Sharon S. Landgraf

Changes in angiotensin receptors expression play a pivotal role in the renal damage observed in spontaneously hypertensive rats

The renal renin-angiotensin system plays a central role in the development of hypertension. The aim of this work was to verify the expression of angiotensin II receptors AT(1)R and AT(2)R in the microsomal fraction of renal cortex and correlate this with the development of hypertension and renal damage in spontaneously hypertensive rats (SHR) using Wistar-Kyoto rats (WKY) as controls. AT(1)R expression increased (126%) and AT(2)R expression decreased (66%) in 4-wk-old SHR; AT(2) expression decreased in 14-wk-old SHR (61%) compared with respective age-matched WKY. These modifications were correlated to the increase in protein kinase C activity and decrease in protein kinase A activity. Four-week-old SHR showed large accumulations of macrophages in kidney glomerulus and the tubulointerstitial area, dense cortical collagen deposition, and arterial proliferative changes in the walls of arterioles and medium-sized vessels. Similar modifications were also observed in 14-wk-old SHR. Four-week-old SHR treated with losartan (30 mg·kg(-1)·day(-1)) or hydralazine (15 and 30 mg·kg(-1)·day(-1)) by gavage for 10 wk did not develop hypertension. The decrease in AT(2)R expression and renal damage observed in SHR remained even after treatment with hydralazine. On the other hand, losartan treatment prevented the modifications observed in 14-wk-old SHR, indicating that renal injuries are caused specifically by AT(1) rather than an increase in blood pressure. Our results indicate that the imbalance in AT(1)R and AT(2)R expression is associated with an inflammatory process that contributes to renal injury in adult SHR and to the development of hypertension.

Na(+)-ATPase in spontaneous hypertensive rats: possible AT(1) receptor target in the development of hypertension.

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na(+)-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na(+)-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10(-12) to 10(-6) mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT(2) receptor, PD123319 (10(-8) mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT(2)-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4-14) prevents the increase in Na(+)-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT(1) receptor activation in SHR and increased ouabain-insensitive Na(+)-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.

Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N‐acylhydrazone derivative, 3,4‐dimethoxyphenyl‐N‐methyl‐benzoylhydrazide (LASSBio‐1359), on monocrotaline (MCT)‐induced pulmonary hypertension in rats.

Sepsis-surviving Mice Are More Susceptible to a Secondary Kidney Insult*

Objective:It is well known that sepsis causes damage in different organs, including kidneys. However, few studies have been conducted on the magnitude of the long-term effects of sepsis on the surviving population, in particular, in relation to kidney disease. In this study, we examined the impact of long-term effects of sepsis on a second kidney insult. Design:Prospective experimental study. Setting:University research laboratory. Interventions:Wild-type mice were subjected to the cecal ligation and puncture sepsis model. Control animals underwent identical laparotomy but without ligation and cecum puncture. On days 0, 7, and 14 after surgery, the ratio between urinary protein and creatinine was measured. Fifteen days after surgery, surviving mice were subjected to a second kidney insult through intraperitoneal injections of bovine serum albumin for 7 days. On day 22 after surgery, urinary protein and creatinine, &ggr;-glutamyl transpeptidase, lactate dehydrogenase, histologic parameters, macrophage infiltration, apoptotic cell, renal and plasmatic cytokines were determined. Measurements and Main Results:On days 7 and 14 after surgery, the urinary protein and creatinine observed in the septic animal group were higher than those observed in the control group. On day 22 after surgery, sepsis-surviving animals that were subjected to a second kidney insult showed more severe tubular injury compared with controls. This process seems to involve an immunosuppressive state because the concentrations of some renal cytokines, such as tumor necrosis factor-&agr;, interleukin 6, interferon-&ggr; and chemokine ligand 2, were decreased and leukocyte numbers were increased. Conclusions:These results suggest that sepsis induces long-term effects in kidney structure aggravating tubule damage in a second kidney insult.

LASSBio-294, A Compound With Inotropic and Lusitropic Activity, Decreases Cardiac Remodeling and Improves Ca2+ Influx Into Sarcoplasmic Reticulum After Myocardial Infarction

