Vitamin C Deficiency Causes Cell Type-Specific Epigenetic Reprogramming and Acute Tubular Necrosis in a Mouse Model

Background: Vitamin C deficiency is found in patients with variable renal diseases. However, the role of vitamin C as an epigenetic regulator in renal homeostasis and pathogenesis remains largely unknown. Methods: We showed that vitamin C deficiency leads to acute tubular necrosis (ATN) using a vitamin C-deficient mouse model (Gulo knock-out). DNA/RNA epigenetic modifications and injured S3 proximal tubule cells were identified in the vitamin C-deficient kidneys using whole-genome bisulfite sequencing, methylated RNA immunoprecipitation sequencing, and single-cell RNA sequencing. Results: Integrated evidence suggested that epigenetic modifications affected the proximal tubule cells and fenestrated endothelial cells, leading to tubule injury and hypoxia through transcriptional regulation. Strikingly, loss of DNA hydroxymethylation and DNA hypermethylation in vitamin C-deficient kidneys preceded the histological sign of tubule necrosis, indicating the causality of vitamin C-induced epigenetic modification in ATN. Consistently, prophylactic supplementation of an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, promoted DNA demethylation and prevented the progression of cisplatin-induced ATN. Conclusions: Vitamin C played a critical role in renal homeostasis and pathogenesis in a mouse model, suggesting vitamin supplementation may be an approach to lower risk of kidney injury.

Autophagy in Cisplatin Nephrotoxicity during Cancer Therapy

Cisplatin is a widely used chemotherapeutic agent but its clinical use is often limited by nephrotoxicity. Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged or dysfunctional cellular organelles for maintaining cell homeostasis. Upon cisplatin exposure, autophagy is rapidly activated in renal tubule cells to protect against acute cisplatin nephrotoxicity. Mechanistically, the protective effect is mainly related to the clearance of damaged mitochondria via mitophagy. The role and regulation of autophagy in chronic kidney problems after cisplatin treatment are currently unclear, despite the significance of research in this area. In cancers, autophagy may prevent tumorigenesis, but autophagy may reduce the efficacy of chemotherapy by protecting cancer cells. Future research should focus on developing drugs that enhance the anti-tumor effects of cisplatin while protecting kidneys during cisplatin chemotherapy.

Proximal Tubule p53 in Cold Storage/Transplantation-Associated Kidney Injury and Renal Graft Dysfunction

Kidney injury associated with cold storage/transplantation is a primary factor for delayed graft function and poor outcome of renal transplants. p53 contributes to both ischemic and nephrotoxic kidney injury, but its involvement in kidney cold storage/transplantation is unclear. Here, we report that p53 in kidney proximal tubules plays a critical role in cold storage/transplantation kidney injury and inhibition of p53 can effectively improve the histology and function of transplanted kidneys. In a mouse kidney cold storage/transplantation model, we detected p53 accumulation in proximal tubules in a cold storage time-dependent manner, which correlated with tubular injury and cell death. Pifithrin-α, a pharmacologic p53 inhibitor, could reduce acute tubular injury, apoptosis and inflammation at 24 h after cold storage/transplantation. Similar effects were shown by the ablation of p53 from proximal tubule cells. Notably, pifithrin-α also ameliorated kidney injury and improved the function of transplanted kidneys in 6 days when it became the sole life-supporting kidney in recipient mice. in vitro, cold storage followed by rewarming induced cell death in cultured proximal tubule cells, which was accompanied by p53 activation and suppressed by pifithrin-α and dominant-negative p53. Together, these results support a pathogenic role of p53 in cold storage/transplantation kidney injury and demonstrate the therapeutic potential of p53 inhibitors.

Protein-Bound Uremic Toxins Induce Reactive Oxygen Species-Dependent and Inflammasome-Mediated IL-1β Production in Kidney Proximal Tubule Cells

