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TGFβ1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFβ. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFβ1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFβ1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFβ1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFβ, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFβ1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFβ1 induced endogenous TGFβ, αSMA, MMP2, MMP9 and Col I mRNA. TGFβ1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFβ1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFβ, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFβ1 was confirmed by the use of a TGFβ receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFβ driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The inhibition of the autophagolysosomal pathway mediated by transcription factor EB (TFEB) inactivation in proximal tubular epithelial cells (TECs) is a key mechanism of TEC injury in diabetic kidney disease (DKD). Acetylation is a novel mechanism that regulates TFEB activity. However, there are currently no studies on whether the adjustment of the acetylation level of TFEB can reduce the damage of diabetic TECs. In this study, we investigated the effect of Trichostatin A (TSA), a typical deacetylase inhibitor, on TFEB activity and damage to TECs in both in vivo and in vitro models of DKD. Here, we show that TSA treatment can alleviate the pathological damage of glomeruli and renal tubules and delay the DKD progression in db/db mice, which is associated with the increased expression of TFEB and its downstream genes. In vitro studies further confirmed that TSA treatment can upregulate the acetylation level of TFEB, promote its nuclear translocation, and activate the expression of its downstream genes, thereby reducing the apoptosis level of TECs. TFEB deletion or HDAC6 knockdown in TECs can counteract the activation effect of TSA on autophagolysosomal pathway. We also found that TFEB enhances the transcription of Tfeb through binding to its promoter and promotes its own expression. Our results, thus, provide a novel therapeutic mechanism for DKD that the alleviation of TEC damage by activating the autophagic lysosomal pathway through upregulating TFEB acetylation can, thus, delay DKD progression., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Bisphenol P (BPP) has been detected in human biological samples; however studies on its nephrotoxicity are scarce. Given the susceptibility of kidneys to endocrine-disrupting chemicals, there is an urgent need to investigate the renal toxicity of BPP. This study aimed to evaluate the effects of different concentrations of BPPs on the kidneys of C57BL/6 mice and elucidate the underlying mechanisms of renal damage using a combination of mouse renal transcriptomic data and human renal proximal tubular epithelial cells (HK-2). Mice were exposed to BPP (0, 0.3, 30, 3000 μg/kg bw/d) via gavage for 5 weeks. Renal injury was assessed based on changes in body and kidney weights, serum renal function indices, and histopathological examination. Transcriptomic analysis identified differentially expressed genes and pathways, whereas cellular assays were used to measure cell viability, reactive oxygen species (ROS), apoptosis, and the expression of key genes and proteins. The results show that BPP exposure induces renal injury, as evidenced by increased body weight, abnormal renal function indices, and renal tissue damage. Transcriptomic analysis revealed alterations in genes and pathways related to oxidative stress, p53 signaling, autophagy, and apoptosis. Cellular experiments confirmed that BPP induces oxidative stress and apoptosis. Furthermore, BPP exposure significantly inhibits autophagy, potentially exacerbating apoptosis and contributing to kidney injury. Treatment with a ROS inhibitor (N-Acetylcysteine, NAC) mitigated BPP-induced autophagy inhibition and apoptosis, implicating oxidative stress as a key factor. BPP exposure may lead to renal injury through excessive ROS accumulation, oxidative stress, inflammatory responses, autophagy inhibition, and increased apoptosis. The effects of NAC highlight the role of oxidative stress in BPP-induced nephrotoxicity. These findings enhance our understanding of BPP-induced nephrotoxicity and underscore the need to control BPP exposure to prevent renal disease. This study emphasized the importance of evaluating the safety of new Bisphenol A analogs, including BPP, in environmental toxicology., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10 -7  M losartan (an antagonist for type 1 angiotensin receptor, AT 1 R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT 1 R axis by HG., 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 © 2024. Published by Elsevier B.V.)

Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.

In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase ( Tnik ) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase ( PARP ) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI. NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase ( Tnik ) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of si TNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research., (© 2024. The Author(s).)

Although recent studies have reported that long non-coding RNA (lncRNA) is involved in the development of ischemic acute kidney injury (AKI), the exact function and regulatory mechanism of lncRNAs in ischemic AKI remain largely unknown. Herein, we found that ischemic injury promoted the expression of lncRNA 148400 in mouse proximal tubule-derived cell line (BUMPT) and C57BL/6J mice. Furthermore, the lncRNA148400 mediates ischemic injury-induced apoptosis of BUMPT cells. Mechanistically, lncRNA 148400 sponged miR-10b-3p to promote apoptosis via GRK4 upregulation. Finally, knockdown of lncRNA 148400 alleviated the I/R-induced deterioration of renal function, renal tubular injury, and cell apoptosis. In addition, cleaved caspase-3 is increased via targeting the miR-10b-3p/GRK4 axis. Collectively, these results showed that lncRNA 148400/miR-10b-3p/GRK4 axis mediated the development of ischemic AKI., Competing Interests: The authors declare that they have no competing interests.

The pathogenesis of crystal nephropathy involves deposition of intratubular crystals, tubular obstruction and cell death. The deposition of 8-dihydroxyadenine (DHA) crystals within kidney tubules, for instance, is caused by a hereditary deficiency of adenine phosphoribosyl transferase in humans or adenine overload in preclinical models. However, the downstream pathobiological patterns of tubular cell attrition in adenine/DHA-induced nephropathy remain poorly understood. In this study, we investigated: (i) the modes of adenine-induced tubular cell death in an experimental rat model and in human primary proximal tubular epithelial cells (PTEC); and (ii) the therapeutic effect of the flavonoid baicalein as a novel cell death inhibitor. In a rat model of adenine diet-induced crystal nephropathy, significantly elevated levels of tubular iron deposition and lipid peroxidation (4-hydroxynonenal; 4-HNE) were detected. This phenotype is indicative of ferroptosis, a novel form of regulated necrosis. In cultures of human primary PTEC, adenine overload-induced significantly increased mitochondrial superoxide levels, mitochondrial depolarisation, DNA damage and necrotic cell death compared with untreated PTEC. Molecular interrogation of adenine-stimulated PTEC revealed a significant reduction in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the significant increase in 4-HNE compared with untreated PTEC, supporting the concept of ferroptotic cell death. Moreover, baicalein treatment inhibited ferroptosis in adenine-stimulated PTEC by selectively modulating the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) and thus, suppressing mitochondrial superoxide production and DNA damage. These data identify ferroptosis as the primary pattern of PTEC necrosis in adenine-induced nephropathy and establish baicalein as a potential therapeutic tool for the clinical management of ferroptosis-associated crystal nephropathies (e.g., DHA nephropathy, oxalate nephropathy)., (© 2022. Crown.)

