Assessment of the progressive nature of cell damage in the pilocarpine model of epilepsy

Pilocarpine-induced (320 mg/kg, ip) status epilepticus (SE) in adult (2-3 months) male Wistar rats results in extensive neuronal damage in limbic structures. Here we investigated whether the induction of a second SE (N = 6) would generate damage and cell loss similar to that seen after a first SE (N = 9). Counts of silver-stained (indicative of cell damage) cells, using the Gallyas argyrophil III method, revealed a markedly lower neuronal injury in animals submitted to re-induction of SE compared to rats exposed to a single episode of pilocarpine-induced SE. This effect could be explained as follows: 1) the first SE removes the vulnerable cells, leaving behind resistant cells that are not affected by the second SE; 2) the first SE confers increased resistance to the remaining cells, analogous to the process of ischemic tolerance. Counting of Nissl-stained cells was performed to differentiate between these alternative mechanisms. Our data indicate that different neuronal populations react differently to SE induction. For some brain areas most, if not all, of the vulnerable cells are lost after an initial insult leaving only relatively resistant cells and little space for further damage or cell loss. For some other brain areas, in contrast, our data support the hypothesis that surviving cells might be modified by the initial insult which would confer a sort of excitotoxic tolerance. As a consequence of both mechanisms, subsequent insults after an initial insult result in very little damage regardless of their intensity.

An extract of Polygonum multiflorum protects against free radical damage induced by ultraviolet B irradiation of the skin

Over the last decades, the incidence of ultraviolet B (UVB)-related skin problems has been increasing. Damages induced by UVB radiation are related to mutations that occur as a result of direct DNA damage and/or the production of reactive oxygen species. We investigated the anti-oxidant effects of a Polygonum multiflorum thumb extract against skin damage induced by UVB irradiation. Female SKH-1 hairless mice were divided into three groups: control (N = 7), distilled water- (N = 10), and P. multiflorum extract-treated (PM, N = 10) groups. The PM (10 g) was extracted with 100 mL distilled water, cryo-dried and 9.8 g was obtained. The animals received a topical application of 500 µL distilled water or PM extract (1, 2, 4, 8, and 16%, w/v, dissolved in distilled water) for 30 min after UVB irradiation (wavelength 280-320 nm, 300 mJ/cm²; 3 min) of the dorsal kin for 14 days, and skin immunohistochemistry and Cu,Zn-superoxide dismutase (SOD1) activity were determined. SOD1 immunoreactivity, its protein levels and activities in the skin were significantly reduced by 70% in the distilled water-treated group after UVB irradiation compared to control. However, in the PM extract-treated groups, SOD1 immunoreactivity and its protein and activity levels increased in a dose-dependent manner (1-16%, w/v, PM extract) compared to the distilled water-treated group. SOD1 protein levels and activities in the groups treated with 8 and 16%, w/v, PM extract recovered to 80-90% of the control group levels after UVB. These results suggest that PM extract strongly inhibits the destruction of SOD1 by UV radiation and probably contains anti-skin photoaging agents.

Molecular and cellular pathogenesis of autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited disease characterized by a malformation complex which includes cystically dilated tubules in the kidneys and ductal plate malformation in the liver. The disorder is observed primarily in infancy and childhood, being responsible for significant pediatric morbidity and mortality. All typical forms of ARPKD are caused by mutations in a single gene, PKHD1 (polycystic kidney and hepatic disease 1). This gene has a minimum of 86 exons, assembled into multiple differentially spliced transcripts and has its highest level of expression in kidney, pancreas and liver. Mutational analyses revealed that all patients with both mutations associated with truncation of the longest open reading frame-encoded protein displayed the severe phenotype. This product, polyductin, is a 4,074-amino acid protein expressed in the cytoplasm, plasma membrane and primary apical cilia, a structure that has been implicated in the pathogenesis of different polycystic kidney diseases. In fact, cholangiocytes isolated from an ARPKD rat model develop shorter and dysmorphic cilia, suggesting polyductin to be important for normal ciliary morphology. Polyductin seems also to participate in tubule morphogenesis and cell mitotic orientation along the tubular axis. The recent advances in the understanding of in vitro and animal models of polycystic kidney diseases have shed light on the molecular and cellular mechanisms of cyst formation and progression, allowing the initiation of therapeutic strategy designing and promising perspectives for ARPKD patients. It is notable that vasopressin V2 receptor antagonists can inhibit/halt the renal cystic disease progression in an orthologous rat model of human ARPKD.

