Proximal Tubular Renal Cells Research Articles

Doxepin as OCT2 inhibitor ameliorates inflammatory response and modulates PI3K/Akt signaling associated with cisplatin-induced nephrotoxicity in rats.

Organic cationic transporter 2 (OCT2) was identified as the main transporter involved in the accumulation of cisplatin (CP) in the proximal tubular renal cells, resulting in nephrotoxicity. Doxepin (DOX) is a tricyclic agent with an inhibitory effect on OCT2. This study aimed to explore the possible mechanisms of the renoprotective role of DOX toward CP-induced nephrotoxicity. Rats were randomly divided into six groups: group 1, control; group 2, CP; groups 3, 4, and 5 were treated with graded doses of DOX (5, 10, and 20 mg/kg, respectively) intraperitoneally (ip) once daily for 10 consecutive days and group 6 was treated only with DOX (20 mg/kg). On the seventh day, a single injected dose of CP (10 mg/kg, ip) was given to the rats in groups 2-5. Seventy-two hours after CP injection, rats were sacrificed, and the kidneys were removed for histological and biochemical measurements. DOX ameliorated the CP-induced histopathological alterations. DOX significantly reduced the expression of OCT2, lipid peroxidation marker (MDA), and inflammatory cytokines, including TNF-α, IL-6, IL-1, IL-2, and IL-1β, and increased the activity of antioxidant enzymes. In addition, pre- and co-treatment with DOX significantly reduced the CP-mediated apoptotic effect by reducing the renal tissue expression of BAX and caspase-3 levels, upregulating the expression of Bcl-2, and modulating the phosphorylation of PI3K/Akt signaling cascade. DOX exerts a nephroprotective impact against CP-mediated nephrotoxicity via the inhibition of OCT2, suppression of inflammation, oxidative stress, and apoptotic markers, and modulation of PI3K/Akt signaling cascade.

New Insights into the Link between SARS-CoV-2 Infection and Renal Cancer.

Cancer has been described as a risk factor for greater susceptibility to SARS-CoV-2 infection and severe COVID-19, mainly for patients with metastatic disease. Conversely, to that reported for most solid and hematological malignancies, the few available clinical studies reported that the infection did not increase the risk of death in renal cancer patients. The expression on proximal tubular renal cells of the key players in cellular viral uptake, ACE2, TMPRSS2, and NRP1, seems to be the mechanism for the direct kidney injury seen in patients with COVID-19. Interestingly, data from The Cancer Genome Atlas and experimental analyses on various renal cancer cell lines demonstrated that the above-reported receptors/cofactors are maintained by renal cancer cells. However, whether SARS-CoV-2 infection directly kills renal cancer cells or generates enhanced immunogenicity is a question worth investigating. In addition, some researchers have further addressed the topic by studying the expression and prognostic significance of gene signatures related to SARS-CoV-2 infection in renal cancer patients. The emerging data highlights the importance of better understanding the existence of a link between renal cancer and COVID-19 since it could lead to the identification of new prognostic factors and the development of new therapeutic targets in the management of renal cancer patients.

Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis.

Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-βgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-βgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.

Change in Attitude in Renal Function in Major Beta Thalassemia

Thalassemia is a multisystemic disease in the field of hemolysis and chronic anemia caused by the erythropoietic disorder. The severe effects of iron overload from continuous blood transfusion iron chelators side effects, and involvement of multiple organs in thalassemias such as heart failure, liver, and endocrine dysfunction can all affect kidney function. Although there has been much debate about changes in renal function in thalassemia for many years, the presence of hyperfiltration and ultimately, decreased renal function in almost all studies. It seems for the researchers to look beyond kidney function in a thalassemia perspective, because of secretory biomarkers of proximal tubular renal cells that are sensitive to pathologic agents, which may be a good indicator of the courses of treatment and prognosis of patients. Future studies will be sooner or later. *Corresponding Author: Malihe Najafpour; Email: malihe_najafpour@modares.ac.ir Please cite this article as: Malaki M, Najafpour M, Talebi M, Azimi A. Change in Attitude in Renal Function in Major Beta Thalassemia. Arch Med Lab Sci. 2020;6:1-5 (e24). https://doi.org/10.22037/amls.v6.33057

