Methylation pattern of urinary DNA as a marker of kidney function decline in diabetes

IntroductionRenal tubular injury contributes to the decline in kidney function in patients with diabetes. Cell type-specific DNA methylation patterns have been used to calculate proportions of particular cell types. In this study, we developed a method to detect renal tubular injury in patients with diabetes by detecting exfoliated tubular cells shed into the urine based on tubular cell-specific DNA methylation patterns.Research design and methodsWe identified DNA methylation patterns specific for human renal proximal tubular cells through compartment-specific methylome analysis. We next determined the methylation levels of proximal tubule-specific loci in urine sediment of patients with diabetes and analyzed correlation with clinical variables.ResultsWe identified genomic loci in SMTNL2 and G6PC to be selectively unmethylated in human proximal tubular cells. The methylation levels of SMTNL2 and G6PC in urine sediment, deemed to reflect the proportion of exfoliated proximal tubular cells due to injury, correlated well with each other. Methylation levels of SMTNL2 in urine sediment significantly correlated with the annual decline in estimated glomerular filtration rate. Moreover, addition of urinary SMTNL2 methylation to a model containing known risk factors significantly improved discrimination of patients with diabetes with faster estimated glomerular filtration rate decline.ConclusionsThis study demonstrates that patients with diabetes with continual loss in kidney function may be stratified by a specific DNA methylation signature through epigenetic urinalysis and provides further evidence at the level of exfoliated cells in the urine that injury of proximal tubular cells may contribute to pathogenesis of diabetic kidney disease.

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Palmitate aggravates proteinuria-induced cell death and inflammation via CD36-inflammasome axis in the proximal tubular cells of obese mice

AJP Renal Physiology ◽

10.1152/ajprenal.00536.2017 ◽

2018 ◽

Vol 315 (6) ◽

pp. F1720-F1731 ◽

Cited By ~ 8

Author(s):

Lung-Chih Li ◽

Jenq-Lin Yang ◽

Wen-Chin Lee ◽

Jin-Bor Chen ◽

Chien-Te Lee ◽

...

Keyword(s):

Cell Death ◽

Tubular Injury ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Caspase 1 ◽

Proximal Tubular ◽

Renal Tubular ◽

Nlrp3 Inflammasomes

High levels of serum free fatty acids (FFAs) and proteinuria have been implicated in the pathogenesis of obesity-related nephropathy. CD36, a class B scavenger receptor, is highly expressed in the renal proximal tubules and mediates FFA uptake. It is not clear whether FFA- and proteinuria-mediated CD36 activation coordinates NLRP3 inflammasomes to induce renal tubular injury and inflammation. In this study, we investigated the roles of CD36 and NLRP3 inflammasomes in FFA-induced renal injury in high-fat diet (HFD)-induced obesity. HFD-fed C57BL/6 mice and palmitate-treated HK2 renal tubular cells were used as in vivo and in vitro models. Immunohistochemical staining showed that CD36, IL-1β, and IL-18 levels increased progressively in the kidneys of HFD-fed mice. Sulfo- N-succinimidyl oleate (SSO), a CD36 inhibitor, attenuated the HFD-induced upregulation of NLRP3, IL-1β, and IL-18 and suppressed the colocalization of NLRP3 and ASC in renal tubular cells. In vitro, SSO abolished the palmitate-induced activation of IL-1β, IL-18, and caspase-1 in HK2 proximal tubular cells. Furthermore, treatment with SSO and the knockdown of caspase-1 expression by siRNA both inhibited palmitate-induced cell death and apoptosis in HK2 cells. Collectively, palmitate causes renal tubular inflammation, cell death, and apoptosis via the CD36/NLRP3/caspase-1 axis, which may explain, at least in part, the mechanism underlying FFA-related renal tubular injury. The blockade of CD36-induced cellular processes is therefore a promising strategy for treating obesity-related nephropathy.

