Early inflammatory changes and CC chemokine ligand-8 upregulation in the heart contribute to uremic cardiomyopathy.

Uremic cardiomyopathy is a common complication in chronic kidney disease (CKD) patients, accounting for a high mortality rate. Several mechanisms have been proposed to link CKD and cardiac alterations; however, the early cardiac modifications that occur in CKD that may trigger cardiac remodeling and dysfunction remain largely unexplored. Here, in a mouse model of CKD induced by 5/6 nephrectomy, we first analyzed the early transcriptional and inflammatory changes that occur in the heart. Five days after 5/6 nephrectomy, RNA-sequencing showed the upregulation of 54 genes in the cardiac tissue of CKD mice and the enrichment of biological processes related to immune system processes. Increased cardiac infiltration of T-CD4+ lymphocytes, myeloid cells, and macrophages during early CKD was observed. Next, since CC chemokine ligand-8 (CCL8) was one of the most upregulated genes in the heart of mice with early CKD, we investigated the effect of acute and transient CCL8 inhibition on uremic cardiomyopathy severity. An increase in CCL8 protein levels was confirmed in the heart of early CKD mice. CCL8 inhibition attenuated the early infiltration of T-CD4+ lymphocytes and macrophages to the cardiac tissue, leading to a protection against chronic cardiac fibrotic remodeling, inflammation and cardiac dysfunction induced by CKD. Altogether, our data show the occurrence of transcriptional and inflammatory changes in the heart during the early phases of CKD and identify CCL8 as a key contributor to the early cardiac inflammatory state that triggers further cardiac remodeling and dysfunction in uremic cardiomyopathy.

Reduced endothelial nitric oxide synthase activation contributes to cardiovascular injury during chronic kidney disease progression.

Major cardiovascular events are a common complication in patients with chronic kidney disease (CKD). Endothelial dysfunction can contribute to the cardiovascular injury observed in CKD. Here, we used a rat model of acute kidney injury to CKD transition to investigate heart alterations in the pathway activating endothelial nitric oxide synthase (eNOS) and its impact on the cardiac injury observed during CKD progression. Fifty male Wistar rats were subjected to sham surgery (n = 25) or bilateral renal ischemia-reperfusion (IR-CKD) for 45 min (n = 25). Rats were studied on a monthly basis up to 5 mo (n = 5). In another set of sham and IR-CKD rats, l-arginine was administered starting on the third month after renal ischemia. CKD development and cardiac alterations were monitored in all groups. CKD was characterized by a progressive increase in proteinuria and renal dysfunction that was evident after the fifth month of followup. Heart hypertrophy was observed starting on the fourth month after ischemia-reperfusion. There was a significant increase in brain natriuretic peptide levels. In the heart, IR-CKD rats had increased eNOS phosphorylation at threonine 495 and reduced eNOS-heat shock protein-90α interactions. l-Arginine administration prevented the heart alterations observed during CKD and increased eNOS coupling/dimerization and activation. In summary, CKD progression is accompanied by cardiac hypertrophy, fibrosis, oxidative stress, and increased brain natriuretic peptide levels. These alterations were associated with limited eNOS activation in the heart, which may result in reduced nitric oxide bioavailability and contribute to cardiac injury during CKD.

Delayed spironolactone administration prevents the transition from acute kidney injury to chronic kidney disease through improving renal inflammation.