Rapamycin-induced autophagy protects proximal tubular renal cells against proteinuric damage through the transcriptional activation of the nerve growth factor receptor NGFR

ABSTRACTExperimental evidence demonstrated that macroautophagy/autophagy exerts a crucial role in maintain renal cellular homeostasis and represents a protective mechanism against renal injuries. Interestingly, it has been demonstrated that in the human proximal tubular renal cell line, HK-2, the MTOR inhibitor rapamycin enhanced autophagy and mitigated the apoptosis damage induced by urinary protein overload. However, the underlying molecular mechanism has not yet been elucidated. In our study we demonstrated, for the first time, that in HK-2 cells, the exposure to low doses of rapamycin transactivated the NGFR promoter, leading to autophagic activation. Indeed, we observed that in HK-2 cells silenced for the NGFR gene, the rapamycin-induced autophagic process was prevented, as the upregulation of the proautophagic markers, BECN1, as well as LC3-II, and the autophagic vacuoles evaluated by transmission electron microscopy, were not found. Concomitantly, using a series of deletion constructs of the NGFR promoter we found that the EGR1 transcription factor was responsible for the rapamycin-mediated transactivation of the NGFR promoter. Finally, our results provided evidence that the cotreatment with rapamycin plus albumin further enhanced autophagy via NGFR activation, reducing the proapoptotic events promoted by albumin alone. This effect was prevented in HK-2 cells silenced for the NGFR gene or pretreated with the MTOR activator, MHY1485. Taken together, our results describe a novel molecular mechanism by which rapamycin-induced autophagy, mitigates the tubular renal damage caused by proteinuria, suggesting that the use of low doses of rapamycin could represent a new therapeutic strategy to counteract the tubule-interstitial injury observed in patients affected by proteinuric nephropathies, avoiding the side effects of high doses of rapamycin.

Fructose, but Not Dextrose, Induces Leukocyte Adherence to the Mesenteric Venule of the Rat by Oxidative Stress

Recent evidence indicates that fructose is a pro-inflammatory molecule. Oral fructose induces serum and kidney inflammatory intercellular adhesion molecule-1 (ICAM-1) in rats. Fructose also induces ICAM-1 expression in human aortic endothelial cells (HAEC) and monocyte chemoattractant protein-1 in proximal tubular renal cells. It is not known whether fructose may directly promote inflammation on the intestinal microcirculation. Accordingly, using intravital microscopy we studied the effect of topical fructose and dextrose on leukocyte adherence to the mesenteric venule of the rat. Leukocyte adherence was determined during a control period and after fructose was added to the mesentery, in the presence or absence of the NO donor spermine NONO-ate (SNO), and after i.v. injection of the antioxidant lipoic acid (LA). In separate experiments, we examined the effect of topical dextrose on leukocyte adherence to the mesenteric venule. Venular shear rate was calculated. Fructose, but not dextrose, induced significant inflammation independent of shear rate. This effect was completely blocked by SNO and LA, suggesting that fructose induces inflammation via reactive oxygen species (ROS) generation. These results suggest that fructose present in formulas may adversely affect the intestinal microcirculation of premature infants and potentially contribute to the pathogenesis of necrotizing enterocolitis (NEC).

Functional TauT Protects Against Acute Kidney Injury

Nephrotoxicity is common with the use of the chemotherapeutic agent cisplatin, but the cellular mechanisms that modulate the extent of injury are unknown. Cisplatin downregulates expression of the taurine transporter gene (TauT) in LLC-PK1 proximal tubular renal cells, and forced overexpression of TauT protects against cisplatin-induced apoptosis in vitro. Because the S3 segments of proximal tubules are the sites of both cisplatin-induced injury and adaptive regulation of the taurine transporter, we hypothesized that TauT functions as an anti-apoptotic gene and protects renal cells from cisplatin-induced nephrotoxicity in vivo. Here, we studied the regulation of TauT in cisplatin nephrotoxicity in a human embryonic kidney cell line and in LLC-PK1 cells, as well as in TauT transgenic mice. Cisplatin-induced activation of p53 repressed TauT and overexpression of TauT prevented the progression of cisplatin-induced apoptosis and renal dysfunction in TauT transgenic mice. Although cisplatin activated p53 and PUMA (a p53-responsive proapoptotic Bcl-2 family protein) in the kidneys of both wildtype and TauT transgenic mice, only wildtype animals demonstrated acute kidney injury. These data suggest that functional TauT plays a critical role in protecting against cisplatin-induced nephrotoxicity, possibly by attenuating a p53-dependent pathway.