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Intracellular prostaglandin E2 contributes to hypoxia-induced proximal tubular cell death

Scientific Reports ◽

10.1038/s41598-021-86219-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Coral García-Pastor ◽

Selma Benito-Martínez ◽

Ricardo J. Bosch ◽

Ana B. Fernández-Martínez ◽

Francisco J. Lucio-Cazaña

Keyword(s):

Hypoxia Inducible Factor ◽

Renal Diseases ◽

Prostaglandin E ◽

Relevant Factor ◽

Tubular Injury ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Proximal Tubular ◽

Cox 2 ◽

Induced Apoptosis

AbstractProximal tubular cells (PTC) are particularly vulnerable to hypoxia-induced apoptosis, a relevant factor for kidney disease. We hypothesized here that PTC death under hypoxia is mediated by cyclo-oxygenase (COX-2)-dependent production of prostaglandin E2 (PGE2), which was confirmed in human proximal tubular HK-2 cells because hypoxia (1% O2)-induced apoptosis (i) was prevented by a COX-2 inhibitor and by antagonists of prostaglandin (EP) receptors and (ii) was associated to an increase in intracellular PGE2 (iPGE2) due to hypoxia-inducible factor-1α-dependent transcriptional up-regulation of COX-2. Apoptosis was also prevented by inhibitors of the prostaglandin uptake transporter PGT, which indicated that iPGE2 contributes to hypoxia-induced apoptosis (on the contrary, hypoxia/reoxygenation-induced PTC death was exclusively due to extracellular PGE2). Thus, iPGE2 is a new actor in the pathogenesis of hypoxia-induced tubular injury and PGT might be a new therapeutic target for the prevention of hypoxia-dependent lesions in renal diseases.

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Tubular Injury and Dendritic Cell Activation Are Integral Components of Light Chain–Associated Acute Tubulointerstitial Nephritis

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0032-oa ◽

2019 ◽

Vol 143 (10) ◽

pp. 1212-1224 ◽

Cited By ~ 2

Author(s):

Mingyu Cheng ◽

Xin Gu ◽

Elba A. Turbat-Herrera ◽

Guillermo A. Herrera

Keyword(s):

Dendritic Cells ◽

Light Chain ◽

Tubulointerstitial Nephritis ◽

Light Chains ◽

Tubular Injury ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Interstitial Inflammation ◽

Biopsy Specimens ◽

Proximal Tubular

Context.— Light chain–associated acute tubulointerstitial nephritis (LC-ATIN) is a variant of light chain proximal tubulopathy (LCPT). It is characterized by interstitial inflammation with tubulitis and deposition of monoclonal light chains in the tubulointerstitium. LC-ATIN is a rather poorly recognized pattern of LCPT and not much is known about this entity. Objective.— To determine the clinicopathologic features of patients with LC-ATIN and investigate the proximal tubular injury and mechanism of interstitial inflammation in LC-ATIN. Design.— A total of 38 cases of LC-ATIN were identified from the archives of 5043 renal biopsy specimens. In all cases, routine light microscopic examination, immunofluorescence, and electron microscopic examination were performed. In selected cases, immunofluorescent staining of dendritic cells and immunohistochemical staining for 4 tubular injury markers—KIM-1, p53, bcl-2, and Ki-67—were performed. Results.— A characteristic finding in LC-ATIN cases was immunofluorescence staining of monoclonal light chains along tubular basement membranes in linear fashion and inside proximal tubular cells with a granular pattern. No monoclonal light chains were present in glomerular or vascular compartments confirmed with immunofluorescence, electron microscopy, and ultrastructural gold labeling. Ten of 15 LC-ATIN cases (67%) were concurrently positive for the 4 tubular injury markers. Dendritic cells were identified within the tubulointerstitium in the renal biopsy specimens, interacting with surrounding tubules with light-chain deposits and inflammatory cells. Conclusions.— Significant proximal tubular injury occurs associated with LC-ATIN, and the monoclonal light chains accumulated in proximal tubular cells contribute to the injury. Dendritic cells are involved in the pathogenesis of interstitial inflammation in LC-ATIN.

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Prolactin and dopamine 1-like receptor interaction in renal proximal tubular cells

AJP Renal Physiology ◽

10.1152/ajprenal.00582.2009 ◽

2010 ◽

Vol 299 (1) ◽

pp. F49-F54 ◽

Cited By ~ 13

Author(s):

Susanne Crambert ◽

Agneta Sjöberg ◽

Ann-Christine Eklöf ◽

Fernando Ibarra ◽

Ulla Holtbäck

Keyword(s):