Kidney hypertrophy is a common clinical feature in patients with diabetes and is associated with poor renal outcomes. Initial cell proliferation followed by cellular hypertrophy are considered the responsible mechanisms for diabetic kidney hypertrophy. However, whether similar responses against hyperglycemia continue in the chronic phase in diabetes is unclear. We performed lineage tracing analysis of proximal tubular epithelia using novel type 2 diabetic mice with a tamoxifen-inducible proximal tubule-specific fluorescent reporter. Clonal analysis of proximal tubular epithelia demonstrated that the labeled epithelia proliferated in type 2 diabetic mice. Based on the histological analysis and protein/DNA ratio of sorted labeled tubular epithelia, there was no evidence of cellular hypertrophy in type 2 diabetic mice. Lineage tracing and histological analyses of streptozocin-induced type 1 diabetes also revealed that cellular proliferation occurs in the chronic phase of type 1 diabetes induction. According to our study, epithelial proliferation accompanied by SGLT2 upregulation, rather than cellular hypertrophy, predominantly occurs in the hypertrophic kidney in both type 1 and type 2 diabetes. An increased number of SGLT2+ tubular epithelia may be an adaptive response against hyperglycemia, and linked to the hyper-reabsorption of sodium and glucose observed in type 2 diabetes patients., (© 2022. The Author(s).)

Ischemia-reperfusion injury is one of the major causes of acute kidney injury (AKI), which is increasingly prevalent in clinical settings. Glaucocalxin A (GLA), a biologically ent-kauranoid diterpenoid, has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. In this study, the effect of GLA on AKI and its mechanism were studied in vitro. HK-2 human renal tubular epithelial cells were exposed to hypoxia/reoxygenation (H/R), which were established as an in vitro AKI model. Subsequently, the mRNA expressions of inflammatory and antioxidant factors were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Reactive oxygen species (ROS) production and cell death were detected by fluorescence-activated cell sorting. GLA pre-treatment improved the cell viability of HK-2 cells exposed to H/R. GLA suppressed the H/R-induced ROS production in HK-2 cells. GLA also elevated the activities of superoxide dismutase of HK-2 cells exposed to H/R. Moreover, GLA prevented H/R-induced cell death in HK-2 cells. Furthermore, GLA ameliorated the activation of the protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in HK-2 cells exposed to H/R. Our findings suggested that GLA protected HK-2 cells from H/R-induced oxidative damage, which was mediated by the Akt/Nrf2/HO-1 signaling pathway. These results indicate that GLA may serve as a promising therapeutic drug for AKI.

Hypoxia occurs in the kidneys of chronic kidney disease (CKD) patients, inducing interstitial fibrosis and tubule cell death. Renal tubule cell death is an important determinant of mortality in CKD. We focused on the regulation of cell-cycle-mediated protein expression to prevent cell death under chronic hypoxia in the kidneys of CKD patients. Paraffin-embedded kidney sections from patients with CKD (diabetes nephropathy, nephrosclerosis, or IgA nephropathy) were analyzed for the expression of hypoxia-inducible factor (HIF), thyroid hormone receptor (TR) β, or p21 and levels of interstitial fibrosis. Human renal proximal tubule cells were exposed to hypoxia and analyzed for the expression of HIF, TRβ, or p21 and the cell-cycle stage. TRβ expression was enhanced early on when fibrosis was not fully developed in the tubule cells of CKD patients. HIF1α bound to the TRβ promoter and directly induced its transcription. Further, HIF1α expression induced the expression of TRβ and inhibited cell-cycle progression. In the early stage of kidney injury, TRβ might act as a guardian to prepare and organize cell-cycle proliferation and prevent cell death. While the molecular mechanism that regulates the expression of cell-cycle regulators in renal tubule cells remains controversial, TRβ has strong potential as a new therapeutic target.

Particulate matter exposure has been known as a potential risk for the global burden of disease, such as respiratory and cardiovascular diseases. Accumulating evidence suggests that PM2.5 (particulate matter with a diameter less than 2.5 μm) is associated with increased risk of kidney disease, but the mechanisms underlying the renal injury caused by PM2.5 remain to be elucidated. This study investigated the effects of PM2.5 on human proximal tubular epithelial (HK-2) cells by monolayer and 3D spheroid cultures and explored the potential mechanisms. The typical morphology of HK-2 cells showed epithelial-mesenchymal transition (EMT), resulting in reduced adhesion and enhanced migration after PM2.5 exposure, and was accompanied by decreased E-cadherin expression and increased vimentin and α-SMA expressions. Exposure to PM2.5 in the HK-2 cells could lead to an increase in interleukin-6 (IL-6) levels and cause the activation of signal transducer and activator of transcription 3 (STAT3), which is involved in EMT features of HK-2 cells. Furthermore, blocking IL-6/STAT3 signaling by an IL-6 neutralizing antibody or STAT3 inhibitor was sufficient to reverse PM2.5-induced EMT characteristics of the HK-2 cells. Our study suggests that PM2.5 could induce early renal tubule cell injury, contributing to EMT change, and the induction of IL-6/STAT3 pathway may play an important role in this process.