Changes in renal sodium transport during a systemic inflammatory response

Sirs, Watanabe et al. [1] reported on the clinical characteristics of patients with hyponatremia in Kawasaki disease (KD). They noted that hyponatremia was associated with a more severe systemic inflammatory response reflected in prolonged pyrexia and higher C-reactive protein levels. We have recently published the result of a prospective study investigating changes in systemic sodium and chloride transport in meningococcal septicemia. Severe meningococcal septicemia was associated with increased fractional renal sodium excretion [2]. Both meningococcal septicemia and Kawasaki disease are associated with an intensive systemic inflammatory response with high circulating levels of interleukin-1 (IL-1) and tumor necrosis factor (TNF) [3– 6]. The mechanism by which these inflammatory mediators are able to induce hyponatremia has been investigated in both animal studies and in vitro. It involves increased renal sodium loss by a reduction in renal tubular sodium absorption. Inflammatory mediators like IL-1 and TNF have been shown to reduce epithelial sodium transport in epithelial cells by a reduction of the expression and function of the apical epithelial sodium channel (ENaC) and/or the sodium potassium ATPase (Na/K ATPase) at the basolateral membrane [7]. IL-1 was hereby found to induce natriuresis in the rat model [8]. The mechanism was investigated in cultures of inner medullary, cortical collecting duct and proximal tubular renal cells in vitro and it was found to involve a reduction of the Na/K ATPase function. The effect was mediated by prostaglandin E2 and inhibitable by the cyclooxygenase inhibitor indomethacin [9–13]. Another pathway by which IL-1 and TNF are involved in a reduction of renal sodium absorption is by increasing tissue levels of nitric oxide, which is a potent suppressor of the epithelial Na/K ATPase function mediated by the intracellular messenger cGMP and via modification of protein kinase G [14, 15]. Future studies into the etiology of hyponatremia in KD need to investigate the effects of therapies directed at a reduction of effects of IL-1 and TNF and their mediators PGE2 and NO on renal fractional sodium excretion and hyponatremia.

Endocytosis of gentamicin in a proximal tubular renal cell line

The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 330/ megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PK 1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 °C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PK 1 cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK 1 cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.

Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells

The capability of cyclosporin to produce direct injury to primary proximal tubular renal cells was studied. These cells, when grown on Millicell inserts, retain the functional polarity of the proximal tubule, i.e., generate a transepithelial pH gradient (apical compartment acidic) that is reversibly blocked by amiloride addition only if it is added to the apical compartment. Administration of ouabain to the basal compartment also blocks the generation of the transepithelial pH gradient. Additionally, the cells were more responsive to parathyroid hormone (PTH), a proximal tubule characteristic, than to arginine vasopressin (AVP), a distal tubule characteristic. The following substances were tested for their effect on the capacity of these cells to generate a pH gradient: Sandimmune, the commercial form of cyclosporin A; the free form of the drug; Cremophor EL, the vehicle used in the commercial preparation; and ethanol, the vehicle used to dissolve the free form. Sandimmune, at 25-50 microM, inhibited the generation of the pH gradient within 24 h. Surprisingly, Cremophor also blocked the development of a pH gradient, although somewhat less effectively. In contrast, 10 microM cyclosporin, regardless of the form tested, had no effect for up to 96 h. These findings show that cyclosporin, in the form of Sandimmune, has a direct toxic effect on these cells; they also suggest that the vehicle, Cremophor, may contribute to the well-established nephrotoxicity of cyclosporin A.