Steven T. Haller

Embolic Protection and Platelet Inhibition During Renal Artery Stenting

Background— Preservation of renal function is an important objective of renal artery stent procedures. Although atheroembolization can cause renal dysfunction during renal stent procedures, whether adjunctive use of embolic protection devices or glycoprotein IIb/IIIa inhibitors improves renal function is unknown. Methods and Results— One hundred patients undergoing renal artery stenting at 7 centers were randomly assigned to an open-label embolic protection device, Angioguard, or double-blind use of a platelet glycoprotein IIb/IIIa inhibitor, abciximab, in a 2×2 factorial design. The main effects of treatments and their interaction were assessed on percentage change in Modification in Diet in Renal Disease–derived glomerular filtration rate from baseline to 1 month using centrally analyzed creatinine. Filter devices were analyzed for the presence of platelet-rich thrombus. With stenting alone, stenting and embolic protection, and stenting with abciximab alone, glomerular filtration rate declined (P<0.05), but with combination therapy, it did not decline and was superior to the other allocations in the 2×2 design (P<0.01). The main effects of treatment demonstrated no overall improvement in glomerular filtration rate; although abciximab was superior to placebo (0±27% versus −10±20%; P<0.05), embolic protection was not (−1±28% versus −10±20%; P=0.08). An interaction was observed between abciximab and embolic protection (P<0.05), favoring combination treatment. Abciximab reduced the occurrence of platelet-rich emboli in the filters from 42% to 7% (P<0.01). Conclusions— Renal artery stenting alone, stenting with embolic protection, and stenting with abciximab were associated with a decline in glomerular filtration rate. An unanticipated interaction between Angioguard and abciximab was seen, with combination therapy better than no treatment or either treatment alone.

Spironolactone Attenuates Experimental Uremic Cardiomyopathy by Antagonizing Marinobufagenin

Spironolactone has been noted to attenuate cardiac fibrosis. We have observed that the cardiotonic steroid marinobufagenin plays an important role in the diastolic dysfunction and cardiac fibrosis seen with experimental renal failure. We performed the following studies to determine whether and how spironolactone might ameliorate these changes. First, we studied rats subjected to partial nephrectomy or administration of exogenous marinobufagenin. We found that spironolactone (20 mg/kg per day) attenuated the diastolic dysfunction as assessed by ventricular pressure-volume loops and essentially eliminated cardiac fibrosis as assessed by trichrome staining and Western blot. Next, we examined the effects of spironolactone and its major metabolite, canrenone (both 100 nM), on marinobufagenin stimulation of rat cardiac fibroblasts. Both spironolactone and canrenone prevented the stimulation of collagen production by 1 nM marinobufagenin but not 100 nM marinobufagenin, as assessed by proline incorporation and procollagen 1 expression, as well as signaling through the sodium-potassium-ATPase, as evidenced by protein kinase C isoform &dgr; translocation and extracellular signal regulated kinase 1/2 activation. Both spironolactone and canrenone also altered ouabain binding to cultured porcine cells in a manner consistent with competitive inhibition. Our data suggest that some of the antifibrotic effects of spironolactone may be attributed to antagonism of marinobufagenin signaling through the sodium-potassium-ATPase.

Monoclonal antibody against marinobufagenin reverses cardiac fibrosis in rats with chronic renal failure

BACKGROUND Cardiotonic steroids (CTS) are implicated in pathophysiology of uremic cardiomyopathy. In the present study, we tested whether a monoclonal antibody (mAb) against the bufadienolide CTS, marinobufagenin (MBG), alleviates cardiac hypertrophy and fibrosis in partially nephrectomized (PNx) rats. METHODS In PNx rats, we compared the effects of 3E9 anti-MBG mAb and of Digibind, an affinity-purified digoxin antibody, on blood pressure and cardiac hypertrophy and fibrosis following 4 weeks after the surgery. RESULTS In PNx rats, a fourfold elevation in plasma MBG levels was associated with hypertension, increased cardiac levels of carbonylated protein, cardiac hypertrophy, a reduction in cardiac expression of a nuclear transcription factor which is a negative regulator of collagen synthesis, Friend leukemia integration-1 (Fli-1), and an increase in the levels of collagen-1. A single intraperitoneal administration of 3E9 mAb to PNx rats reduced blood pressure by 59 mm Hg for 7 days and produced a significant reduction in cardiac weight and cardiac levels of oxidative stress, an increase in the expression of Fli-1, and a reduction in cardiac fibrosis. The effects of Digibind were similar to those of 3E9 mAb, but were less pronounced. CONCLUSIONS In experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy.