Immunomodulation is considered a potential therapeutic approach for chronic kidney disease (CKD). Although it has been previously reported that CD4 + T cells contribute to the development of renal fibrosis, the role of MHC class II (MHCII) in the development of renal fibrosis remains largely unknown. The present study reports that the expression of MHCII molecules in renal cortical tubules is upregulated in mouse renal fibrosis models generated by unilateral ureter obstruction (UUO) and folic acid (FA). Proximal tubule epithelial cells (PTECs) are functional antigen-presenting cells that promote the proliferation of CD4 + T cells in an MHCII-dependent manner. PTECs from mice with renal fibrosis had a stronger ability to induce T cell proliferation and cytokine production than control cells. Global or renal tubule-specific ablation of H2-Ab1 significantly alleviated renal fibrosis following UUO or FA treatment. Renal expression of profibrotic genes showed a consistent reduction in H2-Ab1 gene-deficient mouse lines. Moreover, there was a marked increase in renal tissue CD4 + T cells after UUO or FA treatment and a significant decrease following renal tubule-specific ablation of H2-Ab1. Furthermore, renal tubule-specific H2-Ab1 gene knockout mice exhibited higher proportions of regulatory T cells (Tregs) and lower proportions of Th2 cells in the UUO- or FA-treated kidneys. Finally, Immunohistochemistry (IHC) studies showed increased renal expression of MHCII and the profibrotic gene α smooth muscle actin (α-SMA) in CKD patients. Together, our human and mouse data demonstrate that renal tubular MHCII plays an important role in the pathogenesis of renal fibrosis., (© 2021. The Author(s), under exclusive licence to CSI and USTC.)

Background: Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis., Methods: Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing., Results: A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1-S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters ("new PTC clusters") were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages., Conclusions: These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis., (Copyright © 2021 by the American Society of Nephrology.)

Acute kidney injury (AKI) is one of the most common clinical syndromes. AKI is associated with significant morbidity and subsequent chronic kidney disease (CKD) development. Thus, it is urgent to develop a strategy to hinder AKI progression. Renal tubules are responsible for the reabsorption and secretion of various solutes and the damage to this part of the nephron is a key mediator of AKI. As we know, many common renal insults primarily target the highly metabolically active proximal tubular cells (PTCs). PTCs are the most energy-demanding cells in the kidney. The ATP that they use is mostly produced in their mitochondria by fatty acid β-oxidation (FAO). But, when PTCs face various biological stresses, FAO will shut down for a time that outlives injury. Recent studies have suggested that surviving PTCs can adapt to FAO disruption by increasing glycolysis when facing metabolic constraints, although PTCs do not perform glycolysis in a normal physiological state. Enhanced glycolysis in a short period compensates for impaired energy production and exerts partial renal-protective effects, but its long-term effect on renal function and AKI progression is not promising. Deranged FAO and enhanced glycolysis may contribute to the AKI to CKD transition through different molecular biological mechanisms. In this review, we concentrate on the recent pathological findings of AKI with regards to the metabolic reprogramming in PTCs, confirming that targeting metabolic reprogramming represents a potentially effective therapeutic strategy for the progression of AKI., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis., Methods: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-β1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively., Results: Only the combination of high D-glucose and TGF-β1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this rise was significantly reduced by both SGLT2 and MEK inhibition., Conclusions: We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney., (© 2021 The Author(s).)

Background: Cisplatin-induced injury of renal proximal tubular cells results basically from increased apoptosis via mitochondrial damage, and is mitigated by appropriate enhancement of autophagy. Peroxisome proliferator-activated receptor-delta (PPAR-δ) reportedly protects against not only mitochondrial damages but also enhances autophagy. Thus, PPAR-δ may protect against cisplatin-induced kidney injury., Methods: We examined the protective effects of PPAR-δ activation on cisplatin-induced cellular injury and their detailed mechanisms in a murine renal proximal tubular (mProx) cell line using GW0742, an authentic PPAR-δ activator. Cisplatin-induced cell damages were evaluated by TUNEL assay and immunoblot analyses for p53, 14-3-3, Bax, Bcl2, cytochrome C, and activated caspases. Autophagy status was examined by immunoblot analyses for p62 and LC3., Results: GW0742 suppressed cisplatin-induced apoptosis of mProx cells by reducing the activation of caspase-3 via attenuating the phosphorylation of p53 and 14-3-3, mitochondrial Bax accumulation, cytochrome C release from mitochondria to the cytosol and ensuing cytosolic caspase-9 activation. In contrast, GW0742 did not diminish cisplatin-enhanced activation of caspases-8 or -12 as extrinsic or endothelium reticulum apoptotic pathways, respectively. The inhibitory effect of GW0742 on cisplatin-induced caspase-3 activation was significantly diminished by silencing of the PPAR-δ gene expression. GW0742 itself had no influence on starvation-stimulated or cisplatin-induced autophagy in mProx cells, suggesting that the protective effects were not mediated by autophagy modification., Conclusion: Our results indicate that GW0742 may serve as a candidate agent to mitigate cisplatin nephrotoxicity via inhibiting the mitochondrial apoptotic pathway considerably depending on PPAR-δ, without modulating autophagy.

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFβ1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD.

Purpose: This study aimed to down-regulate LINC00667 and inhibit apoptosis and fibrosis of renal tubular epithelial cells through miR-34c., Methods: Altogether, 98 patients with chronic kidney disease treated in our hospital were selected as the study group, and 67 normal people were selected as the control group. Epithelial cells of proximal convoluted tubules in human renal cortex were purchased. TGF-β1 was used to induce fibrosis of HK-2 renal tubular epithelial cells. The expression of LINC00667, miR-34c, type I collagen (Col 1) and type III collagen (Col 3) were detected by qRT-PCR and WB., Results: LINC00667 was highly expressed in cancer tissues and HK-2, while miR-34c was poorly expressed. Inhibition of LINC00667 and over-expression of miR-34c could inhibit the proliferation and invasion of chronic kidney disease cells, but increase the apoptosis rate. Down-regulation of LINC00667 could significantly reduce of Col 1 and Col 3 in renal interstitial fibroblasts induced by TGF-β1, while up-regulation of miR-34c could also achieve this effect. Double luciferase report confirmed that there was a targeted regulatory relationship between LINC00667 and miR-34c., Conclusion: LINC00667 could reduce the proliferation and invasion of chronic kidney disease cells, increase the apoptosis rate by regulating miR-34c, and improve renal fibrosis.

