Metformin arrests the progression of established kidney disease in the subtotal nephrectomy model of chronic kidney disease.

Metformin, an AMP-activated protein kinase (AMPK) activator, has been shown in previous studies to reduce kidney fibrosis in different models of experimental chronic kidney disease (CKD). However, in all of these studies, the administration of metformin was initiated before the establishment of renal disease, which is a condition that does not typically occur in clinical settings. The aim of the present study was to investigate whether the administration of metformin could arrest the progression of established renal disease in a well-recognized model of CKD, the subtotal kidney nephrectomy (Nx) model. Adult male Munich-Wistar rats underwent either Nx or sham operations. After the surgery (30 days), Nx rats that had systolic blood pressures of >170 mmHg and albuminuria levels of >40 mg/24 h were randomized to a no-treatment condition or to a treatment condition with metformin (300 mg·kg-1·day-1) for a period of either 60 or 120 days. After 60 days of treatment, we did not observe any differences in kidney disease parameters between Nx metformin-treated and untreated rats. However, after 120 days, Nx rats that had been treated with metformin displayed significant reductions in albuminuria levels and in markers of renal fibrosis. These effects were independent of any other effects on blood pressure or glycemia. In addition, treatment with metformin was also able to activate kidney AMPK and therefore improve mitochondrial biogenesis. It was concluded that metformin can arrest the progression of established kidney disease in the Nx model, likely via the activation of AMPK.

NF-κB blockade during short-term l-NAME and salt overload strongly attenuates the late development of chronic kidney disease.

Nitric oxide synthase inhibition by Nω-nitro-l-arginine methyl ester (l-NAME) plus a high-salt diet (HS) is a model of chronic kidney disease (CKD) characterized by marked hypertension and renal injury. With cessation of treatment, most of these changes subside, but progressive renal injury develops, associated with persistent low-grade renal inflammation. We investigated whether innate immunity, and in particular the NF-κB system, is involved in this process. Male Munich-Wistar rats received HS + l-NAME (32 mg·kg-1·day-1), whereas control rats received HS only. Treatment was ceased after week 4 when 30 rats were studied. Additional rats were studied at week 8 (n = 30) and week 28 (n = 30). As expected, HS + l-NAME promoted severe hypertension, albuminuria, and renal injury after 4 wk of treatment, whereas innate immunity activation was evident. After discontinuation of treatments, partial regression of renal injury and inflammation occurred, along with persistence of innate immunity activation at week 8. At week 28, glomerular injury worsened, while renal inflammation persisted and renal innate immunity remained activated. Temporary administration of the NF-κB inhibitor pyrrolidine dithiocarbamate, in concomitancy with the early 4-wk HS + l-NAME treatment, prevented the development of late renal injury and inflammation, an effect that lasted until the end of the study. Early activation of innate immunity may be crucial to the initiation of renal injury in the HS + l-NAME model and to the autonomous progression of chronic nephropathy even after cessation of the original insult. This behavior may be common to other conditions leading to CKD.

Chronic exposure to hypoxia attenuates renal injury and innate immunity activation in the remnant kidney model.

Hypoxia is thought to influence the pathogenesis of chronic kidney disease, but direct evidence that prolonged exposure to tissue hypoxia initiates or aggravates chronic kidney disease is lacking. We tested this hypothesis by chronically exposing normal rats and rats with 5/6 nephrectomy (Nx) to hypoxia. In addition, we investigated whether such effect of hypoxia would involve activation of innate immunity. Adult male Munich-Wistar rats underwent Nx (n = 54) or sham surgery (sham; n = 52). Twenty-six sham rats and 26 Nx rats remained in normoxia, whereas 26 sham rats and 28 Nx rats were kept in a normobaric hypoxia chamber (12% O2) for 8 wk. Hypoxia was confirmed by immunohistochemistry for pimonidazole. Hypoxia was confined to the medullary area in sham + normoxia rats and spread to the cortical area in sham + hypoxia rats, without changing the peritubular capillary density. Exposure to hypoxia promoted no renal injury or elevation of the content of IL-1ß or Toll-like receptor 4 in sham rats. In Nx, hypoxia also extended to the cortical area without ameliorating the peritubular capillary rarefaction but, unexpectedly, attenuated hypertension, inflammation, innate immunity activation, renal injury, and oxidative stress. The present study, in disagreement with current concepts, shows evidence that hypoxia exerts a renoprotective effect in the Nx model instead of acting as a factor of renal injury. The mechanisms for this unexpected beneficial effect are unclear and may involve NF-κB inhibition, amelioration of oxidative stress, and limitation of angiotensin II production by the renal tissue.

