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.

Angiotensin-(3–4) (Val-Tyr) Normalizes the Elevated Arterial Blood Pressure and Abnormal Na+/Energy Handling Associated With Chronic Undernutrition by Counteracting the Effects Mediated by Type 1 Angiotensin II Receptors

Purpose To investigate the mechanisms by which chronic administration of a multideficient diet after weaning alters bodily Na+ handling, and culminates in high systolic blood pressure (SBP) at a juvenile age.Methods From 28 to 93 days of age, weaned male Wistar rats were given a diet with low content and poor-quality protein, low lipid, without vitamin supplementation, which mimics the diets consumed in impoverished regions worldwide. We measured food, energy and Na+ ingestion, together with urinary Na+ excretion, Na+ density (Na+ intake/energy intake), plasma Na+ concentration, SBP), and renal proximal tubule Na+-transporting ATPases. Results Undernourished rats aged 93 days had only one-third of the control body mass, lower plasma albumin, higher SBP, higher energy intake, and higher positive Na+ balance accompanied by decreased plasma Na+ concentration. SBP was normalized with Losartan and with Ang-(3–4), and the combination of the 2 substances induced an accentuated negative Na+ balance as a result of strong inhibition of Na+ ingestion. Na+ density in undernourished rats was higher than in control, irrespective of the treatment, and they had downregulated (Na++K+)ATPase and upregulated Na+-ATPase in proximal tubule cells, which returned to control levels after Losartan or Ang-(3–4).Conclusions Na+ density, not only Na+ ingestion, plays a central role in the pathophysiology of elevated SBP in chronically undernourished rats. The observations that Losartan and Ang-(3–4) normalized SBP together with Na+ distribution and handling give support to the proposal that Ang IIÞAT1R and Ang IIÞAT2R axes have opposite roles within the renin-angiotensin-aldosterone system of undernourished juvenile rats.

Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

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.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

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.