Exploratory calcineurin inhibitor-free regimens in living-related kidney transplant recipients

Chronic allograft nephropathy is among the major causes of graft loss even in low-risk kidney transplant recipients and correlates with acute nephrotoxic events during the first year post-transplant. Therefore, calcineurin inhibitor-free regimens may improve patient and graft survival among recipients of living-related kidney transplants. To confirm this hypothesis, we evaluated the efficacy and safety of two calcineurin inhibitor-free regimens in 92 low-risk recipients of one-haplotype living-related kidney transplants. Immunosuppression consisted of tacrolimus, azathioprine and prednisone (group I, GI, N = 38), 2 doses of daclizumab, mycophenolate mofetil (MMF), and prednisone (GII, N = 33) and 2 doses of daclizumab, MMF, sirolimus and prednisone (GIII, N = 21). At 12 months, treatment failure (biopsy-confirmed acute rejection, graft loss or death) was higher in GII compared to GIII and GI (54.5 vs 24.0 vs 13.1%, P < 0.01, respectively). In patients of black ethnicity the incidence of acute rejection was 25 vs 83.3 vs 20% (P = 0.055), respectively. Patient and graft survival was comparable. There were no differences in mean creatinine or calculated creatinine clearance at 12 months. Overall incidence of post-transplant diabetes mellitus (3.3%) and cytomegalovirus disease (4.3%) was similar in all groups. Further development of effective calcineurin inhibitor-free regimens should exclude patients of black ethnicity and may need full-induction therapy, perhaps with depleting agents, and concentration-controlled use of sirolimus and MMF.

Are the current chronic allograft nephropathy grading systems sufficient to predict renal allograft survival?

A major problem in renal transplantation is identifying a grading system that can predict long-term graft survival. The present study determined the extent to which the two existing grading systems (Banff 97 and chronic allograft damage index, CADI) correlate with each other and with graft loss. A total of 161 transplant patient biopsies with chronic allograft nephropathy (CAN) were studied. The samples were coded and evaluated blindly by two pathologists using the two grading systems. Logistic regression analyses were used to evaluate the best predictor index for renal allograft loss. Patients with higher Banff 97 and CADI scores had higher rates of graft loss. Moreover, these measures also correlated with worse renal function and higher proteinuria levels at the time of CAN diagnosis. Logistic regression analyses showed that the use of angiotensin-converting enzyme inhibitor (ACEI), hepatitis C virus (HCV), tubular atrophy, and the use of mycophenolate mofetil (MMF) were associated with graft loss in the CADI, while the use of ACEI, HCV, moderate interstitial fibrosis and tubular atrophy and the use of MMF were associated in the Banff 97 index. Although Banff 97 and CADI analyze different parameters in different renal compartments, only some isolated parameters correlated with graft loss. This suggests that we need to review the CAN grading systems in order to devise a system that includes all parameters able to predict long-term graft survival, including chronic glomerulopathy, glomerular sclerosis, vascular changes, and severity of chronic interstitial fibrosis and tubular atrophy.

Effect of melatonin and time of administration on irradiation-induced damage to rat testes

The effect of ionizing irradiation on testes and the protective effects of melatonin were investigated by immunohistochemical and electron microscopic methods. Eighty-two adult male Wistar rats were divided into 10 groups. The rats in the irradiated groups were exposed to a sublethal irradiation dose of 8 Gy, either to the total body or abdominopelvic region using a 60Co source at a focus of 80 cm away from the skin in the morning or evening together with vehicle (20% ethanol) or melatonin administered 24 h before (10 mg/kg), immediately before (20 mg/kg) and 24 h after irradiation (10 mg/kg), all ip. Caspace-3 immunoreactivity was increased in the irradiated group compared to control (P < 0.05). Melatonin-treated groups showed less apoptosis as indicated by a considerable decrease in caspace-3 immunoreactivity (P < 0.05). Electron microscopic examination showed that all spermatogenic cells, especially primary spermatocytes, displayed prominent degeneration in the groups submitted to total body and abdominopelvic irradiation. However, melatonin administration considerably inhibited these degenerative changes, especially in rats who received abdominopelvic irradiation. Total body and abdominopelvic irradiation induced identical apoptosis and testicular damage. Chronobiological assessment revealed that biologic rhythm does not alter the inductive effect of irradiation. These data indicate that melatonin protects against total body and abdominopelvic irradiation. Melatonin was more effective in the evening abdominopelvic irradiation and melatonin-treated group than in the total body irradiation and melatonin-treated group.

