Ouabain and Marinobufagenin: Physiological Effects on Human Epithelial and Endothelial Cells.

Long-term study on the identification of Na,K-ATPase endogenous inhibitors in mammalian tissues has resulted in the discovery of ouabain, marinobufagenin (MBG), and other cardiotonic steroids (CTS) in the blood plasma. Production of ouabain and MBG is increased in essential hypertension and other diseases associated with hypervolemia. Here, we compared the effects of ouabain and MBG on the Na,K-ATPase activity (measured as the transport of Na+, K+, and Rb+ ions) and proliferation and death of human renal epithelial cells (HRECs) and human umbilical vein endothelial cells (HUVEC) expressing α1-Na,K-ATPase. Ouabain concentration that provided the half-maximal inhibition of the Rb+ influx (IC50) into HRECs and HUVECs was 0.07 µM. In both types of cells, the IC50 values for MBG were 10 times higher than for ouabain. Incubation of HREC and HUVEC with 0.001-0.01 µM ouabain for 30 h resulted in 40% increase in the [3H]thymidine incorporation into DNA; further elevation of ouabain concentration to 0.1 µM completely suppressed DNA synthesis. MBG at the concentration of 0.1 µM activated DNA synthesis by 25% in HRECs, but not in HUVECs; 1 µM MBG completely inhibited DNA synthesis in HRECs and by 50% in HUVECs. In contrast to HRECs, incubation of HUVECs in the serum-free medium induced apoptosis, which was almost completely suppressed by ouabain and MBG at the concentrations of 0.1 and 3 µM, respectively. Based on these data, we can conclude that (i) the effect of MBG at the concentrations detected in the blood plasma (<0.01 µM) on HRECs and HUVECs was not due to the changes in the [Na+]i/[K+]i ratio; (ii) the effect of physiological concentrations of ouabain on these cells might be mediated by the activation of Na,K-ATPase, leading to cell proliferation.

Prolactin Receptor Isoforms as the Basis of Tissue-Specific Action of Prolactin in the Norm and Pathology.

The review describes functional and structural features of different isoforms of prolactin receptor, mechanisms of signaling pathway activation, and molecular messengers involved in the transmission and termination of signal from the prolactin receptor isoforms. Changes in the ratio between prolactin receptor isoforms, key mediators of prolactin signal transduction and termination in various organs and tissues, are analyzed. Special attention is given to the role of molecular mediators and the ratio between the isoforms in normal physiological functions and pathologies. Approaches for therapeutic correction of prolactin signaling impairments are discussed.

Prolactin Signaling Pathways Determining Its Direct Effects on Kidneys in the Cholestasis of Pregnancy Model.

Cholestasis of pregnancy is a pathology associated with disruptions in the bile flow and dysregulation of salt and water homeostasis. Prolactin is one of the most important regulators of salt and water balance. Changes in the expression of long and short isoforms of the prolactin receptor (PrlR) and mediators of prolactin signaling were studied by immunoblotting and RT-qPCR in the rat kidney cortex and outer medulla in the model of cholestasis of pregnancy. Both PrlR isoforms were shown to participate in the effects of prolactin in cholestasis of pregnancy. Direct impact of prolactin on the kidney has been demonstrated: (i) mRNA expression of both PrlR isoforms in the kidney depended on the physiological conditions and prolactin levels; (ii) expression of pSTAT5, a key mediator of the long PrlR isoform signaling, was increased in animals with cholestasis of pregnancy; (iii) in the case of long PrlR isoform predomination, expression of mRNAs for the prolactin signaling inhibitors SOCS3 and PIAS3 was upregulated (the genes of these regulators contain STAT-responsive elements in their promoters); (iv) expression of the mRNA for galactose-1-phosphate uridyltransferase (GALT), a molecular target of the PrlR short isoform, was decreased in the kidney outer medulla.

Molecular Targets of Natriuretic Action of Prolactin in the Rat Model of Cholestasis of Pregnancy.

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.

Role of Na+/K+-ATPase in Natriuretic Effect of Prolactin in a Model of Cholestasis of Pregnancy.