Protein bound-uremic toxins (PBUTs) are not efficiently removed by hemodialysis in chronic kidney disease (CKD) patients and their accumulation leads to various co-morbidities via cellular dysfunction, inflammation and oxidative stress. Moreover, it has been shown that increased intrarenal expression of the NLRP3 receptor and IL-1β are associated with reduced kidney function, suggesting a critical role for the NLRP3 inflammasome in CKD progression. Here, we evaluated the effect of PBUTs on inflammasome-mediated IL-1β production in vitro and in vivo. Exposure of human conditionally immortalized proximal tubule epithelial cells to indoxyl sulfate (IS) and a mixture of anionic PBUTs (UT mix) increased expression levels of NLRP3, caspase-1 and IL-1β, accompanied by a significant increase in IL-1β secretion and caspase-1 activity. Furthermore, IS and UT mix induced the production of intracellular reactive oxygen species, and caspase-1 activity and IL-1β secretion were reduced in the presence of antioxidant N-acetylcysteine. IS and UT mix also induced NF-κB activation as evidenced by p65 nuclear translocation and IL-1β production, which was counteracted by an IKK inhibitor. In vivo, using subtotal nephrectomy CKD rats, a significant increase in total plasma levels of IS and the PBUTs, kynurenic acid and hippuric acid, was found, as well as enhanced urinary malondialdehyde levels. CKD kidney tissue showed an increasing trend in expression of NLRP3 inflammasome components, and a decreasing trend in superoxide dismutase-1 levels. In conclusion, we showed that PBUTs induce inflammasome-mediated IL-1β production in proximal tubule cells via oxidative stress and NF-κB signaling, suggesting their involvement in disease-associated inflammatory processes.

Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial–Mesenchymal Transition

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK1 cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β1 signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β1-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.

Potential and Limits of Kidney Cells for Evaluation of Renal Excretion

A large number of therapeutic drugs, herbal components and their metabolites are excreted by the kidneys. Therefore, generally applied models for estimating renal excretion, including freshly isolated rat proximal tubule cells, cultured tubule cells and immortalized kidney cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, were investigated regarding their predictive potential for active renal transport. Cultured proximal tubule cells showed an epithelial cell-like morphology and formed tight monolayers. However, mRNA expression analyses and immunohistochemical studies revealed patterns of tight junction proteins that were notably different from freshly isolated cells and distinct from those in vivo. High levels of mannitol permeation were found in NRK-52E, IHKE-1 and Caki-1 cells, suggesting that they are not suitable for bidirectional transport studies. Cultured cells and freshly isolated cells also differed in proximal tubule markers and transport proteins, indicating that cultured primary cells were in a state of dedifferentiation. Cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1 did not accurately reflect the characteristics of proximal tubules. The expression patterns of marker and transport proteins differed from freshly isolated primary cells. In summary, each of these models has profound disadvantages to consider when adopting them reliable models for the in vivo situation. Thus, they should not be used alone but only in combination.

Abstract P238: Regulation Of G Protein-coupled Receptor Kinase 4 Expression By Serum In Breast Cancer And Renal Proximal Tubule Cells

G protein-coupled receptor kinase 4 (GRK4) is a member of the GRK family which play critical role in regulation of the function of G protein-coupled receptors. Our previous studies have shown that GRK4 not only plays a role in regulating sodium excretion in renal proximal tubule cells but also acts as a stimulator on proliferation of breast cancer cells. Uncontrolled proliferation is a characteristics of cancer cells and GRK4 is upregulated in breast cancer cells. We hypothesized that expression of GRK4 may be regulated differently in cancer and non-cancer cells. To test this hypothesis, expression of GRK4 in response to serum was compared in breast cancer cells and renal proximal tubule cells by Western analysis. In three breast cancer cell lines serum withdrawal caused rapid reduction in the levels of GRK4 which occurred as early as 15 min. GRK4 levels correlated with the concentrations of serum added to the culture media. To determine if growth factors were a critical element for maintaining GRK4 levels in the cells, EGF (10-20 ng/ml) was added to serum free medium for 24 h. There was no increase in GRK4 levels in the cells treated with EGF compared with the serum starvation control. Similarly, serum withdrawal (16 h) led to 40-80% decrease of GRK4 levels in renal proximal tubule cells even in the presence of EFG supplement. Serum feeding for 30 min after starvation dramatically increased the levels of GRK4 in both breast cancer cells and RPTC which exceeded the steady state levels. This rapid recovery of GRK4 protein do not need de novo protein synthesis because pretreatment of the cells with protein synthesis inhibitor, cycloheximide (10 μg/ml, 24 h), did not prevent this event. Expression of GRK2, another member of the GRK family, was not affected by serum starvation. Our results have shown that GRK4 is very sensitive to serum concentration in breast cancer cells as well as in RPTC. Preliminary studies suggest that rapid protein degradation rather than shutting down the protein synthesis plays a major role in this kind of GRK4 regulation. The biological significance of serum regulation of GRK4 in cancer and non-cancerous cells needs further investigation.