Endogenous cardiotonic steroids in chronic renal failure

BACKGROUND Previous reports demonstrated that digitalis-like cardiotonic steroids (CTS) contribute to the pathogenesis of end-stage renal disease. The goal of the present study was to define the nature of CTS in patients with chronic kidney disease (CKD) and in partially nephrectomized (PNx) rats. METHODS In patients with CKD and in healthy controls, we determined plasma levels of marinobufagenin (MBG) and endogenous ouabain (EO) and erythrocyte Na/K-ATPase activity in the absence and in the presence of 3E9 anti-MBG monoclonal antibody (mAb) and Digibind. Levels of MBG and EO were also determined in sham-operated Sprague-Dawley rats and in rats following 4 weeks of PNx. RESULTS In 25 patients with CKD plasma, MBG but not EO was increased (0.86 ± 0.07 versus 0.28 ± 0.02 nmol/L, P < 0.01) and erythrocyte Na/K-ATPase was inhibited (1.24 ± 0.10 versus 2.80 ± 0.09 μmol Pi/mL/h, P < 0.01) as compared to that in 19 healthy subjects. Ex vivo, 3E9 mAb restored Na/K-ATPase in erythrocytes from patients with CKD but did not affect Na/K-ATPase from control subjects. Following chromatographic fractionation of uremic versus normal plasma, a competitive immunoassay based on anti-MBG mAb detected a 3-fold increase in the level of endogenous material having retention time similar to that seen with MBG. A similar pattern of CTS changes was observed in uremic rats. As compared to sham-operated animals, PNx rats exhibited 3-fold elevated levels of MBG but not that of EO. CONCLUSIONS In chronic renal failure, elevated levels of a bufadienolide CTS, MBG, contribute to Na/K-ATPase inhibition and may represent a potential target for therapy.

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

Background: Na/K-ATPase signaling regulates sodium reabsorption in renal proximal tubules. Results: Carbonylation modification of the Na/K-ATPase α1 subunit regulates Na/K-ATPase signaling and subsequent transepithelial sodium transport. Conclusion: ROS is involved in the Na/K-ATPase signaling transduction in a feed-forward mechanism. Significance: ROS regulates Na/K-ATPase signaling and sodium transport in LLC-PK1 cells. Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-l-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial 22Na+ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.

Ouabain-Stimulated Trafficking Regulation of the Na/K-ATPase and NHE3 in Renal Proximal Tubule Cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular 22Na+ transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular 22Na+ transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.

Renal Ischemia Regulates Marinobufagenin Release in Humans

Cardiotonic steroids, including marinobufagenin, are a group of new steroid hormones found in plasma and urine of patients with congestive heart failure, myocardial infarction, and chronic renal failure. In animal studies, partial nephrectomy induces marinobufagenin elevation, cardiac hypertrophy, and fibrosis. The objective of this study is to test the effect of renal ischemia on marinobufagenin levels in humans with renal artery stenosis (RAS). To test this, plasma marinobufagenin levels were measured in patients with RAS of the Prospective Randomized Study Comparing Renal Artery Stenting With or Without Distal Protection, non-RAS patient controls who were scheduled for coronary angiography, and normal healthy individuals. Marinobufagenin levels were significantly higher in patients with RAS compared with those of the other 2 groups. Multivariate analysis shows that occurrence of RAS is independently related to marinobufagenin levels. In addition, renal artery revascularization by stenting partially reversed marinobufagenin levels in the patients with RAS (0.77±0.06 nmol/L at baseline; 0.66±0.06 nmol/L at 24 hours; and 0.61±0.05 nmol/L at 1 month). In conclusion, we have found that marinobufagenin levels are increased in patients with RAS, whereas reversal of renal ischemia by stenting treatment reduces marinobufagenin levels. These results suggest that RAS-induced renal ischemia may be a major cause of marinobufagenin release.