Participation of Na+/K+-ATPase in the natriuretic effect of prolactin in a cholestasis of pregnancy model was investigated. The Na+/K+-ATPase activity in rat kidney medulla, where active sodium reabsorption occurs, decreased in the model of cholestasis of pregnancy and other hyperprolactinemia types compared with intact animals. This effect was not connected with the protein level of α1- and ß-subunits of Na+/K+-ATPase measured by Western blotting in the kidney medulla. Decrease in Na+/K+-ATPase activity in the kidney cortex was not significant, as well as decrease in the quantity of mRNA and proteins of the α1- and ß-subunits of Na+/K+-ATPase. There were no correlations between the Na+/K+-ATPase activity and sodium clearance, although sodium clearance increased significantly in the model of cholestasis of pregnancy and other hyperprolactinemia groups under conditions of stable glomerular filtration rate measured by creatinine clearance. We conclude that the Na+/K+-ATPase is not the only mediator of the natriuretic effect of prolactin in the model of cholestasis of pregnancy.

Role of Prolactin in the Regulation of Bicarbonates Biodynamics in Female Rat Model of Cholestasis of Pregnancy.

We studied possible involvement of prolactin in the regulation of bicarbonate biodynamics using female rat model of cholestasis of pregnancy induced by transplantation of the donor pituitary under the renal capsule of a recipient (hyperprolactinemia) and bile duct ligation (cholestasis). The concentration of bicarbonates in the bile and blood, their excretion, clearance, and reabsorption, as well as glomerular filtration rate and excretion of sodium ions were assessed. It was found that the main effect of prolactin was directed to the kidney-regulated pool of bicarbonates and consisted in stimulation of their clearance and inhibition of reabsorption, which led to a decrease in bicarbonate blood concentration. Parallel influence of prolactin on the clearance of bicarbonates and sodium ions was observed.

Decreased renal expression of PAQR5 is associated with the absence of a nephroprotective effect of progesterone in a rat UUO model.

Fibrosis is a severe complication of chronic kidney disease (CKD). Progesterone, like other sex hormones, plays an important role in renal physiology, but its role in CKD is poorly understood. We investigated progesterone effect on renal fibrosis progression in the rat model of unilateral ureteral obstruction (UUO). Female rats were exposed to UUO, ovariectomy and progesterone administration after UUO with ovariectomy. Expression of key fibrosis markers, proinflammatory cytokines, levels of membrane-bound (PAQR5) and nuclear (PGR) progesterone receptors, and matrix metalloproteinase (MMP) activity were analyzed in the obstructed and intact rat kidney. In all groups exposed to UUO, decreased PAQR5 expression was observed in the obstructed kidney while in the contralateral kidney, it remained unaffected. We found increased mRNA levels for profibrotic COL1A1, FN1, MMP2, TIMP1, TIMP2, proinflammatory IL1α, IL1ß, and IL18, as well as elevated α-SMA and MMP9 proteins, collagen deposition, and MMP2 activity in all UUO kidneys. Progesterone had slight or no effect on the change in these markers. Thus, we demonstrate for the first time diminished sensitivity of the kidney to progesterone associated with renal fibrosis due to a severe decrease in PAQR5 expression that was accompanied by the lack of nephroprotection in a rat UUO model.

Effects of caloric restriction and ketogenic diet on renal fibrosis after ischemia/reperfusion injury.

The beneficial effects of caloric restriction (CR) and a ketogenic diet (KD) have been previously shown when performed prior to kidney injury. We investigated the effects of CR and KD on fibrosis development after unilateral kidney ischemia/reperfusion (UIR). Post-treatment with CR significantly (p < 0.05) affected blood glucose (2-fold decrease), ketone bodies (3-fold increase), lactate (1.5-fold decrease), and lipids (1.4-fold decrease). In the kidney, CR improved succinate dehydrogenase and malate dehydrogenase activity by 2-fold each, but worsened fibrosis progression. Similar results were shown for the KD, which restored the post-UIR impaired activities of succinate dehydrogenase, malate dehydrogenase, and α-ketoglutarate dehydrogenase (which was decreased 2-fold) but had no effect on fibrosis progression. Thus, our study shows that the use of CR or KD after UIR did not reduce the development of fibrosis, as shown by hydroxyproline content, western-blotting, and RT-PCR, whereas it caused significant metabolic changes in kidney tissue after UIR.

Na+ i/K+ i imbalance contributes to gene expression in endothelial cells exposed to elevated NaCl.

High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+ i/K+ i-dependent mechanism(s).