BACKGROUND: Acute kidney injury (AKI) is not as harmless as previously thought since it may lead to chronic kidney disease (CKD). Because most of the time ischemic AKI occurs unexpectedly, it is difficult to prevent its occurrence and there are no specific therapeutic approaches to prevent the AKI to CKD transition. We aimed to determine whether mineralocorticoid receptor blockade (MRB) in the first days after ischemia/reperfusion (IR) can prevent progression to CKD. METHODS: Four groups of male Wistar rats were included: sham and three groups of bilateral renal ischemia for 45 min, one without treatment and the other two receiving spironolactone for 5 or 10 days, starting 24 h after IR. The rats were studied at 10 days or 5 months after ischemia induction. RESULTS: After 5 months of follow-up, the untreated group exhibited clear evidence of AKI to CKD progression, such as proteinuria, reduced renal blood flow, tubulointerstitial fibrosis, glomerulosclerosis and glomerular hypertrophy. All these alterations were prevented by both spironolactone treatments initiated 24 h after IR, the 10-day treatment being more effective. Within the early mechanisms of the MRB protective effect are the reduction of inflammation and increased endothelin-B-receptor expression and endothelial nitric oxide synthase activation in the first 10 days after IR. CONCLUSIONS: We propose that MRB, administered 24 h after the ischemic injury that leads to AKI, reduces inflammation and promotes efficient tissue repair that avoids the AKI to CKD transition. These data highlight a therapeutic window to preclude CKD development after AKI.

Intra-renal transfection of heat shock protein 90 alpha or beta (Hsp90α or Hsp90ß) protects against ischemia/reperfusion injury.

BACKGROUND: We previously reported that radicicol (Hsp90 inhibitor) induced a reduction in the renal blood flow and glomerular filtration rate, in part due to a reduction in urinary NO2/NO3 excretion, suggesting that Hsp90 regulates renal vascular tone in physiological conditions. However, there is a lack of information concerning Hsp90α or Hsp90ß role on eNOS activity and their association with acute kidney injury (AKI) characterized by an inadequate NO production. This study evaluated the effects of Hsp90α or Hsp90ß intra-renal transfection under ischemia/reperfusion (IR) injury. METHODS: Uninephrectomized (Nx) rats were intra-renally transfected through injections with Hsp90α or Hsp90ß cloned into pcDNA3.1(+) or empty vector (EV) at 48 h before inducing IR, as indicated in the following groups: (i) Nx+sham, (ii) Nx+IR, (iii) Nx+IR+EV, (iv) Nx+IR+Hsp90α and (v) Nx+IR+Hsp90ß. After 24 h, physiological, histopathological, biochemical and molecular studies were performed. RESULTS: IR-induced renal dysfunction, structural injury, tubular proliferation, the elevation of urinary Hsp72 and the reduction of urinary NO2/NO3 excretion. These alterations were associated with reduced eNOS-Hsp90 coupling and changes in the eNOS phosphorylation state mediated through a reduction in PKCα and increased Rho kinase expression. In contrast, intra-renal transfection of Hsp90α or Hsp90ß prevented IR injury that was associated with the restoration of eNOS-Hsp90 coupling, eNOS activating phosphorylation and PKCα and Rho kinase levels. CONCLUSIONS: Here we showed that eNOS-Hsp90 uncoupling plays a critical role in promoting NO reduction during IR. This effect was effectively reversed through Hsp90α or Hsp90ß intra-renal transfection, suggesting their implication in regulating NO/eNOS pathway and the renal vascular tone.

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family.

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.

Hsp72 is an early and sensitive biomarker to detect acute kidney injury.

This study was designed to assess whether heat shock protein Hsp72 is an early and sensitive biomarker of acute kidney injury (AKI) as well as to monitor a renoprotective strategy. Seventy-two Wistar rats were divided into six groups: sham-operated and rats subjected to 10, 20, 30, 45 and 60 min of bilateral ischemia (I) and 24 h of reperfusion (R). Different times of reperfusion (3, 6, 9, 12, 18, 24, 48, 72, 96 and 120 h) were also evaluated in 30 other rats subjected to 30 min of ischemia. Hsp72 messenger RNA (mRNA) and protein levels were determined in both kidney and urine. Hsp72-specificity as a biomarker to assess the success of a renoprotective intervention was evaluated in rats treated with different doses of spironolactone before I/R. Renal Hsp72 mRNA and protein, as well as urinary Hsp72 levels, gradually increased relative to the extent of renal injury induced by different periods of ischemia quantified by histomorphometry as a benchmark of kidney damage. Urinary Hsp72 increased significantly after 3 h and continued rising until 18 h, followed by restoration after 120 h of reperfusion in accord with histopathological findings. Spironolactone renoprotection was associated with normalization of urinary Hsp72 levels. Accordingly, urinary Hsp72 was significantly increased in patients with clinical AKI before serum creatinine elevation. Our results show that urinary Hsp72 is a useful biomarker for early detection and stratification of AKI. In addition, urinary Hsp72 levels are sensitive enough to monitor therapeutic interventions and the degree of tubular recovery following an I/R insult.