Na/K-ATPase Signaling Regulates Collagen Synthesis Through microRNA-29b-3p in Cardiac Fibroblasts

Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

Rapamycin Attenuates Cardiac Fibrosis in Experimental Uremic Cardiomyopathy by Reducing Marinobufagenin Levels and Inhibiting Downstream Pro‐Fibrotic Signaling

Background Experimental uremic cardiomyopathy causes cardiac fibrosis and is causally related to the increased circulating levels of the cardiotonic steroid, marinobufagenin (MBG), which signals through Na/K‐ATPase. Rapamycin is an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR) implicated in the progression of many different forms of renal disease. Given that Na/K‐ATPase signaling is known to stimulate the mTOR system, we speculated that the ameliorative effects of rapamycin might influence this pathway. Methods and Results Biosynthesis of MBG by cultured human JEG‐3 cells is initiated by CYP27A1, which is also a target for rapamycin. It was demonstrated that 1 μmol/L of rapamycin inhibited production of MBG in human JEG‐2 cells. Male Sprague‐Dawley rats were subjected to either partial nephrectomy (PNx), infusion of MBG, and/or infusion of rapamycin through osmotic minipumps. PNx animals showed marked increase in plasma MBG levels (1025±60 vs 377±53 pmol/L; P<0.01), systolic blood pressure (169±1 vs 111±1 mm Hg; P<0.01), and cardiac fibrosis compared to controls. Plasma MBG levels were significantly decreased in PNx‐rapamycin animals compared to PNx (373±46 vs 1025±60 pmol/L; P<0.01), and cardiac fibrosis was substantially attenuated by rapamycin treatment. Conclusions Rapamycin treatment in combination with MBG infusion significantly attenuated cardiac fibrosis. Our results suggest that rapamycin may have a dual effect on cardiac fibrosis through (1) mTOR inhibition and (2) inhibiting MBG‐mediated profibrotic signaling and provide support for beneficial effect of a novel therapy for uremic cardiomyopathy.

Passive Immunization Against Marinobufagenin Attenuates Renal Fibrosis and Improves Renal Function in Experimental Renal Disease

BACKGROUND We have shown that the cardiotonic steroid marinobufagenin (MBG) is elevated in clinical and experimental renal disease, and significantly contributes to the development of experimental uremic cardiomyopathy induced by removal of five-sixths of the kidney (5/6 nephrectomy; PNx) in the rat. We have demonstrated that both active and passive immunization against MBG with an anti-MBG monoclonal antibody (mAb 3E9) significantly attenuated cardiac fibrosis following PNx. In the present study we sought to determine whether the use of mAb 3E9 could improve renal function following PNx. METHODS Sprague-Dawley rats were treated with either mAb 3E9 or with DigiFab (an affinity-purified anti-digoxin antibody formerly named Digibind) during the fourth week after PNx. Sham-operated animals and PNx animals treated with an IgG antibody served as controls. Plasma, urine, and renal tissue were collected at the completion of the study to determine the effects of antibody treatment on renal function. RESULTS In PNx rats, treatments with mAb 3E9 and DigiFab, respectively, significantly reduced plasma creatinine, improved creatinine clearance, and reduced proteinuria below the values of these three measures in IgG-treated PNx controls. Additionally, treatment with mAb 3E9 and DigiFab significantly reduced renal fibrosis as measured with Western blotting and Sirius red/Fast green staining. CONCLUSIONS Passive immunization against MBG significantly improved renal function and markedly reduced renal fibrosis following the experimental induction of renal disease. The work in the study reported here adds to a growing body of knowledge implicating MBG in the development of chronic renal disease. Passive immunization against cardiotonic steroids may serve as a promising treatment for chronic renal disease.