Protein Kinase ◽

Atpase Activity ◽

Proximal Tubular Cells ◽

Dopamine System ◽

Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Renal Tubular ◽

Renal Dopamine

Prolactin is a natriuretic hormone and acts by inhibiting the activity of renal tubular Na+-K+-ATPase activity. These effects require an intact renal dopamine system. Here, we have studied by which mechanism prolactin and dopamine interact in Sprague-Dawley rat renal tissue. Na+-K+-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in microdissected renal proximal tubular segments. Intracellular signaling pathways were studied by a variety of different techniques, including Western blotting using phosphospecific antibodies, immunoprecipitation, and biotinylation assays. We found that dopamine and prolactin regulated Na+-K+-ATPase activity via similar signaling pathways, including protein kinase A, protein kinase C, and phosphoinositide 3-kinase activation. The cross talk between prolactin and dopamine 1-like receptors was explained by a heterologous recruitment of dopamine 1-like receptors to the plasma membrane in renal proximal tubular cells. Prolactin had no effect on Na+-K+-ATPase activity in spontaneously hypertensive rats, a rat strain with a blunted response to dopamine. These results further emphasize the central role of the renal dopamine system in the interactive regulation of renal tubular salt balance.

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Overexpression of Nrf2 in Renal Proximal Tubular Cells Stimulates Sodium-Glucose Co-Transporter 2 Expression and Exacerbates Dysglycemia and Kidney Injury in Diabetic Mice

10.2337/figshare.14334080.v1 ◽

2021 ◽

Author(s):

Shuiling Zhao ◽

Chao-Sheng Lo ◽

Kana N. Miyata ◽

Anindya Ghosh ◽

Xinping Zhao ◽

...

Keyword(s):

Blood Glucose ◽

Promoter Activity ◽

Kidney Injury ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Patients With Diabetes ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

The Impact

We investigated the impact of nuclear factor erythroid 2-related factor 2 (Nrf2) overexpression in renal proximal tubular cells (RPTCs) on blood glucose, kidney injury and sodium-glucose co-transporter 2 (Sglt2) expression in diabetic Akita Nrf2-/-/Nrf2RPTC transgenic (Tg) mice. Immortalized human RPTCs (HK2) stably transfected with plasmid containing the SGLT2 promoter, human kidneys from patients with diabetes were also studied. Nrf2 overexpression was associated with increased blood glucose, glomerular filtration rate, urinary albumin-creatinine ratio, tubulointerstitial fibrosis and Sglt2 expression in Akita Nrf2-/-/Nrf2RPTC Tg mice compared to their Akita Nrf2-/- littermates. In vitro, oltipraz or transfection of NRF2 cDNA stimulated SGLT2 expression and SGLT2 promoter activity in HK2, and these effects were inhibited by trigonelline or NRF2 small interfering RNA. The deletion of the NRF2-responsive element (NRF2-RE) in the SGLT2 promoter abolished the stimulatory effect of oltipraz on SGLT2 promoter activity. NRF2 binding to the NRF2-RE of the SGLT2 promoter was confirmed by gel mobility shift assay and chromatin immunoprecipitation assays. Kidneys from patients with diabetes exhibited higher levels of NRF2 and SGLT2 in the RPTCs than kidneys from patients without diabetes. These results suggest a link by which NRF2 mediates hyperglycemia-stimulation of SGLT2 expression and exacerbates blood glucose and kidney injury in diabetes.

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The exosomes derived from urine of premature infants target MAPK8 via miR-30a-5p to protect cisplatin-induced acute kidney injury in mice

10.1101/2021.08.29.458088 ◽

2021 ◽

Author(s):

Mingming Ma ◽

Qiao Luo ◽

Lijing Fan ◽

Weilong Li ◽

Qiang Li ◽

...

Keyword(s):

Acute Kidney Injury ◽

Premature Infants ◽

Target Genes ◽

Kidney Injury ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Proximal Tubular ◽