ERK, an extracellular signal-regulated protein kinase, is involved in various biological responses, such as cell proliferation and differentiation, cell morphology maintenance, cytoskeletal construction, apoptosis, and canceration of cells. In this study, we focused on ERK pathway on cellular injury and autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells and explored the potential mechanisms underlying it. By using antioxidants N-acetylcysteine and catalase, we found that ERK pathway was activated by a reactive oxygen species- (ROS-) dependent mechanism after exposure to urinary proteins. What is more, ERK inhibitor U0126 could decrease the release of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and the number of apoptotic cells induced by urinary proteins, indicating the damaging effects of ERK pathway in mediating cellular injury and apoptosis in HK-2 cells. Interestingly, we also found that the increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II (a key marker of autophagy) and the decreased expression of p62 (autophagic substrate) induced by urinary proteins were reversed by U0126, suggesting autophagy was activated by ERK pathway. Furthermore, rapamycin reduced urinary protein-induced NGAL and KIM-1 secretion and cell growth inhibition, while chloroquine played the opposite effect, indicating that autophagy activation by ERK pathway was an adaptive response in the exposure to urinary proteins. Taken together, our results indicate that activated ROS-ERK pathway can induce cellular injury and in the meantime provide an autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Jian-kun Deng et al.)

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection.

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence. To demonstrate the utility of iRno for predicting kidney function, we exposed primary rat renal proximal tubule epithelial cells to four compounds with varying levels of nephrotoxicity (acetaminophen, gentamicin, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six and twenty-four hours, and collected transcriptomics and metabolomics data to measure the metabolic effects of compound exposure. For the transcriptomics data, we observed changes in fatty acid metabolism and amino acid metabolism, as well as changes in existing markers of kidney function such as Clu (clusterin). The iRno metabolic network reconstruction was used to predict alterations in these same pathways after integrating transcriptomics data and was able to distinguish between select compound-specific effects on the proximal tubule epithelial cells. Genome-scale metabolic network reconstructions with coupled omics data can be used to predict changes in metabolism as a step towards identifying novel metabolic biomarkers of kidney function and dysfunction., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Current management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment., Methods: We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments., Results: Our findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors-including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)-and of several novel genes ( Ahnak , Sh3bgrl3 , and Col18a1 ) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a . Moreover, AKI caused the formation of "mixed-identity cells" (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis., Conclusions: The scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury., (Copyright © 2020 by the American Society of Nephrology.)

Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis as well as the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI-specific siRNA in the absence and presence of an antioxidant, tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in PDI-inhibited cells. Also, PDI inhibition caused nuclear factor erythroid 2-related factor 2 (Nrf2; a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase and glutathione- S -transferase activities, and increased oxidative stress. In PDI-inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, whereas Kelch-like ECH-associated protein 1 (an Nrf2 regulatory protein) silencing protected cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation, causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. This study suggests an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.

Background: Chronic kidney disease (CKD) has become a major public health issue, which can lead to renal fibrosis regardless of the initial injury. It has been previously reported that miRNA-1228-3p was correlate with the progression of kidney fibrosis. However, the mechanism by which miRNA-1228-3p regulates renal fibrosis remains unclear., Methods: Renal tubular epithelial cells (HK-2) were treated with TGF-β1 (10 ng/ml) in an in vitro model of renal fibrosis. Gene and protein expressions in HK-2 cells were measured by Western-blot and RT-qPCR, respectively. The relation between miRNA-1228-3p and its target gene was investigated by dual luciferase report analysis., Results: Upregulation of miRNA-1228-3p significantly inhibited TGF-β1-induced fibrosis of HK-2 cells in vitro by targeting GDF11. In addition, miRNA-1228-3p exhibited anti-fibrosis effect through inhibition of the smad2/smad4 signaling pathway., Conclusion: Upregulation of miRNA-1228-3p markedly inhibited the progression of renal fibrosis in vitro, indicating that miRNA-1228-3p may serve as a potential novel target for the treatment of renal fibrosis.

The acyl-CoA synthetase medium-chain family member 2 ( Acsm2 ) gene was first identified and cloned by our group as a kidney-specific " KS " gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A , a homolog of mouse Acsm2 , was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.

Tubular atrophy is a common pathological feature of kidney fibrosis. Although fibroblasts play a predominant role in tissue fibrosis, the role of repairing tubular epithelia in tubular atrophy is unclear. We demonstrated the essential role of focal adhesion kinase (FAK)-mediated intratubular epithelial-mesenchymal transition (EMT) in the pathogenesis of tubular atrophy after severe ischemia-reperfusion injury (IRI). Actively proliferating tubular epithelia undergoing intratubular EMT were noted in the acute phase of severe IRI, resulting in tubular atrophy in the chronic phase, reflecting failed tubular repair. Furthermore, FAK was phosphorylated in the tubular epithelia in the acute phase of severe IRI, and its inhibition ameliorated both tubular atrophy and interstitial fibrosis in the chronic phase after injury. In vivo clonal analysis of single-labeled proximal tubular epithelial cells after IRI using proximal tubule reporter mice revealed substantial clonal expansion after IRI, reflecting active epithelial proliferation during repair. The majority of these proliferating epithelia were located in atrophic and nonfunctional tubules, and FAK inhibition was sufficient to prevent tubular atrophy. In vitro, transforming growth factor-β induced FAK phosphorylation and an EMT phenotype, which was also prevented by FAK inhibition. In an in vitro tubular epithelia gel contraction assay, transforming growth factor-β treatment accelerated gel contraction, which was suppressed by FAK inhibition. In conclusion, injury-induced intratubular EMT is closely related to tubular atrophy in a FAK-dependent manner.