Pathogenic role of innate immunity in a model of chronic NO inhibition associated with salt overload.

Nitric oxide inhibition with Nω-nitro-l-arginine methyl ester (l-NAME), along with salt overload, leads to hypertension, albuminuria, glomerulosclerosis, glomerular ischemia, and interstitial fibrosis, characterizing a chronic kidney disease (CKD) model. Previous findings of this laboratory and elsewhere have suggested that activation of at least two pathways of innate immunity, Toll-like receptor 4 (TLR4)/NF-κB and nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome/IL-1ß, occurs in several experimental models of CKD and that progression of renal injury can be slowed with inhibition of these pathways. In the present study, we investigated whether activation of innate immunity, through either the TLR4/NF-κB or NLRP3/IL-1ß pathway, is involved in the pathogenesis of renal injury in chronic nitric oxide inhibition with the salt-overload model. Adult male Munich-Wistar rats that received l-NAME in drinking water with salt overload (HS + N group) were treated with allopurinol (ALLO) as an NLRP3 inhibitor (HS + N + ALLO group) or pyrrolidine dithiocarbamate (PDTC) as an NF-κB inhibitor (HS + N + PDTC group). After 4 wk, HS + N rats developed hypertension, albuminuria, and renal injury along with renal inflammation, oxidative stress, and activation of both the NLRP3/IL-1ß and TLR4/NF-κB pathways. ALLO lowered renal uric acid and inhibited the NLRP3 pathway. These effects were associated with amelioration of hypertension, albuminuria, and interstitial inflammation/fibrosis but not glomerular injury. PDTC inhibited the renal NF-κB system and lowered the number of interstitial cells staining positively for NLRP3. PDTC also reduced renal xanthine oxidase activity and uric acid. Overall, PDTC promoted a more efficient anti-inflammatory and nephroprotective effect than ALLO. The NLRP3/IL-1ß and TLR4/NF-κB pathways act in parallel to promote renal injury/inflammation and must be simultaneously inhibited for best nephroprotection.

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.

TLR2 and TLR4 play opposite role in autophagy associated with cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is considered an inflammatory disease in which toll-like receptors (TLRs) signaling pathways play an important role. The activation of TLRs results in production of several inflammatory cytokines leading to further renal damage. In contrast, TLRs are key players on autophagy induction, which is associated with a protective function on cisplatin-induced AKI. Hence, the present study aimed to evaluate the specific participation of TLR2 and TLR4 molecules on the development of cisplatin-induced AKI. Complementarily, we also investigated the link between TLRs and heme oxygenase-1 (HO-1), a promisor cytoprotective molecule. First, we observed that only the absence of TLR2 but not TLR4 in mice exacerbated the renal dysfunction, tissue injury and mortality rate, even under an immunologically privileged microenvironment. Second, we demonstrated that TLR2 knockout (KO) mice presented lower expression of autophagy-associated markers when compared with TLR4 KO animals. Similar parameter was confirmed in vitro, using tubular epithelial cells derived from both KO mice. To test the cross-talking between HO-1 and TLRs, hemin (an HO-1 internal inducer) was administrated in cisplatin-treated TLR2 and TLR4 KO mice and it was detected an improvement in the global renal tissue parameters. However, this protection was less evident at TLR2 KO mice. In summary, we documented that TLR2 plays a protective role in cisplatin-induced AKI progression, in part, by a mechanism associated with autophagy up-regulation, considering that its interplay with HO-1 can promote renal tissue recover.