Abstract MP61: Sex Difference In The Response Of Human Renal Proximal Tubule Cells To A Nephrotoxic Agent.

Our previous work indicated that the renal dopamine D2 receptor (D2R) has a significant role in regulating renal inflammation and injury, as well as in blood pressure control. In mice, D2R has protective effects in the kidney by limiting the inflammatory and fibrotic reaction; impaired D2R function results in renal inflammation and damage. Some common single nucleotide polymorphisms (SNPs; rs 6276 and 6277) in the human DRD2 gene are associated with decreased D 2 R expression and function and high blood pressure. To determine the effects of the presence of SNPs in the response to the nephrotoxic aristolochic acid (AA, 5μg/ml, 24 h), we studied immortalized human renal proximal tubule cells isolated from normal tissue of nephrectomies and genotyped for DRD2 SNPs and DRD2 wild-type (WT). We also determined whether this response is sex dependent. D2R protein was higher in male than in female WT (135±5 vs 100±4%; n=3/group; P<0.04) and lower in males with SNPs (43±2%, P<0.05) and females with SNPs (23±2%,P<0.05), compared with their respective WT counterparts. In both male groups (WT and SNPs), AA increased D2R protein by 80-100% but had no effect in WT females and increased ~50% in females with SNPs. The TNFα mRNA was higher in males with WT and SNPs which was increased by AA 9-10-fold in WT males and females but only 2-3-fold in those with SNPs. The TGFβ mRNA was similar in WT males and females and increased to the same extent in both groups with SNPs and was not affected by AA in all groups. Col1a1 mRNA was higher (30%) in WT males and females than those with SNPs; AA decreased Col1a1 mRNA in all groups. FN1 mRNA was higher (30-40%) in males and females with SNPs than WT; AA increased FN1 mRNA only in males and females with SNPs. The mRNA expression of the cell proliferation marker Ki-67 was higher in WT females than WT males (1.5-2-fold) and higher with SNPs than WT in both groups; AA increased Ki-67 mRNA in both groups and to a greater extent in males than in females. Taken together our data indicate that the presence of DRD2 SNPs affects the baseline expression of inflammatory and fibrotic factors and the response to AA is dependent on both sex and the presence of DRD2 SNPs. These data may have potential clinical translation since rs6276/6277 is commonly expressed (42%/23%) in the human population.

Abstract MP11: Decreased Expression Of Ace2 Is Associated Urine Derived Human Renal Proximal Tubule Cells From Inverse Salt Sensitivity Participants Cultured In Low Salt Conditions

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. Inverse salt sensitivity (ISS) is the paradoxical increase in blood pressure of individuals to a low sodium diet. Our group previously reported decreased expression of dopamine type 2 receptor (D 2 R), increased expression Aminopeptidase N, and increased Ang II dependent sodium transport in human urine derived renal proximal tubule cells isolated from ISS participants. In an attempt to understand the increased Ang II sensitivity demonstrated in ISS cells, we examined angiotensin converting enzyme 2 (ACE2), a membrane associated enzyme involved in the metabolism of Ang II. Urine derived renal proximal tubule cells grown and immortalized from ISS participants were compared to cells from salt resistant (SR) participants cultured in iso-osmotic media with low salt (LS, 90 mM NaCl) normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl). Cells were incubated in LS, NS, and HS media with and without losartan (LOS,1 μM) overnight (18 hours) and ACE2 expression levels determined by in-cell western blot. A monoclonal antibody specific to an extracellular epitope of ACE2 was used as the primary antibody and an Alexa-647 anti-mouse secondary antibody. ACE2 expression was only reduced in ISS cells in LS condition (28.7±2.1 % reduction, ISS LS vs SR LS, N=4 per group, p<0.05). Addition of losartan completely blocked the decrease in ACE2 expression in low salt conditions in ISS in urine derived human renal proximal tubule cells. No other changes in ACE2 expression were found between ISS and SR in either NS or HS conditions and with or without losartan. In conclusion, a decreased expression of ACE2 in ISS urine proximal tubule cells could explain the previously reported increased sensitivity of ISS cells to Ang II by increasing the half-life of Ang II under low salt conditions.