The E-liquid flavoring vanillin alters energy and autophagic pathways in human proximal tubule (HK-2) epithelial cells.

The use of flavored e-liquids in electronic nicotine delivery systems (ENDS) has become very popular in recent years, but effects of these products have not been well characterized outside the lung. In this study, acute exposure to the popular flavoring vanillin (VAN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0-1000 µM VAN for 24 or 48 h and cellular stress responses were determined. Mitochondrial viability using MTT assay showed a significant decrease between the control and 1000 µM group by 48 h. Seahorse XFp analysis showed significantly increased basal respiration, ATP production, and proton leak after 24 h exposure. By 48 h exposure, these parameters remained significantly increased in addition to non-mitochondrial respiration and maximal respiration. Glycolytic activity after 24 h exposure showed significant decreases in glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. The autophagy markers microtubule-associated protein 1A/1B light chain 3 (LC3B-I and LC3B-II) were probed via western blotting. The ratio of LC3B-II/LC3B-I was significantly increased after 24 h exposure to VAN, but by 48 h this ratio significantly decreased. The mitophagy marker PINK1 showed an increasing trend at 24 h, and its downstream target Parkin was significantly increased between the control and 750 µM group only. Finally, the oxidative stress marker 4-HNE was significantly decreased after 48 h exposure to VAN. These results indicate that acute exposure to VAN in the kidney HK-2 model can induce energy and autophagic changes within the cell.

Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection.

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.

Notch1 signaling is limited in healthy mature kidneys in vivo.

OBJECTIVE: A Delta-Notch signaling component, Notch1, is involved in the normal development and multiple disorders of the kidney. Although the increase in Notch1 signaling is crucial to these pathogeneses, the basal signaling level in 'healthy' mature kidneys is still unclear. To address this question, we used an artificial Notch1 receptor fused with Gal4/UAS components in addition to the Cre/loxP system and fluorescent proteins in mice. This transgenic reporter mouse system enabled labeling of past and ongoing Notch1 signaling with tdsRed or Cre recombinase, respectively. RESULTS: We confirmed that our transgenic reporter mouse system mimicked the previously reported Notch1 signaling pattern. Using this successful system, we infrequently observed cells with ongoing Notch1 signaling only in Bowman's capsule and tubules. We consider that Notch1 activation in several lines of disease model mice was pathologically significant itself.

Bone marrow mesenchymal stem cell-derived exosomal miR-30e-5p ameliorates high-glucose induced renal proximal tubular cell pyroptosis by inhibiting ELAVL1.

BACKGROUND: The rapid increase in the prevalence of diabetes has resulted in more cases of diabetic kidney disease (DKD). Treatment with bone marrow mesenchymal stem cells (BMSCs) may represent an alternative strategy to manage DKD. METHODS: HK-2 cells were treated with 30 mM high glucose (HG). Bone marrow MSC-derived exosomes (BMSC-exos) were isolated and internalized into HK-2 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays were used to measure viability and cytotoxicity. The secretion of IL-1ß and IL-18 was measured by ELISA. Pyroptosis was assessed by flow cytometry. Quantitative RT-PCR was used to measure the levels of miR-30e-5p, ELAV like RNA binding protein 1 (ELAVL1), IL-1ß, and IL-18. The expression of ELAVL1 and pyroptosis-associated cytokine proteins was determined by western blot analysis. A dual-luciferase reporter gene assay was conducted to confirm the relationship between miR-30e-5p and ELAVL1. RESULTS: BMSC-exos decreased LDH, IL-1ß, and IL-18 secretion and inhibited the expression of the pyroptosis-related factors (IL-1ß, caspase-1, GSDMD-N, and NLRP3) in HG-induced HK-2 cells. Moreover, miR-30e-5p depletion derived from BMSC-exos promoted HK-2 cell pyroptosis. Besides, miR-30e-5p over-expression or ELVAL1 knockdown could directly inhibit pyroptosis. ELAVL1 was a target of miR-30e-5p and knocking down ELAVL1 reversed the effect of miR-30e-5p inhibition in BMSC-exos-treated HK-2 cells. CONCLUSIONS: BMSC-derived exosomal miR-30e-5p inhibits caspase-1-mediated pyroptosis by targeting ELAVL1 in HG-induced HK-2 cells, which might provide a new strategy for treating DKD.

Ablation of TRPC3 compromises bicarbonate and phosphate transporter activity in mice proximal tubular cells.

Proximal tubular (PT) cells reabsorb most calcium (Ca2+ ), phosphate (PO4 3- ), bicarbonate (HCO3 - ), and oxalate (C2 O4 2- ) ions. We have shown that mice lacking Transient Receptor Potential Canonical 3 (TRPC3-/- ) channel are moderately hypercalciuric with presentation of luminal calcium phosphate (CaP) crystals at the loop of Henle (LOH). However, other predisposing factors for such crystal deposition are unknown. Thus, we examined the distinctions in functional status of HCO3 - , PO4 3- , and C2 O4 2- transporters in PT cells of wild type (WT) and TRPC3-/- mice by whole-cell patch clamp techniques to assess their contribution in the development of LOH CaP crystals. Here we show the development of concentration dependent HCO3 - -induced currents in all PT cells, which was confirmed by using specific HCO3 - channel inhibitor, S0859. Interestingly, such activities were diminished in PT cells from TRPC3-/- mice, suggesting reduced HCO3 - transport in absence of TRPC3. While PO4 3- -induced currents were also concentration dependent in all PT cells (confirmed by PO4 3- channel inhibitor, PF-06869206), those activities were reduced in absence of TRPC3, suggesting lower PO4 3- reabsorption that can leave excess luminal PO4 3- . Next, we applied thiosulfate (O3 S2 2 - ) as a competitive inhibitor of the SLC26a6 transporter upon C2 O4 2- current activation and observed a reduced C2 O4 2- -induced conductance which was greater in TRPC3-/- PT cells. Together, these results suggest that the reduced activities of HCO3 - , PO4 3- , and C2 O4 2- transporters in moderately hypercalciuric (TRPC3-/- ) PT cells can create a predisposing condition for CaP and CaP tubular crystallization, enabling CaP crystal formation in LOH of TRPC3-/- mice.

Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells.

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, ß-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule.

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Fucoxanthin Attenuates Oxidative Damage by Activating the Sirt1/Nrf2/HO-1 Signaling Pathway to Protect the Kidney from Ischemia-Reperfusion Injury.

Oxidative stress is a key component of renal ischemia/reperfusion (I/R) injury. Fucoxanthin (Fx), a marine carotenoid with enhanced antioxidant capacity, acts as a ROS inhibitor in diseases such as ischemic stroke and acute lung injury. We hypothesized that fucoxanthin could attenuate renal I/R-induced oxidative damage. C57BL/6 mice (n = 30) were randomly assigned to sham, IR, IR + DMSO, and IR + Fx (25, 50, and 100 mg/kg) groups. The renal I/R injury was induced by clamping the left kidney nephron tip in mice. Fucoxanthin was injected intraperitoneally 24 hours before surgery. Compared with the IR group, pretreatment with fucoxanthin significantly improved renal dysfunction and tissue structural damage and inhibited ROS levels and apoptosis. Consistent results were observed in HK-2 cells. Besides, we found that renal I/R resulted in decreased expression of Sirt1, Nrf2, and HO-1, while fucoxanthin upregulated the expression of Sirt1, Nrf2, and HO-1. The protective effects of fucoxanthin were significantly reversed by EX527 (a selective inhibitor of Sirt1) or si-Sirt1. In conclusion, our study investigated the protective effect of fucoxanthin against renal I/R injury, and the underlying mechanism may be related to the activation of the Sirt1/Nrf2/HO-1 signaling pathway by fucoxanthin to attenuate oxidative stress-induced apoptosis.

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system.

OBJECTIVE: To provide a standardized protocol for large-scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC). METHODS: The hPSC were expanded and differentiated into PTEC on matrix-coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin-treatment was assessed to check the potential use of PTEC to model drug-induced nephrotoxicity. RESULTS: hPSC expansion and PTEC differentiation could be performed directly on matrix-coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10-fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico-basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM-1 after Cisplatin treatment. CONCLUSION: We demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix-coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.

Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin.

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as "albumin-induced albumin endocytosis". Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.

Direct evidence of proximal tubular proliferation in early diabetic nephropathy.

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.

LncRNA RPSAP52 regulates miR-423-5p/GSTM1 axis to suppress hypoxia-induced renal proximal tubular epithelial cell apoptosis.

This study aimed to investigate the roles of RPSAP52 in renal failure. Our results showed that RPSAP52 was upregulated in plasma of renal failure patients in comparison to healthy controls. Dual luciferase reporter assay showed that RPSAP52 could interact with miR-423-5p, while overexpression of RPSAP52 and miR-423-5p did not alter the expression of each other in human renal proximal tubular epithelial cells (HRPTEpCs). In addition, overexpression of RPSAP52 increased the expression levels of GSTM1 in HRPTEpCs. Cell apoptosis assay showed that overexpression of RPSAP52 and GSTM1 decreased the apoptotic rate of HRPTEpCs under hypoxia conditions. MiR-423-5p played an opposite role and attenuated the effects of overexpressing RPSAP52 and GSTM1. Therefore, RPSAP52 may regulate miR-423-5p/GSTM1 axis to suppress hypoxia-induced HRPTEpC apoptosis.

MicroRNA 1228 Mediates the Viability of High Glucose-Cultured Renal Tubule Cells through Targeting Thrombospondin 2 and PI3K/AKT Signaling Pathway.

AIM: The present study aimed to elucidate the potential function of microRNA 1228 (miR-1228) on the high glucose (HG)-damaged human renal proximal tubule cells (HK-2) and the underlying mechanism. METHODS: The datasets GSE47185 and GSE51674 were downloaded from the Gene Expression Omnibus database for mining differently expressed mRNAs and miRNAs, respectively. Bioinformatics online tools were applied to predict the binding sites between miR-1228 and thrombospondin 2 (THBS2), which was confirmed by dual-luciferase assay. Real-time quantitative polymerase chain reaction was used to detect the mRNA level of miR-1228/THBS2. Western blot was used to detect the protein level of THBS2 and the PI3K/AKT signaling pathway-associated markers. HK-2 cells were cultured in HG (30 mM) to mimic hyperglycemia. Cell counting kit 8 and flow cytometry assays were utilized to determine the cell proliferation and apoptosis. RESULTS: The expression of THBS2 was significantly upregulated in diabetic nephropathy (DN) based on bioinformatics tools and identified as a direct target of miR-1228. miR-1228 was downregulated in DN and HG-damaged HK-2 cells. HG notably reduced HK-2 cell proliferation. This negative effect was attenuated by transfecting with an miR-1228 mimic and aggravated by transfecting with an miR-1228 inhibitor. However, under basal condition, there was no significant effect on the HK-2 cell proliferation among blank control, mimic, and inhibitor groups. Overexpression of THBS2 abolished the elevating effect of the miR-1228 mimic on the HG-damaged HK-2 cell proliferation, while restored the inhibitory effects of the miR-1228 mimic on the cell apoptosis. On the contrary, the suppressive effects on the proliferation and the enhancive effects on the apoptosis by silencing miR-1228 in HK-2 cells stimulated with HG can be weakened by recommendation of THBS2 small interference RNAs. Furthermore, we also found that HG significantly enhanced the phosphorylation levels of PI3K and AKT. In terms of overexpression and knockdown experiments, Western blot analysis further revealed that miR-1228 inhibited the activation of the PI3K/AKT signaling pathway in HG-damaged HK-2 cells by regulating THBS2. CONCLUSION: The findings illustrated that miR-1228 improved survivability and inhibited apoptosis in HK-2 cells stimulated with HG partly by restraining the activation of the PI3K/AKT signaling pathway.

The polypeptide antibiotic polymyxin B acts as a pro-inflammatory irritant by preferentially targeting macrophages.

Polymyxin B (PMB) is an essential antibiotic active against multidrug-resistant bacteria, such as multidrug-resistant Pseudomonas aeruginosa (MDRP). However, the clinical use of PMB is limited, because PMB causes serious side effects, such as nephrotoxicity and neurotoxicity, probably due to its cytotoxic activity. However, cytotoxic mechanisms of PMB are poorly understood. In this study, we found that macrophages are particularly sensitive to PMB, when compared with other types of cells, including fibroblasts and proximal tubule (PT) cells. Of note, PMB-induced necrosis of macrophages allowed passive release of high mobility group box 1 (HMGB1). Moreover, upon exposure of PMB to macrophages, the innate immune system mediated by the NLR family pyrin domain containing 3 (NLRP3) inflammasome that promotes the release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) was stimulated. Interestingly, PMB-induced IL-1ß release occurred in the absence of the pore-forming protein gasdermin D (GSDMD), which supports the idea that PMB causes plasma membrane rupture accompanying necrosis. Emerging evidence has suggested that both HMGB1 and IL-1ß released from macrophages contribute to excessive inflammation that promote pathogenesis of various diseases, including nephrotoxicity and neurotoxicity. Therefore, these biochemical properties of PMB in macrophages may be associated with the induction of the adverse organ toxicity, which provides novel insights into the mechanisms of PMB-related side effects.

Empagliflozin Ameliorates Free Fatty Acid Induced-Lipotoxicity in Renal Proximal Tubular Cells via the PPARγ/CD36 Pathway in Obese Mice.

High serum levels of free fatty acids (FFAs) could contribute to obesity-induced nephropathy. CD36, a class B scavenger receptor, is a major receptor mediating FFA uptake in renal proximal tubular cells. Empagliflozin, a new anti-diabetic agent, is a specific inhibitor of sodium-glucose co-transporter 2 channels presented on renal proximal tubular cells and inhibits glucose reabsorption. In addition, empagliflozin has shown renoprotective effects. However, the mechanism through which empagliflozin regulates CD36 expression and attenuates FFA-induced lipotoxicity remains unclear. Herein, we aimed to elucidate the crosstalk between empagliflozin and CD36 in FFA-induced renal injury. C57BL/6 mice fed a high-fat diet (HFD) and palmitic acid-treated HK-2 renal tubular cells were used for in vivo and in vitro assessments. Empagliflozin attenuated HFD-induced body weight gain, insulin resistance, and inflammation in mice. In HFD-fed mice, CD36 was upregulated in the tubular area of the kidney, whereas empagliflozin attenuated CD36 expression. Furthermore, empagliflozin downregulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ. Treatment with a PPARγ inhibitor (GW9662) did not further decrease PPARγ expression, whereas a PPARγ antagonist reversed this effect; this suggested that empagliflozin may, at least partly, decrease CD36 by modulating PPARγ. In conclusion, empagliflozin can ameliorate FFA-induced renal tubular injury via the PPARγ/CD36 pathway.

Interleukin-7 Is Associated with Clinical and Pathological Activities in Immunoglobulin A Nephropathy and Protects the Renal Proximal Tubule Epithelium from Cellular Fibrosis.

OBJECTIVE: Diagnosis of immunoglobulin A nephropathy (IgAN) requires the evaluation of renal biopsy specimens. However, renal biopsy is an invasive procedure and is not frequently performed for various reasons. Thus, recognized noninvasive biomarkers for predicting IgAN progression are urgently needed. METHODS: In the present study, we included 86 IgAN patients with renal biopsy from June 2015 to May 2016 and had their plasma interleukin-7 (IL-7) level measured with ELISA. The association between the plasma IL-7 level and clinico-pathological characteristics was analyzed. Immunohistochemical staining was used to assay the in situ expression of IL-7 in vivo. Western blotting was performed to examine the production of extracellular matrix, p-mTOR and the markers of autophagy under the treatment of IL-7 after TGF-ß1 stimulation in renal tubular epithelial cells. RESULTS: IL-7 was significantly decreased in patients with IgAN compared to healthy subjects (2.3077 vs. 8.6294 pg/mL, P<0.0001). There was a significant difference in the plasma IL-7 level between tubular atrophy/interstitial fibrosis T0 and T2 classes (P=0.0064). A lower plasma IL-7 value in patients at the time of biopsy indicated a poor renal outcome. In addition, IL-7 was over-expressed in renal tubular epithelial cells and significantly attenuated transforming growth factor ßl-induced extracellular matrix production by suppression of cellular autophagy via activation of mTOR1 signaling. CONCLUSION: These results suggested that IL-7 might be a noninvasive biomarker for predicating IgAN. It protected renal proximal tubular epithelial cells from cellular fibrosis by inhibiting autophagy via mTORl signaling.

miR-23a-3p regulates the inflammatory response and fibrosis in diabetic kidney disease by targeting early growth response 1.

Diabetic kidney disease (DKD) has become the most common cause of chronic kidney disease. Proteinuria is generally considered one of the clinical indicators of renal damage, and it is also closely related to the progression of DKD. Accumulating evidence indicates that proteinuria induces an upregulation of the expression levels of inflammatory cytokines and fibrosis markers in renal tubular epithelial cells, but the mechanism remains unclear. Previously, we showed that early growth response 1 (Egr1) played a key role in renal tubular injury. However, the upstream mechanism of Egr1 in the development of DKD is poorly understood. In this study, we found that albumin stimulation significantly increased the expression levels of Egr1, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and fibronectin (FN) in HK-2 cells but decreased miR-23a-3p levels. We then identified that miR-23a-3p targeted the 3' untranslated region (UTR) of Egr1 and directly suppressed the expression of Egr1. Moreover, we found that overexpression and inhibition of miR-23a-3p in HK-2 cells attenuated and promoted the expression of IL-6, TNF-α, and FN, respectively. Additionally, Egr1 silencing reversed the inflammation and fibrosis caused by the miR-23a-3p inhibitor. Thus, we conclude that miR-23a-3p attenuates the development of DKD through Egr1, suggesting that targeting miR-23a-3p may be a novel therapeutic approach for DKD.

Selective Activation of Endoplasmic Reticulum Stress by Reactive-Oxygen-Species-Mediated Ochratoxin A-Induced Apoptosis in Tubular Epithelial Cells.

Ochratoxin A (OTA), one of the major food-borne mycotoxins, impacts the health of humans and livestock by contaminating food and feed. However, the underlying mechanism of OTA nephrotoxicity remains unknown. This study demonstrated that OTA induced apoptosis through selective endoplasmic reticulum (ER) stress activation in human renal proximal tubular cells (HK-2). OTA increased ER-stress-related JNK and precursor caspase-4 cleavage apoptotic pathways. Further study revealed that OTA increased reactive oxygen species (ROS) levels, and N-acetyl cysteine (NAC) could reduce OTA-induced JNK-related apoptosis and ROS levels in HK-2 cells. Our results demonstrate that OTA induced ER stress-related apoptosis through an ROS-mediated pathway. This study provides new evidence to clarify the mechanism of OTA-induced nephrotoxicity.

Adenosine A1 Receptor Deficiency Aggravates Extracellular Matrix Accumulation in Diabetic Nephropathy through Disturbance of Peritubular Microenvironment.

BACKGROUND: We previously observed that adenosine A1 receptor (A1AR) had a protective role in proximal tubular megalin loss associated with albuminuria in diabetic nephropathy (DN). In this study, we aimed to explore the role of A1AR in the fibrosis progression of DN. METHODS: We collected DN patients' samples and established a streptozotocin-induced diabetes model in wild-type (WT) and A1AR-deficient (A1AR-/-) mice. The location and expression of CD34, PDGFRß, and A1AR were detected in kidney tissue samples from DN patients by immunofluorescent and immunohistochemical staining. We also analyzed the expression of TGFß, collagen (I, III, and IV), α-SMA, and PDGFRß using immunohistochemistry in WT and A1AR-/- mice. CD34 and podoplanin expression were analyzed by Western blotting and immunohistochemical staining in mice, respectively. Human renal proximal tubular epithelial cells (HK2) were cultured in medium containing high glucose and A1AR agonist as well as antagonist. RESULTS: In DN patients, the expression of PDGFRß was higher with the loss of CD34. The location of PDGFRß and TGFß was near to each other. The A1AR, which was colocalized with CD34 partly, was also upregulated in DN patients. In WT-DN mice, obvious albuminuria and renal pathological leisure were observed. In A1AR-/- DN mice, more severe renal tubular interstitial fibrosis and more extracellular matrix deposition were observed, with lower CD34 expression and pronounced increase of PDGFRß. In HK2 cells, high glucose stimulated the epithelial-mesenchymal transition (EMT) process, which was inhibited by A1AR agonist. CONCLUSION: A1AR played a critical role in protecting the tubulointerstitial fibrosis process in DN by regulation of the peritubular microenvironment.