BACKGROUND Myocardial infarction (MI) is commonly associated with cardiac hypertrophy, reduced Ca²(+) uptake into the sarcoplasmic reticulum (SR) and impaired myocardial relaxation. Treatment to prevent MI-associated complications is currently lacking. The purpose of the present study was to investigate the remodeling and function of hearts subjected to experimental MI and to evaluate the response to treatment with a new thienylhydrazone: 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294), which has demonstrated positive inotropic properties. METHODS LASSBio-294 (2 mg/kg) or vehicle (dimethyl sulfoxide) was administered daily by intraperitoneal injection for 4 weeks in sham-operated rats and rats with MI. Cardiac remodeling and hemodynamic parameters were monitored through histological and intraventricular pressure analyses. Intracellular Ca²(+) regulation (uptake and release) and the sensitivity of contractile proteins to Ca²(+) were evaluated by determining the contractile response of saponin-skinned cardiac cells from infarcted hearts. RESULTS Cardiac hypertrophy occurred at 4 weeks post-MI and was partially reverted by treatment with LASSBio-294. LASSBio-294 treatment also reduced the nuclear density, collagen volume fraction, and left ventricular end-diastolic pressure (LV EDP) induced by MI. MI led to reduced Ca²(+) uptake from the SR, but did not modify the Ca²(+) release or the Ca²(+)-force relationship. LASSBio-294 restored SR function and enhanced the sensitivity of contractile proteins to Ca²(+). CONCLUSION LASSBio-294 is a promising candidate for improving intracellular Ca²(+) regulation and preventing MI-induced cardiac dysfunction, which could potentially prevent heart failure (HF).

(Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin

Background: Albumin interacts with megalin and triggers cellular responses in proximal tubule cells. Results: Albumin modulates the PI3K/protein kinase B, protein kinase C, and protein kinase A pathways promoting the regulation of the (Na+ + K+)-ATPase expression. Conclusion: Variation in the albumin concentration in the proximal tubule affects sodium reabsorption. Significance: These results open new avenues to understanding the role of albumin in proximal tubule cells. In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion.

5-Lypoxygenase products are involved in renal tubulointerstitial injury induced by albumin overload in proximal tubules in mice.

The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO–/–). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO–/– mice. The levels of urinary protein observed in the 5-LO–/– mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO–/– mice. LTB4 and LTD4, 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-α and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO–/– mice. However, 5-LO–/– mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload.

Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: implications for modulation of the membrane-associated protein kinase A.

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [(35)S] GTPgammaS binding to the BLM and it was inhibited by 10(-6)M DPCPX, an A(1) receptor-selective antagonist. Inosine (10(-7)M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G(alphai-3) protein-coupled A(1) receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A(1) receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A(1) receptor-mediated inhibition of adenylyl cyclase.

N-acylhydrazone improves exercise intolerance in rats submitted to myocardial infarction by the recovery of calcium homeostasis in skeletal muscle.

AIMS This work investigated the effects of 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294) treatment on the contractile response of soleus (SOL) muscle from rats submitted to myocardial infarction (MI). MAIN METHODS Following coronary artery ligation, LASSBio-294 (2mg/kg, i.p.) or vehicle was administrated once daily for 4 weeks. KEY FINDINGS The run time to fatigue for sham rats was 17.9 ±2.6 min, and it was reduced to 3.3 ± 0.8 min (P<0.05) in MI rats. In MI rats treated with LASSBio-294, the time to fatigue was 15.1 ± 3.6 min. During the contractile test, SOL muscles from sham rats showed a response of 7.12 ± 0.54N/cm(2) at 60 Hz, which was decreased to 5.45 ± 0.49 N/cm(2) (P<0.05) in MI rats. The contractility of SOL muscles from the MI-LASSBio-294 group was increased to 9.01 ± 0.65N/cm(2). At 16 mM caffeine, the contractility was reduced from 2.31 ± 0.33 to 1.60 ± 0.21 N/cm(2) (P<0.05) in the MI group. In SOL muscles from MI-LASSBio-294 rats, the caffeine response was increased to 2.62 ± 0.33 N/cm(2). Moreover, SERCA2a expression in SOL muscles was decreased by 0.31-fold (31%) in the MI group compared to the Sham group (P<0.05). In the MI-LASSBio-294 group, it was increased by 1.53-fold (153%) compared to the MI group (P<0.05). Meanwhile, the nuclear density in SOL muscles was increased in the MI group compared to the Sham group. Treatment with LASSBio-294 prevented this enhancement of cellular infiltrate. SIGNIFICANCE LASSBio-294 treatment prevented the development of muscular fatigue and improved exercise intolerance in rats submitted to MI.

Prostaglandin E2 modulates proximal tubule Na+-ATPase activity: Cooperative effect between protein kinase A and protein kinase C

Previous data showed that prostaglandin E₂ (PGE₂) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na+-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE₂ on PT Na+-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE₂ on PT Na+-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE₂ increased [³⁵S]GTPγS binding; (2) GDPβS abolished the inhibitory effect of PGE₂; (3) PGE₂ increased PKA activity; (4) the inhibitory effect of PGE₂ was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE₂ on PT Na+-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE₂ stimulates Ca²+-independent phospholipase A₂ activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE₂ on proximal tubule sodium reabsorption.