Simone Costa Alarcon Arias

Regression of Albuminuria and Hypertension and Arrest of Severe Renal Injury by a Losartan-Hydrochlorothiazide Association in a Model of Very Advanced Nephropathy

Treatments that effectively prevent chronic kidney disease (CKD) when initiated early often yield disappointing results when started at more advanced phases. We examined the long-term evolution of renal injury in the 5/6 nephrectomy model (Nx) and the effect of an association between an AT-1 receptor blocker, losartan (L), and hydrochlorothiazide (H), shown previously to be effective when started one month after Nx. Adult male Munich-Wistar rats underwent Nx, being divided into four groups: Nx+V, no treatment; Nx+L, receiving L monotherapy; Nx+LH, receiving the L+H association (LH), and Nx+AHHz, treated with the calcium channel blocker, amlodipine, the vascular relaxant, hydralazine, and H. This latter group served to assess the effect of lowering blood pressure (BP). Rats undergoing sham nephrectomy (S) were also studied. In a first protocol, treatments were initiated 60 days after Nx, when CKD is at a relatively early stage. In a second protocol, treatments were started 120 days after Nx, when glomerulosclerosis and interstitial fibrosis are already advanced. In both protocols, L treatment promoted only partial renoprotection, whereas LH brought BP, albuminuria, tubulointerstitial cell proliferation and plasma aldosterone below pretreatment levels, and completely detained progression of renal injury. Despite normalizing BP, the AHHz association failed to prevent renal damage, indicating that the renoprotective effect of LH was not due to a systemic hemodynamic action. These findings are inconsistent with the contention that thiazides are innocuous in advanced CKD. In Nx, LH promotes effective renoprotection even at advanced stages by mechanisms that may involve anti-inflammatory and intrarenal hemodynamic effects, but seem not to require BP normalization.

NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy

Adenine overload promotes intratubular crystal precipitation and interstitial nephritis. We showed recently that these abnormalities are strongly attenuated in mice knockout for Toll-like receptors-2, -4, MyD88, ASC, or caspase-1. We now investigated whether NF-κB activation also plays a pathogenic role in this model. Adult male Munich-Wistar rats were distributed among three groups: C (n = 17), receiving standard chow; ADE (n = 17), given adenine in the chow at 0.7% for 1 wk and 0.5% for 2 wk; and ADE + pyrrolidine dithiocarbamate (PDTC; n = 14), receiving adenine as above and the NF-κB inhibitor PDTC (120 mg·kg⁻¹·day⁻¹ in the drinking water). After 3 wk, widespread crystal deposition was seen in tubular lumina and in the renal interstitium, along with granuloma formation, collagen accumulation, intense tubulointerstitial proliferation, and increased interstitial expression of inflammatory mediators. Part of the crystals were segregated from tubular lumina by a newly formed cell layer and, at more advanced stages, appeared to be extruded to the interstitium. p65 nuclear translocation and IKK-α increased abundance indicated activation of the NF-κB system. PDTC treatment prevented p65 migration and normalized IKK-α, limited crystal shift to the interstitium, and strongly attenuated interstitial fibrosis/inflammation. These findings indicate that the complex inflammatory phenomena associated with this model depend, at least in part, on NF-κB activation, and suggest that the NF-κB system may become a therapeutic target in the treatment of chronic kidney disease.

Chronic VEGF Blockade Worsens Glomerular Injury in the Remnant Kidney Model

VEGF inhibition can promote renal vascular and parenchymal injury, causing proteinuria, hypertension and thrombotic microangiopathy. The mechanisms underlying these side effects are unclear. We investigated the renal effects of the administration, during 45 days, of sunitinib (Su), a VEGF receptor inhibitor, to rats with 5/6 renal ablation (Nx). Adult male Munich-Wistar rats were distributed among groups S+V, sham-operated rats receiving vehicle only; S+Su, S rats given Su, 4 mg/kg/day; Nx+V, Nx rats receiving V; and Nx+Su, Nx rats receiving Su. Su caused no change in Group S. Seven and 45 days after renal ablation, renal cortical interstitium was expanded, in association with rarefaction of peritubular capillaries. Su did not worsen hypertension, proteinuria or interstitial expansion, nor did it affect capillary rarefaction, suggesting little angiogenic activity in this model. Nx animals exhibited glomerulosclerosis (GS), which was aggravated by Su. This effect could not be explained by podocyte damage, nor could it be ascribed to tuft hypertrophy or hyperplasia. GS may have derived from organization of capillary microthrombi, frequently observed in Group Nx+Su. Treatment with Su did not reduce the fractional glomerular endothelial area, suggesting functional rather than structural cell injury. Chronic VEGF inhibition has little effect on normal rats, but can affect glomerular endothelium when renal damage is already present.