Advanced oxidative protein products (AOPPs) are novel uremic toxins whose concentrations continuously increases in patients with chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) of tubular cells is the main mechanism underlying CKD pathogenesis. Studies have shown that AOPPs can induce EMT and promote renal fibrosis. However, the mechanism through which AOPPs induce tubular cell-EMT is poorly understood. In this study, we aimed to clarify the mechanisms underlying AOPP-induced EMT in human kidney proximal tubular (HKC-8) epithelial cells. Small molecule inhibitor, CRISPR-Cas9 knockout technology, siRNA knockdown technology, western blot, and reverse transcription-quantitative polymerase chain reaction were applied to investigate the mechanisms underlying AOPP-induced EMT in HKC-8 cells. AOPP treatment was found to significantly induce EMT, as evidenced by increased α-smooth muscle actin (α-SMA) and decreased E-cadherin levels, and upregulated Wnt1, β-catenin, Tcf4, and Gsk-3β expression. Conversely, blockade of Wnt/β-catenin signaling using small molecule inhibitor ICG-001 hindered AOPP-induced EMT. Moreover, knockout of receptor of advanced glycation end-products (RAGE) reversed these aforementioned effects, whereas AGE receptor 1 (AGER1)-specific siRNA transfection enhanced them. Taken together, these data suggested that AOPPs could induce HKC-8 cell EMT by activating the RAGE/Wnt/β-catenin signaling pathway and AGER1 could restore EMT by antagonizing the role of RAGE. These results may provide a new theoretical basis for EMT and help identify new therapeutic targets for suppressing CKD progression., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Renal tubulointerstitial fibrosis (TIF) is a common pathological feature of aristolochic acid (AA) nephropathy (AAN). G2/M arrest of proximal tubular cells (PTCs) is implicated in renal fibrosis of AAN, but the upstream regulatory molecule remains unknown. Hypoxia inducible factor-1α (HIF-1α) promotes renal fibrosis in kidney disease, but the role of HIF-1α in AAN is unclear. Evidence shows that HIF-1α and p21, a known inducer of cellular G2/M arrest, are closely related to each other. To investigate the role of HIF-1α in renal fibrosis of AAN and its effects on p21 expression and PTCs G2/M arrest, mice with HIF-1α gene knockout PTCs (PT-HIF-1α-KO) were generated, and AAN was induced by AA. In vitro tests were conducted on the human PTCs line HK-2 and primary mouse PTCs. HIF-1α and p21 expression, fibrogenesis, and G2/M arrest of PTCs were determined. Results showed that HIF-1α was upregulated in the kidneys of wild-type (WT) AAN mice, accompanied by p21 upregulation, PTCs G2/M arrest and renal fibrosis, and these alterations were reversed in PT-HIF-1α-KO AAN mice. Similar results were observed in HK-2 cells and were further confirmed in primary PTCs from PT-HIF-1α-KO and WT mice. Inhibiting p21 in HK-2 cells and primary PTCs did not change the expression of HIF-1α, but G2/M arrest and fibrogenesis were reduced. These data indicate that HIF-1α plays a key role in renal fibrosis in AAN by inducing PTCs G2/M arrest modulated through p21. HIF-1α may serve as a potential therapeutic target for AAN., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Diabetic nephropathy (DN), characterized by the chronic loss of kidney function during diabetes, is a long-term kidney disease that affects millions of populations. However, the etiology of DN remains unclear. DN cell model was established by treating HK-2 cells with high glucose (HG) in vitro. Expression of metastasis-associated lung adenocarcinoma transcript-1 (MALAT1), miR-30c, nucleotide binding and oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, IL-1β, and IL-18 in treated HK-2 cells were tested by quantitative polymerase chain reaction. HK-2 cell pyroptosis was assessed using flow cytometry analysis. Lactate dehydrogenase (LDH) activity was examined with a LDH assay kit. Correlation among MALAT1, miR-30c, and NLRP3 was examined via dual-luciferase reporter assay. Here, we revealed that MALAT1 was upregulated, but miR-30c was downregulated in HG-treated HK-2 cells, leading to upregulation of NLRP3 expression and cell pyroptosis. Knockdown of MALAT1 or overexpression of miR-30c protected HK-2 cells from HG-induced pyroptosis. Meanwhile, we found that MALAT1 promoted NLRP3 expression by sponging miR-30c through dual-luciferase reporter assay. Moreover, the co-transfection of sh-MALAT1 and miR-30c inhibitor could reverse the protective effects of the sh-MALAT1 on the HG-induced pyroptosis. These results confirmed that MALAT1 regulated HK-2 cell pyroptosis by inhibiting miR-30c targeting for NLRP3, contributing to a better understanding of DN pathogenesis and help to find out the effective treatment for DN., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Oxidative stress is associated with many renal disorders, both acute and chronic, and has also been described to contribute to the disease progression. Therefore, oxidative stress is a potential therapeutic target. The human antioxidant α 1 -microglobulin (A1M) is a plasma and tissue protein with heme-binding, radical-scavenging and reductase activities. A1M can be internalized by cells, localized to the mitochondria and protect mitochondrial function. Due to its small size, A1M is filtered from the blood into the glomeruli, and taken up by the renal tubular epithelial cells. A1M has previously been described to reduce renal damage in animal models of preeclampsia, radiotherapy and rhabdomyolysis, and is proposed as a pharmacological agent for the treatment of kidney damage. In this paper, we examined the in vitro protective effects of recombinant human A1M (rA1M) in human proximal tubule epithelial cells. Moreover, rA1M was found to protect against heme-induced cell-death both in primary cells (RPTEC) and in a cell-line (HK-2). Expression of stress-related genes was upregulated in both cell cultures in response to heme exposure, as measured by qPCR and confirmed with in situ hybridization in HK-2 cells, whereas co-treatment with rA1M counteracted the upregulation. Mitochondrial respiration, analyzed with the Seahorse extracellular flux analyzer, was compromised following exposure to heme, but preserved by co-treatment with rA1M. Finally, heme addition to RPTE cells induced an upregulation of the endogenous cellular expression of A1M, via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-pathway. Overall, data suggest that A1M/rA1M protects against stress-induced damage to tubule epithelial cells that, at least partly, can be attributed to maintaining mitochondrial function.