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.

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.

The Protein-Independent Role of Phosphate in the Progression of Chronic Kidney Disease.

Several factors contribute to renal-function decline in CKD patients, and the role of phosphate content in the diet is still a matter of debate. This study aims to analyze the mechanism by which phosphate, independent of protein, is associated with the progression of CKD. Adult Munich-Wistar rats were submitted to 5/6 nephrectomy (Nx), fed with a low-protein diet, and divided into two groups. Only phosphate content (low phosphate, LoP, 0.2%; high phosphate, HiP, 0.95%) differentiated diets. After sixty days, biochemical parameters and kidney histology were analyzed. The HiP group presented worse renal function, with higher levels of PTH, FGF-23, and fractional excretion of phosphate. In the histological analysis of the kidney tissue, they also showed a higher percentage of interstitial fibrosis, expression of α-actin, PCNA, and renal infiltration by macrophages. The LoP group presented higher expression of beclin-1 in renal tubule cells, a marker of autophagic flux, when compared to the HiP group. Our findings highlight the action of phosphate in the induction of kidney interstitial inflammation and fibrosis, contributing to the progression of renal disease. A possible effect of phosphate on the dysregulation of the renal cell autophagy mechanism needs further investigation with clinical studies.

Renal Inflammation and Innate Immune Activation Underlie the Transition From Gentamicin-Induced Acute Kidney Injury to Renal Fibrosis.

Subjects recovering from acute kidney injury (AKI) are at risk of developing chronic kidney disease (CKD). The mechanisms underlying this transition are unclear and may involve sustained activation of renal innate immunity, with resulting renal inflammation and fibrosis. We investigated whether the NF-κB system and/or the NLRP3 inflammasome pathway remain activated after the resolution of AKI induced by gentamicin (GT) treatment, thus favoring the development of CKD. Male Munich-Wistar rats received daily subcutaneous injections of GT, 80 mg/kg, for 9 days. Control rats received vehicle only (NC). Rats were studied at 1, 30, and 180 days after GT treatment was ceased. On Day 1, glomerular ischemia (ISCH), tubular necrosis, albuminuria, creatinine retention, and tubular dysfunction were noted, in association with prominent renal infiltration by macrophages and myofibroblasts, along with increased renal abundance of TLR4, IL-6, and IL1ß. Regression of functional and structural changes occurred on Day 30. However, the renal content of IL-1ß was still elevated at this time, while the local renin-angiotensin system remained activated, and interstitial fibrosis became evident. On Day 180, recurring albuminuria and mild glomerulosclerosis were seen, along with ISCH and unabated interstitial fibrosis, whereas macrophage infiltration was still evident. GT-induced AKI activates innate immunity and promotes renal inflammation. Persistence of these abnormalities provides a plausible explanation for the transition of AKI to CKD observed in a growing number of patients.

Early brief treatment with losartan plus mycophenolate mofetil provides lasting renoprotection in a renal ablation model.

BACKGROUND: Inflammatory events antecede established renal injury in rats with 5/6 renal ablation (Nx), as indicated by the beneficial effects of early, uninterrupted treatment with mycophenolate mofetil (MMF). Angiotensin II also exerts a major pathogenic role at this initial phase. We investigated whether losartan (L) or L+MMF treatment, started early, and L+MMF treatment, started late, would exert lasting renoprotection in Nx even after being discontinued. METHODS: Adult male Munich-Wistar rats underwent Nx and were divided into three groups: Nx (untreated), Nx(L) (given L), and Nx(LMMF) (given L and MMF). Protocol 1: treatments began on day 1, and ceased on day 30, after Nx. Protocol 2: L+MMF treatment began on day 30 and ceased on day 60. RESULTS: Protocol 1: on day 30, hypertension, albuminuria and renal injury were strongly attenuated in Groups Nx(L) and Nx(LMMF). On day 120, these abnormalities were still attenuated in group Nx(LMMF). Protocol 2: on day 120, all parameters were similar between this late Nx(LMMF) group and untreated Nx. CONCLUSION: In Nx, temporary suppression of early, transitory hemodynamic/inflammatory phenomena affords relatively durable renoprotection even after treatment discontinuation. This effect is not obtained with similar temporary treatment initiated later in the course of renal disease.