An association of losartan-hydrochlorothiazide, but not losartan-furosemide, completely arrests progressive injury in the remnant kidney.

We have previously shown that an association of losartan and hydrochlorothiazide, initiated 1 mo after 5/6 nephrectomy (Nx), reversed hypertension and albuminuria and promoted lasting renoprotection. In this new study, we investigated whether equal or even better protection could be obtained by combining losartan and furosemide. Nx was performed in 58 Munich-Wistar rats. One month later, tail-cuff pressure and albuminuria were markedly elevated. At this time, Nx rats were distributed among the following four groups: untreated Nx rats, Nx rats that received losartan, Nx rats that received losartan + hydrochlorothiazide, and Nx rats that received losartan + furosemide. Seven months later, Nx rats exhibited high mortality, severe hypertension, albuminuria, glomerulosclerosis, and interstitial fibrosis. Losartan treatment limited mortality and attenuated the renal and hemodynamic abnormalities associated with Nx. As previously shown, the losartan + hydrochlorothiazide association normalized tail-cuff pressure and albumin, prevented renal injury, and reduced mortality to zero. The losartan + furosemide treatment failed to reduce tail-cuff pressure or albumin to normal and prevented renal injury less efficiently than the losartan and hydrochlorothiazide regimen. The reasons for the differing efficacies of the losartan + furosemide and losartan + hydrochlorothiazide schemes are unclear and may include beneficial nondiuretic actions of thiazides, such as vasorelaxation and antiproliferative activity. These results refute the established concept that thiazides and thiazide-like diuretics are ineffective at advanced chronic kidney disease stages. Rather, they suggest that, in view of their renoprotective action, these compounds may even be preferable to loop diuretics in the management of hypertension in advanced chronic kidney disease.

NLRP3 inflammasome inhibition ameliorates tubulointerstitial injury in the remnant kidney model

Recent studies suggest that NLRP3 inflammasome activation is involved in the pathogenesis of chronic kidney disease (CKD). Allopurinol (ALLO) inhibits xanthine oxidase (XOD) activity, and, consequently, reduces the production of uric acid (UA) and reactive oxygen species (ROS), both of which can activate the NLRP3 pathway. Thus, ALLO can contribute to slow the progression of CKD. We investigated whether inhibition of XOD by ALLO reduces NLRP3 activation and renal injury in the 5/6 renal ablation (Nx) model. Adult male Munich–Wistar rats underwent Nx and were subdivided into the following two groups: Nx, receiving vehicle only, and Nx + ALLO, Nx rats given ALLO, 36 mg/Kg/day in drinking water. Rats undergoing sham operation were studied as controls (C). Sixty days after surgery, Nx rats exhibited marked albuminuria, creatinine retention, and hypertension, as well as glomerulosclerosis, tubular injury, and cortical interstitial expansion/inflammation/fibrosis. Such changes were accompanied by increased XOD activity and UA renal levels, associated with augmented heme oxigenase-1 and reduced superoxide dismutase-2 renal contents. Both the NF-κB and NLRP3 signaling pathways were activated in Nx. ALLO normalized both XOD activity and the parameters of oxidative stress. ALLO also attenuated hypertension and promoted selective tubulointerstitial protection, reducing urinary NGAL and cortical interstitial injury/inflammation. ALLO reduced renal NLRP3 activation, without interfering with the NF-κB pathway. These observations indicate that the tubulointerstitial antiinflammatory and antifibrotic effects of ALLO in the Nx model involve inhibition of the NLRP3 pathway, and reinforce the view that ALLO can contribute to arrest or slow the progression of CKD.

Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model

The mechanisms triggering renal inflammation in chronic kidney disease (CKD) are unclear. We performed a detailed analysis of the time course of innate and adaptive immunity activation in the 5/6 renal ablation (Nx) model. Munich-Wistar rats undergoing Nx were studied 15, 60 and 120 days after ablation. Hypertension, albuminuria, creatinine retention, interstitial expansion and infiltration by macrophages and T-lymphocytes were already evident 15 days after Nx. PCR-array was used to screen for altered gene expression, whereas gene and protein expressions of TLR4, CASP1, IL-1β and NLRP3 were individually assessed. Tlr4, Tlr5, Lbp, Nlrp3, Casp1, Irf7 and Il1b were already upregulated 15 days after Nx, while activation of Tlr2, Tlr7, Tlr9, Nod2, Tnf and Il6 was seen after 60 days post-ablation. The number of genes related to innate or adaptive immunity grew steadily with time. These observations indicate that parallel activation of innate and adaptive immunity antecedes glomerular injury and involves a growing number of intricate signaling pathways, helping to explain the difficulty in detaining renal injury in Nx as CKD advances, and, stressing the need for early treatment. Additionally, these findings may contribute to the search of therapeutic targets specific for advanced phases of CKD.