In the present study, we examined the molecular mechanism of astragaloside IV (AS-IV) in high glucose (HG)-induced epithelial-to-mesenchymal transition (EMT) in renal proximal tubular epithelial cells (PTCs). NRK-52E cell viability and apoptosis were determined by the cell counting kit-8 (CCK-8) assay and flow cytometric analysis, respectively. Expressions of E-cadherin, N-cadherin, vimentin, and occludin were measured by Western blot, and those of E-cadherin and N-cadherin were additionally measured by immunofluorescence analysis. Transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The expressions of Smad2, Smad3, phosphorylated-Smad2 (p-Smad2), and p-Smad3 were measured using Western blot. We found that AS-IV could recover NRK-52E cell viability and inhibit HG-induced cell apoptosis. TGF-β1, α-SMA, Smad2, Smad3, p-Smad2, and p-Smad3 expressions were decreased in the AS-IV-treated groups compared with the HG group. Moreover, the expressions of E-cadherin and occludin were remarkably up-regulated and those of N-cadherin and vimentin were down-regulated in the AS-IV-treated groups compared with the HG group. Interestingly, the TGF-β1 activator SRI-011381 hydrochloride had an antagonistic effect to AS-IV on HG-induced EMT behavior. In conclusion, AS-IV attenuates HG-induced EMT by inhibiting the TGF-β/Smad pathway in renal PTCs., (© 2020 The Author(s).)

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-β1 (TGF-β1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-β1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor + CD4 - CD8 - double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI.

Astragalus polysaccharide (APS) was modified using the Na 2 SeO 3 /HNO 3 method to obtain selenized APS (Se-APS) with a selenium content of 1.75 mg/g. The structure and physicochemical properties of APS and Se-APS were investigated through transmission electron microscopy-energy dispersive spectroscopy mapping, fourier transform infrared spectroscopy, nuclear magnetic resonance, nano-zetasizer analysis, atomic force microscopy, and scanning electron microscopy. APS and Se-APS did not exhibit toxic effects on human kidney proximal tubular epithelial (HK-2) cells and were able to remove hydroxyl and DPPH radicals, alleviate the damage caused by calcium oxalate (CaOx) monohydrate (COM) crystals to HK-2 cells, reduce intracellular reactive oxygen species levels, and restore cell viability and morphology. Both APS and Se-APS could inhibit COM growth, induce calcium oxalate dihydrate formation, and increase the absolute zeta potential of the crystals to inhibit crystal aggregation. However, the ability of Se-APS to regulate CaOx crystals and protect the cells from COM-induced damage was better than that of APS. These results suggested that Se-APS might be a candidate drug for the treatment and prevention of kidney stones., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10 -5 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK-2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1β, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD., Competing Interests: Declaration of Competing Interest Funding was provided, in part, by Boehringer Ingelheim Pharma to VGD., (Copyright © 2019. Published by Elsevier Inc.)

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic nephropathy (DN). However, the molecular mechanisms of EMT remain largely unknown. Tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling, and the loss of TSC1 or TSC2 function leads to a spectrum of diseases that underlie abnormalities in cell growth, proliferation, differentiation, and migration. In this study, we investigated the effects of TSC1 on high glucose (HG)-induced EMT of human proximal tubular epithelial HK-2 cells in vitro and renal fibrosis in TSC1 -/- and db/db mice. We found that the exposure of HK-2 cells to HG (30 mM) time-dependently decreased TSC1 expression, increased the phosphorylation of mTORC1, P70S6K, and 4E-BP-1, and promoted cell migration, resulting in EMT. Transfection of the cells with TSC1 mimic significantly ameliorated HG-induced EMT of HK-2 cells. The tubules-specific TSC1 knockout mice (TSC1 -/- ) displayed a significant decline in renal function. TSC1 -/- mice, similar to db/db mice, showed greatly activated mTORC1 signaling and EMT process in the renal cortex and exacerbated renal fibrosis. Overexpression of TSC1 through LV-TSC1 transfection significantly alleviated the progression of EMT and renal fibrosis in the renal cortex of db/db mice. Taken together, our results suggest that TSC1 plays a key role in mediating HG-induced EMT, and inhibition of TSC1-regulated mTORC1 signaling may be a potential approach to prevent renal fibrosis in DN.

The Wnt/β-catenin pathway is one of the most conserved signaling pathways across species with essential roles in development, cell proliferation, and disease. Wnt signaling occurs at the protein level and via β-catenin-mediated transcription of target genes. However, little is known about the underlying mechanisms regulating the expression of the key Wnt ligand Wnt3a or the modulation of its activity. Here, we provide evidence that there is significant cross-talk between the dopamine D 2 receptor (D2R) and Wnt/β-catenin signaling pathways. Our data suggest that D2R-dependent cross-talk modulates Wnt3a expression via an evolutionarily-conserved TCF/LEF site within the WNT3A promoter. Moreover, D2R signaling also modulates cell proliferation and modifies the pathology in a renal ischemia/reperfusion-injury disease model, via its effects on Wnt/β-catenin signaling. Together, our results suggest that D2R is a transcriptional modulator of Wnt/β-catenin signal transduction with broad implications for health and development of new therapeutics.

Dyslipidemia is a common risk factor of chronic kidney disease (CKD). Current notion suggests that insufficient intracellular fatty acid oxidation (FAO) and subsequently enhanced fatty acid esterification within renal resident cells, a process termed as renal lipotoxicity, is the key pathogenic event responsible for dyslipidemia-induced kidney injury. However, the detailed mechanism is not fully elucidated. Recently, accumulating data indicated that acetylation modification is an important regulating manner for both mitochondrial function and energy metabolism, while whether acetylation modification is involved in renal lipotoxicity is of little known. In the present study, the expression level of global lysine acetylation was detected by immunohistochemistry in high-fat diet mice and western blot in palmitic acid (PA) stimulated HK-2 cells. The acetylation levels of long-chain acyl-CoA dehydrogenases (LCAD) and β-hydroxyacyl-CoA dehydrogenase (β-HAD) were measured by immunoprecipitation. And a multifunction microplate reader was applied to detect FAO rate, triglyceride and acyl-CoA contents, and the enzyme activities, with cellular lipid accumulation identified by Oil Red O staining. We evidenced the acetylation levels of LCAD and β-HAD that were enhanced, which led to decreased enzymatic activities and impaired FAO rate. Furthermore, renal protein hyperacetylation induced by lipid overload was associated with increased expression of GCN5L1. And the silence of GCN5L1 in tubular epithelial cells resulted in deacetylation and activation of LCAD and β-HAD. Finally, excess lipids induced lipotoxicity and epithelial-mesenchymal transition (EMT) were ameliorated by GCN5L1 suppression, suggesting GCN5L1-mediated mitochondrial LCAD and β-HAD acetylation might be a key pathogenic event underlying excess lipids induced FAO impairment.

Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.

Epithelial-to-mesenchymal transition (EMT) and injury of tubular cells play critical roles in the pathogenesis of diabetic nephropathy (DN). lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been shown to be involved in DN progression. However, whether MALAT1 induces EMT and injury in tubular cells is unclear. Here, we investigated the effects of MALAT1 on human proximal tubular cells (HK-2 cells) and the underlying mechanism. We performed qPCR to detect MALAT1, E-cadherin, α-smooth muscle actin (α-SMA), kidney injury molecule 1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL). Additionally, we conducted Western blot analyses to measure E-cadherin, α-SMA, cyclin D1, c-Myc, and β-catenin in HK-2 cells cultured with normal glucose and high glucose (HG) and in transfected cells or cells treated with LiCl and DKK-1. The β-catenin localization was observed using immunofluorescence, and the protein levels of NGAL and KIM-1 were evaluated by ELISA. We found that HG-upregulated MALAT1 decreased E-cadherin and increased α-SMA, KIM-1, NGAL, cyclin D1, c-Myc, and β-catenin in HK-2 cells. LiCl exposure increased the expression of α-SMA but decreased that of E-cadherin on the base of knocking down MALAT1, and decreased NGAL and KIM-1 expression. DKK-1 showed the opposite effects. Our results suggested that upregulated MALAT1 induced EMT in HG-treated HK-2 cells through activating the Wnt/β-catenin pathway. However, MALAT1-mediated injury in HK-2 cells was not mediated by activation of the Wnt/β-catenin pathway. Our results indicate that MALAT1 might serve as a novel therapeutic target for suppressing the progression of DN.

Background: Dyslipidemia causes renal damage; however, the detailed molecular mechanism has not been clarified. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. In the present study, we investigated whether the accumulation of lipids induced by 7-ketocholesterol (7-KC) in tubular epithelial cells produce a fibrotic and inflammatory response through CPT1a., Methods: Using an epithelial cell line, NRK-52E, we determine if intracellular accumulation of 7-KC modulates profibrotic and inflammatory events through CPT1a gene expression. In addition, the direct effects of CPT1a genetic modification has been studied., Results: Our results revealed that high levels of 7-KC induce increased expression of CPT1a, TGF-β1, α- SMA and NLRP3 that was correlated with lipid content. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC laden cells and decreased the expression of CPT1a, TGF-β1, α- SMA and NLRP3. Furthermore, CPT1a Knockdown and C75 pre-treatment increased lipid content but decreased TGF-β1, α- SMA and NLRP3., Conclusions: Our findings reveal that the profibrotic effect of 7-KC on renal epithelial cells are mediated by CPT1a overexpression, which acts on TGF-β1, α-SMA and NLRP3. Thus, CPT1a downregulation protects against 7-KC-induced fibrosis in tubular epithelial cells by downregulating TGF-β1, α- SMA and NLRP3., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The antiviral drug Telbivudine (LdT) has an extrahepatic pharmaceutical effect that improves renal inflammation and tubulointerstitial fibrosis. However, the exact mechanism of action requires further investigation. Toll-like receptor 4 (TLR4) is involved in several physiological processes, including inflammation, fibrosis, innate immunity, and hepatitis B virus-associated glomerulonephritis. The epithelial-to-mesenchymal transition (EMT) is the characteristic pathological change in tubulointerstitial fibrosis. In this study, we used transforming growth factor-β (TGF-β) to stimulate human proximal tubular epithelial (HK-2) cells to investigate the effects of LdT in EMT. In addition, we treated HK-2 cells with a TLR4 agonist, lipopolysaccharide, to determine the effect of LdT on TLR4. The results indicated that LdT inhibited the expression of TLR4 and its downstream proteins. It also decreased the release of inflammatory factors, downregulated the TGF-β/Smad signaling pathway, and reversed the EMT changes seen in HK-2 cells. In conclusion, LdT antagonized TLR4 to inhibit EMT in proximal tubular epithelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tubular epithelial cell-derived profibrotic factors are known as the driving force in renal fibrosis for their roles in activating the surrounding fibroblast. However, the mechanisms driving their expressions remain undefined. Here, we find that kidney human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, significantly increased in unilateral ureteral obstruction-induced renal fibrosis, in type 1 and type 2 diabetic nephropathy, and in kidney biopsies from patients with renal fibrosis. Notably, the upregulation of HER2 mainly occurred in proximal tubular epithelial cells (PTECs). In vivo, the ectopic expression of HER2 in these cells was sufficient to activate the interstitial fibroblast and initiate interstitial fibrosis, whereas inhibiting HER2 reduced the accumulation of myofibroblasts and the extent of renal fibrosis in the mouse obstruction model and in streptozotocin (STZ)-induced diabetic mice. We also generated a tubular epithelial cell subline stably expressing HER2 and performed transcriptome RNA sequence analysis. This showed that sustained HER2 expression significantly induced the expression of profibrotic connective tissue growth factor (CTGF). Mechanistically, the induction of CTGF depended on the HER2-mediated activation of Stat3 in the tubular epithelium. In vitro, the incubation of kidney fibroblasts with culture medium from HER2-overexpressed tubular epithelial cells promoted fibroblast proliferation and activation, whereas silencing CTGF impeded the profibrotic effects of the tubular epithelial cell preconditioned media. Thus, our results highlight the significance of HER2 in tubular injury and characterize its role in promoting surrounding fibroblast activation and renal fibrosis in a paracrine manner., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to elucidate the role of microRNA-216a in microvesicles (MVs) during the process of renal interstitial fibrosis and to investigate its underlying mechanism., Materials and Methods: Unilateral ureteral occlusion (UUO) model was first established in mice, and kidney tissues and urine in the obscured kidney were collected. NRK-52E cells were induced with 5 ng/mL transforming growth factor-β1 (TGF-β1) for constructing the renal interstitial fibrosis model in vitro. Subsequently, the expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin (FN) in NRK-52E cells induced with or without TGF-β1 were determined, respectively. The culture medium was collected from NRK-52E cells of the control group (without TGF-β1 induction) and the TGF-β1 group (TGF-β1 induction), and MVs were observed. Afterward, NRK-52E cells were treated with MVs isolated from the control group or the TGF-β1 group, followed by detecting the expressions of E-cadherin, α-SMA and FN. Meanwhile, the expression levels of CD63, microRNA-216a, PTEN and p-AKT were determined as well. The microRNA-216 level in kidney tissues and urine of UUO mice were determined. Furthermore, the expressions of PTEN and p-AKT in mouse kidney tissues were accessed by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)., Results: TGF-β1 induction in NRK-52E cells gradually downregulated E-cadherin, whereas upregulated α-SMA and FN with the prolongation of induction time. MVs isolated from the culture medium of the TGF-β1 group downregulated E-cadherin, and upregulated FN and α-SMA. The expression levels of CD63 and microRNA-216a were markedly higher in the TGF-β1 group compared with the control group. Downregulated PTEN and upregulated p-AKT were observed in TGF-β1-induced cells at both mRNA and protein levels. Besides, microRNA-216a expression in mouse kidney tissues and urine from obscured kidney was remarkably increased with the prolongation of UUO. Consistent with those in NRK-52E cells, the protein level of PTEN was significantly decreased, whereas p-AKT was markedly increased with the prolongation of UUO., Conclusions: MVs containing microRNA-216a secreted by injured proximal tubular epithelial cells participate in renal interstitial fibrosis by activating the PTEN/AKT pathway.