NF-κB System Is Chronically Activated and Promotes Glomerular Injury in Experimental Type 1 Diabetic Kidney Disease.

High glucose concentration can activate TLR4 and NF-κB, triggering the production of proinflammatory mediators. We investigated whether the NF-κB pathway is involved in the pathogenesis and progression of experimental diabetic kidney disease (DKD) in a model of long-term type 1 diabetes mellitus (DM). Adult male Munich-Wistar rats underwent DM by a single streptozotocin injection, and were kept moderately hyperglycemic by daily insulin injections. After 12 months, two subgroups - progressors and non-progressors - could be formed based on the degree of glomerulosclerosis. Only progressors exhibited renal TLR4, NF-κB and IL-6 activation. This scenario was already present in rats with short-term DM (2 months), at a time when no overt glomerulosclerosis can be detected. Chronic treatment with the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), prevented activation of renal TLR4, NF-κB or IL-6, without interfering with blood glucose. PDTC prevented the development of glomerular injury/inflammation and oxidative stress in DM rats. In addition, the NF-κB p65 component was detected in sclerotic glomeruli and inflamed interstitial areas in biopsy material from patients with type 1 DM. These observations indicate that the renal NF-κB pathway plays a key role in the development and progression of experimental DKD, and can become an important therapeutic target in the quest to prevent the progression of human DKD.

l-Arginine supplementation blunts resistance exercise improvement in rats with chronic kidney disease.

Chronic kidney disease (CKD) patients present L-arginine (L-arg) deficiency and L-arg supplementation has been used as a treatment. In addition, sarcopenia is another common problem in CKD population, resistance training (RT) is one of the conservative strategies developed to prevent CKD progression, and however there are no evidences of a combination of these two strategies to treat CKD outcomes. The aim of this study was to evaluate the effects of oral L-arg supplementation combined with RT in an experimental model of CKD. Twenty-five Munich-Wistar male rats, 8-week-old were divided in 5 groups: Sham (sedentary control), Nx (CKD sedentary), Nx L-arg (CKD sedentary supplemented with 2% of L-arg), Nx RT (CKD exercised) Nx RT + L-arg (CKD exercised and supplemented with 2% of L-arg). CKD model was obtained by a subtotal 5/6 nephrectomy. RT was performed on a ladder climbing, three weekly sessions on non-consecutive days, with an intensity of 70% maximum carrying capacity. They were submitted to RT and/or L-arg supplementation for 10 weeks. There was a significant improvement in muscle strength, renal function, anti-inflammatory cytokines, arginase metabolism and renal fibrosis after RT. However, the combination of RT and L-arg impaired all the improvements promoted by RT alone. The L-arg supplementation alone did not impair renal fibrosis and renal function. In conclusion, RT improved inflammatory balance, muscle strength, renal function and consequently decreased renal fibrosis. Nevertheless, the association with L-arg supplementation prevented all these effects promoted by RT.

Demonstration of epithelial-mesenchymal transition in kidney. The contribution of a coupled histochemical and immunohistochemical staining with Periodic Acid-thionin Schiff.