Opposite effect of Hsp90α and Hsp90ß on eNOS ability to produce nitric oxide or superoxide anion in human embryonic kidney cells.

Heat shock protein 90 subfamily is composed by two cytosolic isoforms known as Hsp90α and Hsp90ß. Endothelial nitric oxide synthase (eNOS) is regulated by Hsp90, however the specific role of each Hsp90 isoform on NO production has not been established. This study was designed to evaluate the effect of Hsp90α and Hsp90ß over-expression on eNOS/NO pathway. Rat Hsp90α and Hsp90ß were cloned into pcDNA3.1(+) and transfected in human embryonic kidney cells (HEK-293). Hsp90α and Hsp90ß transfection was corroborated by Western blot analysis and their effect on NO production (NO(2)/NO(3)), eNOS protein and its phosphorylation at Ser1177 and Thr495, as well as Akt/PKB Ser473 phosphorylation was determined. The interaction of Hsp90α and Hsp90ß with eNOS and the dimer/monomer ratio of Hsp90, as well as O(2)(-) generation were also assessed. After transfection, Hsp90α and Hsp90ß levels were significantly increased in HEK-293 cells. The Hsp90α over-expression induced a significant increase in NO(2)/NO(3) levels, an effect that was associated with increased phosphorylation of eNOS Ser 1177 and Akt/PKB Ser473, as well as with a greater Hsp90α dimerization. Noteworthy, pcHsp90ß transfection reduced significantly NO(2)/NO(3) and increased O(2)(-) generation. These effects were associated with a reduction of eNOS dimeric conformation, increased eNOS Thr495 phosphorylation, reduced Akt/PKB phosphorylation, and by a greater amount of monomeric Hsp90ß conformation. These data show for first time that Hsp90α and Hsp90ß differentially modulate NO and O(2)(-) generation by eNOS through promoting changes in eNOS conformation and phosphorylation state.

Effects of losartan pretreatment in an experimental model of ischemic acute kidney injury.

BACKGROUND/AIMS: Contributions to the understanding of acute renal failure (ARF) pathogenesis have not been translated into an effective clinical therapy. We studied the effects of pretreatment with the angiotensin II type 1 (AT1) receptor blocker, losartan, on renal function, tissue injury, inflammatory response and serum aldosterone levels in a model of ischemic ARF. METHODS: Rats underwent unilateral renal ischemia followed by 24 h of reperfusion (IR), and were pretreated or not with 8 (IRL8) or 80 (IRL80) mg/kg/day of losartan for 3 days. RESULTS: IR kidneys showed marked renal dysfunction, epithelial damage, capillary congestion, increased myeloperoxidase (MPO) activity and increased TNF-alpha, IL1-beta and IL-6 mRNA levels. IRL80 kidneys showed protection against dysfunction and tissue injury, associated with normal MPO activity and cytokine mRNA levels. The lower dose was not able to achieve the same degree of functional renoprotection and could not prevent an increase of MPO or proinflammatory cytokine mRNA levels. The high losartan dose completely prevented an increase of serum aldosterone levels induced by IR. CONCLUSION: Renoprotection of the high losartan dose would be mainly mediated by its anti-inflammatory actions. Our results show a potential pathophysiological role of AT1 activation in promoting renal dysfunction, structural injury, inflammation and aldosterone elevation after IR injury.