Stress granules (SGs) are a type of cytoplasmic structures formed in eukaryotic cells upon cell stress, which mainly contain RNA-binding proteins and RNAs. The formation of SGs is generally regarded as a mechanism for cells to survive a harsh insult. However, little is known about SG formation and function in kidneys. To address this, we applied different kinds of stressors to cultured proximal tubular cells as well as a short period of ischemia-reperfusion to mouse kidneys. It was found that glycolytic inhibitors such as 2-deoxy-d-glucose and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one induced SG formation within 30 min in these cells. Similarly, SGs were induced by inhibitors of mitochondrial respiration such as sodium azide and CCCP. Renal ischemia-reperfusion induced SG formation in the cells of proximal tubules. To test the role of SGs, we stably knocked down G3bp1, a SG core protein, in renal tubular cells by shRNA viral transduction. As expected, knockdown of G3bp1 largely disrupted the assembly of SGs. After azide or cisplatin treatment, more dead cells were found in knockdown cells compared with controls, accompanied by increases in cleaved/active caspase-3. Reintroduction of exogenous G3bp1 into knockdown cells could rescue the cell death phenotype. Taken together, our data provide the first evidence of SG formation in renal tubular cells during metabolic stress and acute kidney injury. SGs are formed to protect proximal tubular cells under these conditions. Modulation of SG biogenesis may provide a novel approach to lessen the severity of renal diseases.

In immune-mediated glomerular diseases like crescentic glomerulonephritis (cGN), inflammatory CD4 + T cells accumulate within the tubulointerstitial compartment in close contact to proximal and distal tubular epithelial cells and drive renal inflammation and tissue damage. However, whether renal epithelial cell populations play a role in the pathogenesis of cGN by modulating CD4 + T cell responses is less clear. In the present study, we aimed to investigate the potential of renal epithelial cells to function as antigen-presenting cells, thereby stimulating CD4 + T cell responses. Using a FACS-based protocol that allowed comparative analysis of cortical epithelial cell populations, we showed that particularly proximal tubular epithelial cells (PTECs) express molecules linked with antigen-presenting cell function, including major histocompatibility complex class II (MHCII), CD74, CD80, and CD86 in homeostasis and nephrotoxic nephritis, a murine model of cGN. Protein expression was visualized at the PTEC single cell level by imaging flow cytometry. Interestingly, we found inflammation-dependent regulation of epithelium-expressed CD74, CD80, and CD86, whereas MHCII expression was not altered. Antigen-specific stimulation of CD4 + T cells by PTECs in vitro supported CD4 + T cell survival and induced CD4 + T cell activation, proliferation, and inflammatory cytokine production. In patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis, MHCII and CD74 were expressed by both proximal and distal tubules, whereas CD86 was predominantly expressed by proximal tubules. Thus, particularly PTECs have the potential to induce an inflammatory phenotype in CD4 + T cells in vitro, which might also play a role in the pathology of immune-mediated kidney disease.

The kidney is a frequent target for organ-specific toxicity as a result of its primary function in controlling body fluids, for example, via resorption of amino acids, peptides, nutrients, ions, xenobiotics and water from the primary urine as well as excretion of metabolic waste products and hydrophilic and amphiphilic xenobiotics. Compounds exhibiting dose-limiting nephrotoxicity include drugs from highly diverse classes and chemical structures, e.g., antibiotics (gentamicin), chemotherapeutics (cisplatin), immunosuppressants (cyclosporine A and tacrolimus) or bisphosphonates (zoledronate). All of these compounds elicit nephrotoxicity primarily by injuring renal proximal tubule epithelial cells (RPTECs). However, prediction of a compound's nephrotoxic potential in humans to support early unmasking of risk-bearing drug candidates remains an unmet challenge, mainly due to the complex kidney anatomy as well as pronounced inter- and intraspecies differences and lack of relevant and validated human in vitro models. Accordingly, we used the recently established human RPTEC/TERT1 cell line to carry out toxicity studies with a focus on impairment of functional characteristics, i.e., transepithelial electrical resistance (TEER), vectorial transport of water, cations, and anions. Results were compared to real-time cytotoxicity assessments using cellular impedance (xCELLigence assay) and the routine cell viability readout (MTT). As expected, most toxins caused exposure time- and concentration-dependent cytotoxicity. However, for some compounds (cyclosporine A and tacrolimus), transport processes were strongly impaired in absence of a concomitant decrease in cell viability. In conclusion, these data demonstrate that functional parameters are important, highly sensitive and meaningful additional readouts for nephrotoxicity assessment in human renal proximal tubule epithelial cells.