet its role in kidney fibrosis remains unclear. PTEC-specific Pacs-2 knockout mice were generated by utilizing LoxP-Cre recombination system and subjected to unilateral ureteral obstruction (UUO) and aristolochic acid to induce kidney fibrosis. Cultured human and mouse tubular epithelial cells were treated with TGF-β1 to analyze the underlying cellular mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CO-IP/MS), molecular cloning and genetic manipulation were used to investigate PACS-2 interactions and specific binding domains. PACS-2 expression was significantly lower in the cortex of fibrotic kidney from UUO mouse. PACS-2 deficiency in PTECs exacerbated G2/M cell cycle arrest and kidney fibrosis in murine UUO and aristolochic acid nephropathy models, two independent models for CKD. In vitro, overexpression of PACS-2 alleviated TGF-β1-induced fibrogenic responses in PTECs through inhibiting cell cycle arrest at G2/M phase. By CO-IP/MS, we identified cyclin-dependent kinase-like 1 (CDKL1) as the key molecule linking PACS-2 to cell cycle progression in PTECs. Knockdown of CDKL1 partially reversed the anti-fibrotic effects of PACS-2 by promoting G2/M cell cycle arrest in TGF-β1-stimulated HK-2 cells. Mechanistically, we demonstrated that PACS-2 interacted with kinase domain of CDKL1 and modulated its kinase activity, thereby regulating cell cycle, rather than affecting its subcellular translocation or protein expression. Our study demonstrates that renal tubular PACS-2 alleviated G2/M cell cycle arrest and kidney fibrosis by interacting with CDKL1 and modulating its kinase activity.

A growing number of clinical and experimental studies show that the renin–angiotensin system (RAS) is involved in the progression of CKD.1 In fact, pharmacological inhibitors of the RAS such as angiotensin-converting enzyme inhibitors and angiotensin II (Ang II) type I receptor (AT1R) blockers are the most reliable and effective tools known to attenuate progression of parenchymal changes in CKD.1 However, despite the clinical evidence, understanding how RAS inhibition exerts its renoprotective function at the molecular level remains unclear.

Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.

Critical role of transcription factor SOX4 in tubular epithelial cell dedifferentiation and fibroblast activation during kidney fibrosis.

Background/Aims: The interactions between calcium oxalate monohydrate (COM) crystals and renal tubular epithelial cells are important for renal stone formation but still unclear. This study aimed to investigate changes of epithelial cell phenotype after COM attachment and whether L-carnitine could protect cells against subsequent COM crystals adhesion. Methods: Cultured MDCK cells were employed and E-cadherin and Vimentin were used as markers to estimate the differentiate state. AlexaFluor-488-tagged COM crystals were used in crystals adhesion experiment to distinguish from the previous COM attachment, and adhesive crystals were counted under fluorescence microscope, which were also dissolved and the calcium concentration was assessed by flame atomic absorption spectrophotometry. Results: Dedifferentiated MDCK cells induced by transforming growth factor β1 (TGF-β1) shown higher affinity to COM crystals. After exposure to COM for 48 hours, cell dedifferentiation were observed and more subsequent COM crystals could bind onto, mediated by Akt/GSK-3β/Snail signaling. L-carnitine attenuated this signaling, resulted in inhibition of cell dedifferentiation and reduction of subsequent COM crystals adhesion. Conclusions: COM attachment promotes subsequent COM crystals adhesion, by inducing cell dedifferentiation via Akt/GSK-3β/Snail signaling. L-carnitine partially abolishes cell dedifferentiation and resists COM crystals adhesion. L-carnitine, may be used as a potential therapeutic strategy against recurrence of urolithiasis.

Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

Tubular hypoxia has a major pathogenic role in diabetic kidney disease (DKD). Hypoxia is in strong association with elevated glucose reabsorption and increased protein O-GlcNAcylation which could contribute to renal fibrosis. We recently showed that SGLT2 inhibitors (SGLT2i) are renoprotective in experimental type 1 diabetes. Considering emerging evidence of proximal tubular involvement in DKD and the major role of SGLT2 in glucose metabolism, here we investigated the direct effects of SGLT2i on hypoxia and O-GlcNAcylation. Diabetes (D) was induced by streptozotocin in adult, male Wistar rats. Rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day). Renal function and fibrosis were evaluated. The effect of hyperglycaemia was tested in human proximal tubular epithelial cells (HK-2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions. HG cells were treated with 10 µM DAPA. O-GlcNAc, O GlcNAc transferase (OGT) and O GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 µM DAPA and were placed in a hypoxic chamber for 2 hours. HIF1-α, EPO, VEGFA and PAI-1 were measured. Immunocytochemistry (ICC) of HIF1-α was performed. DAPA improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p<0.01) and decreased fibrosis. DAPA minimized hyperglycemia-induced total protein O-GlcNAcylation and OGT, while OGA was elevated in HK-2 cells. Hypoxia-induced HIF-1α elevation was suspended by DAPA treatment. Abolishment of HIF-1α upregulation by DAPA was confirmed by ICC staining. EPO, VEGFA and PAI-1 levels were also increased in hypoxia and DAPA prevented EPO and PAI-1 elevation. Here we identified a novel mechanism of SGLT2i by which the direct reduction of tubular hypoxia and inhibition of OGT by DAPA results in decreased O-GlcNAcylation in proximal tubular cells. These processes contribute to improved renal function and alleviated kidney fibrosis. Disclosure D.B. Balogh: None. J. Hodrea: None. L. Lenart: None. A. Hosszu: None. C. Mezei: None. L. Wagner: None. A.J. Szabo: None. A. Fekete: None. Funding Hungarian Academy of Sciences (LP008/2017, OTKA-K112629-FK124491-NN-114607, VKE-2017-00006); Semmelweis University; New National Excellence Program of the Hungarian Ministry of Human Capacities (?NKP-18-3)

Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells