Calcineurin inhibitors ameliorate PAN-induced podocyte injury through the NFAT-Angptl4 pathway.

Podocyte injury plays a vital role in proteinuria and nephrotic syndrome. Calcineurin (CaN) inhibitors are effective in reducing proteinuria. However, their molecular mechanism is still not fully understood. Angiopoietin-like-4 (ANGPTL4) is a secreted protein that mediates proteinuria in podocyte-related nephropathy. In this study, we established a puromycin aminonucleoside (PAN)-induced minimal-change disease (MCD) rat model and a cultured podocyte injury model. We found that CaN inhibitors protected against PAN-induced podocyte injury, accompanied by an inhibition of Nfatc1 and Angptl4 both in vivo and in vitro. Nfatc1 overexpression and knockdown experiments indicated that Angptl4 was regulated by Nfatc1 in podocytes. ChIP assays further demonstrated that Nfatc1 increased Angptl4 expression by binding to the Angptl4 promoter. In addition, overexpression and knockdown of Angptl4 revealed that Angptl4 directly induced rearrangement of the cytoskeleton of podocytes, reduced the expression of synaptopodin, and enhanced PAN-induced podocyte apoptosis. Furthermore, in a cohort of 83 MCD and 94 membranous nephropathy (MN) patients, we found increased expression of serum ANGPTL4 compared to 120 healthy controls, and there were close correlations between serum ANGPTL4 and Alb, urinary protein, urinary Alb, eGFR, Scr, and BUN in MCD patients. No obvious correlation was found in MN patients. Immunofluorescence studies indicated that increased ANGPTL4 in MCD and MN patients was located mostly in podocytes. In conclusion, our results demonstrate that CaN inhibitors ameliorate PAN-induced podocyte injury by targeting Angptl4 through the NFAT pathway, and Angptl4 plays a vital role in podocyte injury and is involved in human podocyte-related nephropathy. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ADAM10 mediates ectopic proximal tubule development and renal fibrosis through Notch signalling.

Disturbed intrauterine development increases the risk of renal disease. Various studies have reported that Notch signalling plays a significant role in kidney development and kidney diseases. A disintegrin and metalloproteinase domain 10 (ADAM10), an upstream protease of the Notch pathway, is also reportedly involved in renal fibrosis. However, how ADAM10 interacts with the Notch pathway and causes renal fibrosis is not fully understood. In this study, using a prenatal chlorpyrifos (CPF) exposure mouse model, we investigated the role of the ADAM10/Notch axis in kidney development and fibrosis. We found that prenatal CPF-exposure mice presented overexpression of Adam10, Notch1 and Notch2, and led to premature depletion of Six2+ nephron progenitors and ectopic formation of proximal tubules (PTs) in the embryonic kidney. These abnormal phenotypic changes persisted in mature kidneys due to the continuous activation of ADAM10/Notch and showed aggravated renal fibrosis in adults. Finally, both ADAM10 and NOTCH2 expression were positively correlated with the degree of renal interstitial fibrosis in IgA nephropathy patients, and increased ADAM10 expression was negatively correlated with decreased kidney function evaluated by serum creatinine, cystatin C, and estimated glomerular filtration rate. Regression analysis also indicated that ADAM10 expression was an independent risk factor for fibrosis in IgAN. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Plasma metabolite biomarkers related to secondary hyperparathyroidism and parathyroid hormone.

BACKGROUND: Hyperphosphatemia, hypocalcemia, and elevation of parathyroid hormone (PTH) are typical abnormalities of uremic patients with Secondary hyperparathyroidism (SHPT). However, metabolic imbalance associated with SHPT is not well understood. METHODS: A total of 15 SHPT patients with an intact parathyroid hormone (iPTH) level > 600 pg/mL were set as preoperative (PR) group, 15 age- and gender-matched controls who had undergone parathyroidectomy plus forearm transplantation because of hyperparathyroidism and achieved an iPTH level <150 pg/mL were set as postoperative (PO) group. Metabolite profiling of these 30 uremic patients and five healthy controls (HC) was performed using ultra performance liquid chromatography-mass spectrometry. RESULTS: Five differential metabolites, including allyl isothiocyanate, L-phenylalanine, D-Aspartic acid, indoleacetaldehyde, and D-galactose correlated with PTH were identified in this study. Taking them as a biomarker signature, PR group can be distinguished from HC group with an area under the curve (AUC) of 0.947 (95% CI, 0.76-1) and PO group with an AUC of 0.6 (95% CI, 0.38-0.807). CONCLUSIONS: The serum metabolome correlated with PTH is successfully demonstrated for a better understanding of the pathogenesis of SHPT.

The Mitochondria-Targeted Metabolic Tubular Injury in Diabetic Kidney Disease.

BACKGROUND/AIMS: Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD) worldwide, and the importance of tubular injury has been highlighted in recent years. However, the underlying mechanisms and effective therapeutic targets are still unclear. In this study, we investigated mtDNA, mitochondrial dynamics, function and metabolic pathways to determine if mitochondrial damage plays a critical role in the development of tubular injury in DKD patients. METHODS: A cross-sectional study was carried out among healthy controls (HCs, n = 65), diabetes patients without kidney disease (DCs, n = 48) and DKD patients (n = 60). Serum, peripheral blood mononuclear cells (PBMCs) and kidney biopsy specimens were obtained from participants. Metabolomics was employed to investigate cellular metabolism. RESULTS: DKD patients had decreased mtDNA copy numbers and increased mtDNA damage compared to DCs. Mitochondrial fragmentation was specifically presented in tubules, but not in podocytes of DKD patients. The accumulation of damaged mtDNA and fragmented mitochondria resulted in increased reactive oxygen species (ROS) generation, activation of apoptosis and loss of mitochondrial membrane potential (ΔΨm) in tubules and PBMCs. Furthermore, glycolysis and tricarboxylic acid (TCA) cycle was perturbed, and increased dihydroxyacetone phosphate (DHAP) and decreased succinyl-CoA synthetase (SCS) respectively in these two metabolic pathways were identified as potential biomarkers for tubular injury in DKD. CONCLUSION: Our study indicates that mitochondrial damage could be the hallmark of tubular injury in DKD patients, and this would provide a novel and attractive therapeutic target to improve this disease.

NMDA receptors participate in the progression of diabetic kidney disease by decreasing Cdc42-GTP activation in podocytes.

Podocytes play important roles in the progression of diabetic kidney disease (DKD) and these roles are closely associated with cytoskeletal actin dynamics. N-Methyl-d-aspartate receptors (NMDARs), which consist of two functional NR1 subunits and two regulatory NR2 subunits, are widely expressed in the brain but are also found in podocytes. Here, we found increased NR1 expression in two diabetic mouse models and in podocytes incubated in high glucose (HG). In diabetic mice, knockdown of NR1 using lentivirus carrying NR1-shRNA ameliorated the pathological features associated with DKD, and reversed the decreased expression of synaptopodin and Wilms' tumour-1. In podocytes incubated with HG, NR1 was secreted from the endoplasmic reticulum and this was blocked by bisindolylmaleimide I. NR1 knockdown decreased the cell shape remodelling, cell collapse, bovine serum albumin permeability, and migration induced by HG. After HG incubation, levels of cell division control protein 42 (Cdc42) and its active form increased, and a significantly higher Cdc42-GTP level, increased Cdc42 translocation onto the leading edges, and lower migration ability were found in podocytes with NR1 knockdown. Increases in the number and length of filopodia were found in podocytes with NR1 knockdown but these were abolished by Cdc42-GTP blockade with ML141. In conclusion, the activation of NMDARs plays an important role in DKD by reducing Cdc42-GTP activation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Serum Matrix Metalloproteinase-7 Level is Associated with Fibrosis and Renal Survival in Patients with IgA Nephropathy.

BACKGROUND/AIMS: In view of the latest findings that matrix metalloproteinase-7 (MMP-7) acted as a vital marker and pathogenic mediator of renal fibrosis in a murine model, we hypothesized that serum MMP-7 level might serve as a noninvasive prognostic biomarker in IgA nephropathy (IgAN) patients. METHODS: We conducted a retrospective follow-up study of 244 IgAN patients for a median of 81.9 months. Serum MMP-7 was detected at the time of diagnosis, and renal progression was assessed by Cox proportional hazards method. RESULTS: Compared with healthy populations, the serum levels of MMP-7 were significantly elevated in IgAN patients. Besides, serum MMP-7 levels were well correlated with renal scarring lesions characterized by glomerular sclerosis and interstitial fibrosis. Follow-up analyses revealed that increased serum MMP-7 levels were linked with a greater risk of poor renal outcome with a hazard ratio of 1.898 per doubling MMP-7 concentration. By contrast with the first quartile, the risk of deterioration in renal function elevated such that the hazard ratio for the second quartile was 1.805, 3.383 for the third, and 5.173 for the fourth quartile of the MMP-7 level. CONCLUSIONS: This study showed that the higher serum MMP-7 levels were independently associated with renal fibrosis and poor prognosis in IgAN.

Lipopolysaccharide-induced podocyte injury is regulated by calcineurin/NFAT and TLR4/MyD88/NF-κB signaling pathways through angiopoietin-like protein 4.

Podocyte injury is an important cause of proteinuria. Angiopoietin-like protein 4 (Angptl4) is a secreted glycoprotein and has a role in proteinuria. However, the exact role of Angptl4 in podocyte injury and its upstream regulators has not been clarified. In this study, we used lipopolysaccharide (LPS)-induced mice and cultured podocytes as podocyte injury models. Our results indicated that LPS increased the expression of podocyte Angptl4 in vivo and in vitro. Furthermore, we showed that Angptl4 overexpression deteriorated LPS-induced podocyte injury by inducing podocyte cytoskeleton rearrangement, reducing the expression of synaptopodin while Angptl4 knockdown alleviated LPS-induced podocyte injury. In addition, we found that inhibitors and siRNA targeting TLR4/MyD88/NF-κB signaling inhibited the upregulation of Angptl4 in LPS-induced podocytes. Moreover, inhibitors and siRNA targeting calcineurin/NFAT signaling also relieved LPS-induced Angptl4 expression and podocyte injury in vivo and in vitro. Taken together, our study has elucidated that both of the TLR4/MyD88/NF-κB and calcineurin/NFAT signaling mediate the upregulation of Angptl4 in LPS-induced podocytes, which has important implications for further understanding the molecular mechanism of LPS-induced podocyte injury.

Corrigendum to "Lipopolysaccharide-induced podocyte injury is regulated by calcineurin/NFAT and TLR4/MyD88/NF-κB signaling pathways through Angiopoietin-like protein 4" [Genes Dis DOI: 10.1016/j.gendis.2020.07.005].

[This corrects the article DOI: 10.1016/j.gendis.2020.07.005.].

Corrigendum to "Lipopolysaccharide-induced podocyte injury is regulated by calcineurin/NFAT and TLR4/MyD88/NF-κB signaling pathways through Angiopoietin-like protein 4" [Genes & Diseases 9(2022) 443-455].

[This corrects the article DOI: 10.1016/j.gendis.2020.07.005.][This corrects the article DOI: 10.1016/j.gendis.2021.09.001.].

[Involvement of LMO4 in tumorigenesis associated epithelial-mesenchymal transition].

LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors, so named because they are composed almost entirely of two tandem LIM domains. This subgroup of LIM proteins has 4 members: LMO-1, LMO-2, LMO-3 and LMO-4. They all play important roles in the normal mammalian development, functioning as an important regulator of cell proliferation. LMO4 is highly expressed in the epithelial compartments at locations of active epithelial-mesenchymal interactions, and can interact with some signaling pathways involved in epithelial-mesenchymal signaling. Thus the disregulation of LMO4 expression may be involved in tumorigenesis. In this paper, we will at first expound LMO4 in detail, based on which the possible mechanisms for its interaction with TGF-ß signaling and the roles of this cross-talk between them in the vital process of cell will be introduced. All of those will add to our understanding of tumorigenesis and contribute to the search of new targets for the treatment of cancer.

Caspase 3/GSDME-dependent pyroptosis contributes to chemotherapy drug-induced nephrotoxicity.

Chemotherapy drug-induced nephrotoxicity limits clinical applications for treating cancers. Pyroptosis, a newly discovered programmed cell death, was recently reported to be associated with kidney diseases. However, the role of pyroptosis in chemotherapeutic drug-induced nephrotoxicity has not been fully clarified. Herein, we demonstrate that the chemotherapeutic drug cisplatin or doxorubicin, induces the cleavage of gasdermin E (GSDME) in cultured human renal tubular epithelial cells, in a time- and concentration-dependent manner. Morphologically, cisplatin- or doxorubicin-treated renal tubular epithelial cells exhibit large bubbles emerging from the cell membrane. Furthermore, activation of caspase 3, not caspase 9, is associated with GSDME cleavage in cisplatin- or doxorubicin-treated renal tubular epithelial cells. Meanwhile, silencing GSDME alleviates cisplatin- or doxorubicin-induced HK-2 cell pyroptosis by increasing cell viability and decreasing LDH release. In addition, treatment with Ac-DMLD-CMK, a polypeptide targeting mouse caspase 3-Gsdme signaling, inhibits caspase 3 and Gsdme activation, alleviates the deterioration of kidney function, attenuates renal tubular epithelial cell injury, and reduces inflammatory cytokine secretion in vivo. Specifically, GSDME cleavage depends on ERK and JNK signaling. NAC, a reactive oxygen species (ROS) inhibitor, reduces GSDME cleavage through JNK signaling in human renal tubular epithelial cells. Thus, we speculate that renal tubular epithelial cell pyroptosis induced by chemotherapy drugs is mediated by ROS-JNK-caspase 3-GSDME signaling, implying that therapies targeting GSDME may prove efficacious in overcoming chemotherapeutic drug-induced nephrotoxicity.

The Study of Angptl4-Modulated Podocyte Injury in IgA Nephropathy.

BACKGROUND: Increasing evidence shows that Angptl4 affects proteinuria in podocytes injured kidney disease, however, whether there is a relationship between Angptl4 and IgA nephropathy (IgAN) has not been studied yet. METHODS: Plasma and urine samples were obtained from 71 patients with IgAN and 61 healthy controls. Glomeruli from six renal biopsy specimens (three IgAN patients and three healthy controls) were separated by RNA-Seq. Differentially expressed genes (DEGs) related to podocytes and Angptl4 between IgAN patients and healthy controls were performed using the Limma package. Gene set enrichment analysis was used to determine whether there was a statistically significant difference between the two groups. STRING was used to create a protein-protein interaction network of DEGs. Association analysis between Angptl4 levels and clinical features of IgAN was performed. RESULTS: Thirty-three podocyte-related and twenty-three Angpt4-related DEGs were found between IgAN patients and healthy controls. By overlapping the genes, FOS and G6PC were found to be upregulated in IgAN patients, while MMP9 was downregulated in IgAN patients. Plasma and urine Angptl4 levels were closely related to the degree of podocyte injury and urine protein, but not to the protein-creatine ratio. CONCLUSION: Our findings show that Angptl4 levels in plasma and urine are related to podocyte damage and, therefore, may be a promising tool for assessing the severity of IgAN patients to identify and reverse the progression to ESRD.

Correction: Tacrolimus dose requirement based on the CYP3A5 genotype in renal transplant patients.

[This corrects the article DOI: 10.18632/oncotarget.18150.].

[Fibroblasts-colorectal cancer cells interaction induces the expression of IGFBP7].

OBJECTIVE: To investigate the impact of intracellular interaction between fibroblasts and colorectal cancer cells on the expression of insulin growth factor binding protein7 (IGFBP7). METHODS: Colorectal cancer cells SW480 were cultured with or without fibroblasts HELF cells in RPMI 1640 medium for 0 h, 48 h, 7 2 h and 96 h, respectively. By RT-PCR and immunohistochemical staining methods,the expression of IGFBP7 was detected in mono-and co-cultured cells. RESULT: When SW480 cells were co-cultured with HELF cells, IGFBP7 expression in SW480 cells was significantly upregulated. Furthermore, IGFBP7 was induced in HELF cells both at mRNA and protein levels, which did not express when cells were mono-cultured. CONCLUSION: Fibroblasts-colorectal cancer cells interaction induces the expression of IGFBP7, which indicates tumor-stroma interactions may play an important role in colorectal cancer development.

The pathogenic AGT c.856+1G>T mutation of a patient with multiple renal cysts and hypertension.

Angiotensinogen (AGT) is an essential member of the renin-angiotensin system (RAS); this system regulates blood pressure and affects the physiological function of the kidney. Studies found that mutations of the human AGT gene are involved in diseases such as recessive renal tubular dysgenesis (RTD) and essential hypertension (EHT). Here, we report a 29-year-old male Chinese with essential hypertension and cystic kidney disease. Exome sequencing analysis of the patient and his parents revealed a mutation in the splice donor site of intron 2 of the AGT gene, c.856+1G>T. This mutation was a heterozygous form and inherited from his mother, and the mother was diagnosed with essential hypertension lasting over 20 years. Function prediction of c.856+1G>T mutation using online software showed this intron mutation may affect protein features by destroying the normal splice site. These findings suggest that this intron mutation of the AGT gene is related to the patient's essential hypertension and cystic kidney disease.

Evaluation of crescent formation as a predictive marker in immunoglobulin A nephropathy: a systematic review and meta-analysis.

The 2009 Oxford Classification of immunoglobulin A (IgA) nephropathy (IgAN) identifies four histological features as predictors of renal prognosis: mesangial hypercellularity (M), endocapillary hypercellularity (E), segmental glomerulosclerosis (S), and tubular atrophy/interstitial fibrosis (T). However, the clinical and prognostic significance of crescent formation still remains controversial. Therefore, we performed a meta-analysis to evaluate the association between crescents and kidney outcome in IgAN. A total of 20 studies published from January 2009 to July 2016 involving 5,285 patients were included after systematic searches of PubMed and EMBASE databases. Pooled results showed that crescent lesions were associated with kidney failure (HR, 1.93; 95% CI, 1.49-2.50; P < 0.001). IgAN patients with crescents had lower eGFR levels (SMD, -0.21; 95% CI, -0.40--0.03; P = 0.023); higher proteinuria levels (SMD, 0.87; 95% CI, 0.11-1.63; P = 0.024); a larger number of patients with M1 (RR, 1.22; 95% CI, 1.07-1.40; P = 0.003), E1 (RR, 4.83; 95% CI, 3.04-7.66;P < 0.001), S1 (RR, 1.76; 95% CI, 1.11-2.80; P = 0.016) and T1/2 (RR, 2.74; 95% CI, 2.10-3.57; P < 0.001) lesions; and received immunosuppressive therapy more frequently (RD, 0.17; 95% CI, 0.11-0.23; P < 0.001). Our results suggest that crescent formation represents an efficient prognostic factor associated with progression to kidney failure and thus could be considered into the new Oxford Classification.

Tacrolimus dose requirement based on the CYP3A5 genotype in renal transplant patients.

Tacrolimus (FK506) and cyclosporine A (CsA) are widely used to protect graft function after renal transplantation. The aim of the present study is to determine whether the single nucleotide polymorphism of CYP3A5 is a predictive index of FK506 dose requirement, and also the selection yardstick of FK506 or CsA treatment.We tested archival peripheral blood of 218 kidney recipients for CYP3A5 genotyping with PCR-SSP. Meanwhile, the dose of FK506 and CsA was recorded, blood concentration of the drugs was measured, and graft outcome was monitored.These results indicate that CYP3A5*AA/AG carriers need higher FK506 dose than CYP3A5*GG homozygote to achieve the target blood concentration. For CYP3A5*GG carriers, taking FK506 or CsA are both advisable. CYP3A5*AA/AG carriers preferred to CsA treatment depending on the graft outcomes and drug costs. CYP3A5 genotyping is a new approach to detecting FK506 dose requirement and a predictive index for the FK506 or CsA treatment selection in kidney recipients.

Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models.

Podocyte injury and the appearance of proteinuria are features of minimal-change disease (MCD). Cyclosporin A (CsA) and tacrolimus (FK506) has been reported to reduce proteinuria in patients with nephrotic syndrome, but mechanisms remain unknown. We, therefore, investigated the protective mechanisms of CsA and FK506 on proteinuria in a rat model of MCD induced by puromycin aminonucleoside (PAN) and in vitro cultured mouse podocytes. Our results showed that CsA and FK506 treatment decreased proteinuria via a mechanism associated to a reduction in the foot-process fusion and desmin, and a recovery of synaptopodin and podocin. In PAN-treated mouse podocytes, pre-incubation with CsA and FK506 restored the distribution of the actin cytoskeleton, increased the expression of synaptopodin and podocin, improved podocyte viability, and reduced the migrating activities of podocytes. Treatment with CsA and FK506 also inhibited PAN-induced podocytes apoptosis, which was associated with the induction of Bcl-xL and inhibition of Bax, cleaved caspase 3, and cleaved PARP expression. Further studies revealed that CsA and FK506 inhibited PAN-induced p38 and JNK signaling, thereby protecting podocytes from PAN-induced injury. In conclusion, CsA and FK506 inhibit proteinuria by protecting against PAN-induced podocyte injury, which may be associated with inhibition of the MAPK signaling pathway.

Effects of rapamycin against paraquat-induced pulmonary fibrosis in mice.

BACKGROUND AND AIMS: Ingestion of paraquat (PQ), a widely used herbicide, can cause severe toxicity in humans, leading to a poor survival rate and prognosis. One of the main causes of death by PQ is PQ-induced pulmonary fibrosis, for which there are no effective therapies. The aim of this study was to evaluate the effects of rapamycin (RAPA) on inhibiting PQ-induced pulmonary fibrosis in mice and to explore its possible mechanisms. METHODS: Male C57BL/6J mice were exposed to either saline (control group) or PQ (10 mg/kg body weight, intraperitoneally; test group). The test group was divided into four subgroups: a PQ group (PQ-exposed, non-treated), a PQ+RAPA group (PQ-exposed, treated with RAPA at 1 mg/kg intragastrically), a PQ+MP group (PQ-exposed, treated with methylprednisolone (MP) at 30 mg/kg intraperitoneally), and a PQ+MP+RAPA group (PQ-exposed, treated with MP at 30 mg/kg intraperitoneally and with RAPA at 1 mg/kg intragastrically). The survival rate and body weight of all the mice were recorded every day. Three mice in each group were sacrificed at 14 d and the rest at 28 d after intoxication. Lung tissues were excised and stained with hematoxylin-eosin (H&E) and Masson's trichrome stain for histopathological analysis. The hydroxyproline (HYP) content in lung tissues was detected using an enzyme-linked immunosorbent assay (ELISA) kit. The expression of transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) in lung tissues was detected by immunohistochemical staining and Western blotting. RESULTS: A mice model of PQ-induced pulmonary fibrosis was established. Histological examination of lung tissues showed that RAPA treatment moderated the pathological changes of pulmonary fibrosis, including alveolar collapse and interstitial collagen deposition. HYP content in lung tissues increased soon after PQ intoxication but had decreased significantly by the 28th day after RAPA treatment. Immunohistochemical staining and Western blotting showed that RAPA treatment significantly down-regulated the enhanced levels of TGF-ß1 and α-SMA in lung tissues caused by PQ exposure. However, RAPA treatment alone could not significantly ameliorate the lower survival rate and weight loss of treated mice. MP treatment enhanced the survival rate, but had no significant effects on attenuating PQ-induced pulmonary fibrosis or reducing the expression of TGF-ß1 and α-SMA. CONCLUSIONS: This study demonstrates that RAPA treatment effectively suppresses PQ-induced alveolar collapse and collagen deposition in lung tissues through reducing the expression of TGF-ß1 and α-SMA. Thus, RAPA has potential value in the treatment of PQ-induced pulmonary fibrosis.