Traditional Periodic Acid Schiff has been extensively used, coupled with immunohistochemistry for epithelia or mesenchymal cells, to highlight renal tubular basement membrane (TBM). We recently tried to perform such technique in a 5/6 nephrectomy model of progressive renal fibrosis to demonstrate TBM disruption as an evidence for epithelial-mesenchymal transdifferentiation. Despite excellent basement membrane staining with traditional fuchsin-Periodic Acid Schiff, the interface between epithelial and mesenchymal cells was frequently blurred when revealed with 3'3 diaminobenzidine tetrachloride-peroxidase. Also, it was inadequate when revealed with alkaline phosphatase-fast red. We devised a triple staining method with Periodic Acid-Thionin Schiff to highlight basement membrane in blue, after double immunostaining for epithelium and mesenchymal cells. Blue basement membrane rendered a brisk contrast and highlighted boundaries between epithelial-mesenchymal interfaces. This method was easy to perform and useful to demonstrate the TBM, yield a clear demonstration of the very focal TBM disruption found in this model of progressive renal fibrosis.

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

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 animal's 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-1ß, 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.

Persistent hypertension and progressive renal injury induced by salt overload after short term nitric oxide inhibition.

INTRODUCTION: Administration of the NO inhibitor N(wdelta)-nitro-L-arginine methyl ester (NAME) and a high-salt diet (HS) promotes severe albuminuria and renal injury, which regresses upon discontinuation of treatments. OBJECTIVE: We investigated whether these changes reappear after reinstitution of HS, and whether they are prevented by treatment with the antilymphocyte agent mycophenolate mofetil (MMF) or the AT-1 receptor blocker losartan (L). Adult male Munich-Wistar rats received NAME and HS. A control Group (C) received only HS. After 20 days, rats receiving HS and NAME exhibited severe hypertension and albuminuria. After a 30-day recovery period, hypertension was attenuated and albuminuria had virtually disappeared. MATERIAL AND METHODS: Rats were then distributed among the following groups: HS, receiving HS; NS, receiving a normal salt (NS) diet; HS-MMF, receiving HS and MMF; HS-LOS, receiving HS and L; HS-HDZ, receiving HS and hydralazine (HDZ). Sixty days later, NS rats showed only slight albuminuria and renal damage or inflammation. In contrast, HS rats developed severe hypertension, marked glomerulosclerosis with interstitial expansion and renal infiltration by macrophages and angiotensin II-positive cells. The group treated with losartan had lowered blood pressure and a lack of albuminuria or renal injury. MMF provided similar protection without altering blood pressure, suggesting a nonhemodynamic effect, a hypothesis reinforced by the finding that HDZ lowered blood pressure without preventing renal injury. RESULTS: These results indicate that treatment with HS and NAME predisposes to the development of hypertension and renal injury upon salt overload, characterizing a new model of chronic nephropathy. CONCLUSION: The response to MMF or L, but not HDZ, suggests a key role for inflammatory rather than hemodynamic factors.

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats.

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.

Mechanisms of progressive renal disease: role of angiotensin II, cyclooxygenase products and nitric oxide.

There is mounting evidence that a number of inflammatory mechanisms play a crucial role in the pathogenesis of both immune- and non-immune-mediated glomerulopathies. These mechanisms include T lymphocyte activation, macrophage infiltration and the expression of several cytokines, growth factors and adhesion molecules. Inflammation may also be strongly influenced by three, until recently, unsuspected mediators: angiotensin II, cyclooxygenase derivatives and nitric oxide. Angiotensin II exerts several biological actions that are completely unrelated to its well-known haemodynamic effects, and can mediate cell proliferation, renal fibrosis and the synthesis of other proinflammatory compounds. Cyclooxygenase products have been long associated with non-renal inflammatory phenomena such as arthritis, and could mediate several steps of chronic renal inflammation. Although nitric oxide is generally regarded as a physiological vasodilator with irreplaceable homeostatic effects, it is possible that it participates in the pathogenesis of progressive nephropathies. Treatment with antagonists of these three compounds, alone or in combination, may represent a valuable therapeutic tool in the struggle to arrest or attenuate progressive renal disease.