A Novel Aldosterone Antagonist Limits Renal Injury in 5/6 Nephrectomy

Aldosterone antagonists slow the progression of chronic kidney disease (CKD), but their use is limited by hyperkalemia, especially when associated with RAS inhibitors. We examined the renoprotective effects of Ly, a novel non-steroidal mineralocorticoid receptor (MR) blocker, through two experimental protocols: In Protocol 1, male Munich-Wistar rats underwent 5/6 renal ablation (Nx), being divided into: Nx+V, receiving vehicle, Nx+Eple, given eplerenone, 150 mg/kg/day, and Nx+Ly, given Ly, 20 mg/kg/day. A group of untreated sham-operated rats was also studied. Ly markedly raised plasma renin activity (PRA) and aldosterone, and exerted more effective anti-albuminuric and renoprotective action than eplerenone. In Protocol 2, Nx rats remained untreated until Day 60, when they were divided into: Nx+V receiving vehicle; Nx+L treated with losartan, 50 mg/kg/day; Nx+L+Eple, given losartan and eplerenone, and Nx+L+Ly, given losartan and Ly. Treatments lasted for 90 days. As an add-on to losartan, Ly normalized blood pressure and albuminuria, and prevented CKD progression more effectively than eplerenone. This effect was associated with strong stimulation of PRA and aldosterone. Despite exhibiting higher affinity for the MR than either eplerenone or spironolactone, Ly caused no hyperkalemia. Ly may become a novel asset in the effort to detain the progression of CKD.

Resistance training attenuates inflammation and the progression of renal fibrosis in chronic renal disease

&NA; Patients with chronic kidney disease (CKD) have progressive renal fibrosis, inflammation, and reduced muscle mass and strength. Resistance training (RT) has been suggested to mitigate the loss of muscle mass, of strength and the inflammation in CKD, but the mechanisms are unknown. The aim of this study was to evaluate the influence of RT on renal fibrosis, renal cytokine expression, creatine kinase levels, and muscle mass and strength in CKD rats. A CKD model was obtained by 5/6 nephrectomy (Nx). Fifteen 8‐week‐old male rats were divided into 3 groups: Sham (control), Nx SED (CKD sedentary) and Nx RT (CKD trained). The RT consisted of ladder climbing at 70% of the animals maximal carrying capacity for 10 weeks. Muscle strength, creatine kinase levels, renal fibrosis and mRNA interleukin (IL)‐4, IL‐6 and IL‐10 were analyzed after the RT protocol. There was significant improvement in the muscle strength and creatine kinase levels in the Nx RT group. Moreover, renal fibrosis and inflammation were attenuated, with increased IL‐4 and IL‐10 expression and reduced IL‐6 expression in the Nx RT group compared with that in the Nx SED group. No difference in muscle mass was observed among the groups. In conclusion, RT was effective in reducing fibrosis and inflammation, in addition to increasing muscle strength and creatine kinase levels, in rats with CKD, independent of muscle mass.

Simultaneous activation of innate and adaptive immunity participates in the development of renal injury in a model of heavy proteinuria

Protein overload of proximal tubular cells (PTCs) can promote interstitial injury by unclear mechanisms that may involve activation of innate immunity. We investigated whether prolonged exposure of tubular cells to high protein concentrations stimulates innate immunity, triggering progressive interstitial inflammation and renal injury, and whether specific inhibition of innate or adaptive immunity would provide renoprotection in an established model of massive proteinuria, adriamycin nephropathy (ADR). Adult male Munich–Wistar rats received a single dose of ADR (5 mg/kg, iv), being followed for 2, 4, or 20 weeks. Massive albuminuria was associated with early activation of both the NF-κB and NLRP3 innate immunity pathways, whose intensity correlated strongly with the density of lymphocyte infiltration. In addition, ADR rats exhibited clear signs of renal oxidative stress. Twenty weeks after ADR administration, marked interstitial fibrosis, glomerulosclerosis, and renal functional loss were observed. Administration of mycophenolate mofetil (MMF), 10 mg/kg/day, prevented activation of both innate and adaptive immunity, as well as renal oxidative stress and renal fibrosis. Moreover, MMF treatment was associated with shifting of M from the M1 to the M2 phenotype. In cultivated NRK52-E cells, excess albumin increased the protein content of Toll-like receptor (TLR) 4 (TLR4), NLRP3, MCP-1, IL6, IL-1β, Caspase-1, α-actin, and collagen-1. Silencing of TLR4 and/or NLRP3 mRNA abrogated this proinflammatory/profibrotic behavior. Simultaneous activation of innate and adaptive immunity may be key to the development of renal injury in heavy proteinuric disease. Inhibition of specific components of innate and/or adaptive immunity may be the basis for future strategies to prevent chronic kidney disease (CKD) in this setting.