Renal Tubular ◽

Hk2 Cells

Aim: Acute kidney injury (AKI), a global public health issue, not only causes millions of deaths every year, but is also a susceptible factor for chronic kidney disease (CKD). Nephrotoxic drugs are an important cause of AKI. There is still a lack of effective and satisfactory prevention method in clinical practice. This study investigated the protective effect of the exosomes derived from urine of premature infants on cisplatin-induced acute kidney injury. Methods: Isolation of exosomes from fresh urine of premature infants: The characteristics of exosomes were determined by flow cytometry, transmission electron microscopy and Western blotting. A C57BL/6 mice model of cisplatin-induced acute kidney injury was established. The mice in the experimental group were given 100ug exosomes dissolved in 200ul solution. The mice in the control group were given normal saline (200ul). These treatments were performed 24 hours after AKI was induced by intraperitoneal injection of cisplatin. To evaluate renal function, blood was drawn 24 hours after AKI model was established and serum creatinine (sCr) was measured. The mice were euthanized 72 hours after exosome treatment. The kidneys were collected for pathological examination, RNA and protein extraction, and the evaluation of renal tubular damage and apoptosis. In the in-vitro experiment, human renal cortex/proximal tubular cells (HK2) was induced by cisplatin to assess the protective ability of the exosomes derived from urine of premature infants. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blotting were used to evaluate the effect of exosomes treatment on the apoptosis of HK2 cells induced by cisplatin. Exosome microRNA sequencing technology and bioinformatics analysis method were applied to investigate the miRNAs enriched in exosomes and their target genes. The dual luciferase gene reporter system was used to detect the interaction of target genes. Results: Treatment of exosomes derived from urine of premature infants could decrease the level of serum creatinine and the apoptosis of renal tubular cell, inhibit the infiltration of inflammatory cell, protect mice from acute kidney injury induced by cisplatin and reduce mortality. In addition, miR-30a-5p was the most abundant miRNA in the exosomes derived from urine of premature infants. It protected HK2 cells from cisplatin-induced apoptosis by targeting and down-regulating the 3'UTR of mitogen-activated protein kinases (MAPK8) mRNA. Conclusions: According to our results, the exosomes derived from urine of premature infants alleviated cisplatin-induced acute kidney injury in mice and inhibited the apoptosis of human proximal tubular cells (HK2) induced by cisplatin in vitro. MiR-30a-5p in exosomes inhibited cisplatin-induced MAPK activation, ameliorated apoptosis, and protected renal function. The exosomes derived from urine of premature infants provided a promising acellular therapy for AKI.

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Activation of COX-2/mPGES-1/PGE2 Cascade via NLRP3 Inflammasome Contributes to Albumin-Induced Proximal Tubule Cell Injury

Cellular Physiology and Biochemistry ◽

10.1159/000478070 ◽

2017 ◽

Vol 42 (2) ◽

pp. 797-807 ◽

Cited By ~ 12

Author(s):

Yibo Zhuang ◽

Fei Zhao ◽

Jing Liang ◽

Xu Deng ◽

Yue Zhang ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Cell Apoptosis ◽

Cell Injury ◽

Renal Tubular Cell ◽

Proximal Tubule Cell ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Proximal Tubular ◽

Cox 2 ◽

Renal Tubular

Background/Aims: The activation of NOD-like receptor family, pyrin domain containing3 (NLRP3) inflammasome has been shown to be positively correlated with the severity of proteinuria in chronic kidney disease (CKD) patients. Prostaglandin E2 (PGE2), an important inflammatory mediator, is also involved in various kidney injuries. The aim of the present study was to investigate the involvement of NLRP3 inflammasome and PGE2 synthetic pathway in albumin-induced renal tubular injury. Methods: Murine proximal tubular cells (mPTCs) were treated with albumin to induce cell injury. NLRP3 siRNA and specific COX-2 inhibitor NS398 were used to define their roles in mediating albumin-induced mPTC injury or the activation of COX-2/mPGES-1/PGE2 cascade. Results: In mPCTs, inhibition of NLRP3 by a small interfering RNA (siRNA) blocked albumin-induced kidney injury molecule 1 (KIM-1) upregulation, inflammatory response, and cell apoptosis. Albumin markedly activated cyclooxygenase-2 (COX-2)/ microsomal prostaglandin E synthase-1 (mPGES-1)/PGE2 pathway in this cell line, an effect largely abolished by NLRP3 silencing at both mRNA and protein levels. More interestingly, blockade of COX-2 using a specific COX-2 inhibitor NS398 markedly inhibited the upregulation of KIM-1 and inflammatory cytokines, and attenuated cell apoptosis in line with blunted PGE2 release following albumin treatment. Conclusions: The findings suggest that COX-2/mPGES-1/PGE2 axis could be activated by albumin in the proximal tubular cells via a NLRP3 inflammasome-mediated mechanism and could thus contribute to proteinuria-related renal tubular cell injury.

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Stress granules are formed in renal proximal tubular cells during metabolic stress and ischemic injury for cell survival

AJP Renal Physiology ◽

10.1152/ajprenal.00139.2019 ◽

2019 ◽

Vol 317 (1) ◽

pp. F116-F123 ◽

Cited By ~ 1

Author(s):

Shixuan Wang ◽

Sang-Ho Kwon ◽

Yunchao Su ◽

Zheng Dong

Keyword(s):

Rna Binding ◽

Ischemia Reperfusion ◽

Metabolic Stress ◽

Stress Granules ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Short Period ◽

Proximal Tubular ◽

Renal Tubular

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.

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