Raising awareness of chronic kidney disease in a Brazilian urban population

The incidence and prevalence of chronic kidney disease have been increasing in recent years in developing countries. The aim of this study was to report the results of a general chronic kidney disease awareness program applied to an urban population in a large Brazilian city. From January 2002 to January 2005 a total of 8883 individuals in the city of Curitiba (PR, Brazil) were screened for hypertension, body mass index, hematuria, and proteinuria. A family history and previous medical diagnosis of hypertension and diabetes mellitus (DM) were also recorded. Of the 8883 individuals assessed, 56% were women, subject median age was 47 years (range: 17-93 years) and more than 90% were Caucasian. Thirty percent had signs of hematuria, 6% had proteinuria, and 3% had hematuria and proteinuria. The median of mean arterial pressure values was 93 mmHg (range: 71-135 mmHg) and 16% of the population screened had a history of hypertension. A significant positive family history of both hypertension or DM was present in 42% (P < 0.0001; chi-square = 83.18) and 7% (P < 0.0001; chi-square = 161.31) of the hypertensive group, respectively. Finally, the prevalence of hypertension and DM was significantly higher in older individuals with proteinuria. In the present study, a higher prevalence of hematuria and proteinuria was found in older individuals with hypertension and diabetes compared to the general population. These data confirm the need for public awareness of renal disease in high-risk individuals.

Cystatin C and renal function in pediatric kidney transplant recipients

In clinical practice, the glomerular filtration rate (GFR) is often determined with serum creatinine. However, studies have shown cystatin C to be a better parameter for the diagnosis of impaired renal function. We compared GFR estimated by plasma cystatin C with GFR estimated by serum creatinine in a sample of 50 pediatric renal transplant recipients and 24 healthy children. The correlation between GFR estimated by serum creatinine and by cystatin C was significant (r = 0.75; P < 0.001, Person’s correlation); however, in pediatric kidney transplant recipients, the GFR was 6.7 mL/min lower when determined using cystatin C rather than serum creatinine. Moreover, using GFR estimated by cystatin C we found that 42% of the pediatric kidney transplant recipients had an estimated GFR <60 mL·min-1·1.73 (m²)-1, whereas when GFR was estimated by the serum creatinine formula only 16% of the children had values below this cutoff point indicative of chronic kidney disease (P < 0.001). We conclude that, in pediatric kidney transplant recipients, estimation of GFR yields lower values when cystatin C is used rather than serum creatinine.

Cross-transmission of vancomycin-resistant Enterococcus in patients undergoing dialysis and kidney transplant

The objective of this study was to investigate the occurrence of vancomycin-resistant Enterococcus (VRE) cross-transmission between two patient groups (long-term dialysis and kidney transplant patients). Molecular typing, by automated ribotyping with the RiboPrinter Microbial Characterization System (Qualicon, USA), was used to analyze VRE isolates from 31 fecal samples of 320 dialysis patients and 38 fecal samples of 280 kidney transplant patients. Clonal spread of E. faecalis and E. casseliflavus was observed intragroup, but not between the two groups of patients. In turn, transmission of E. gallinarum and E. faecium between the groups was suggested by the finding of vancomycin-resistant isolates belonging to the same ribogroup in both dialysis and transplant patients. The fact that these patients were colonized by VRE from the same ribogroup in the same health care facility provides evidence for cross-transmission and supports the adoption of stringent infection control measures to prevent dissemination of these bacteria.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Hypomagnesemia is a risk factor for nonrecovery of renal function and mortality in AIDS patients with acute kidney injury

The objective of the present study was to determine the prevalence of electrolyte disturbances in AIDS patients developing acute kidney injury in the hospital setting, as well as to determine whether such disturbances constitute a risk factor for nephrotoxic and ischemic injury. A prospective, observational cohort study was carried out. Hospitalized AIDS patients were evaluated for age; gender; coinfection with hepatitis; diabetes mellitus; hypertension; time since HIV seroconversion; CD4 count; HIV viral load; proteinuria; serum levels of creatinine, urea, sodium, potassium and magnesium; antiretroviral use; nephrotoxic drug use; sepsis; intensive care unit (ICU) admission, and the need for dialysis. Each of these characteristics was correlated with the development of acute kidney injury, with recovery of renal function and with survival. Fifty-four patients developed acute kidney injury: 72% were males, 59% had been HIV-infected for >5 years, 72% had CD4 counts <200 cells/mm³, 87% developed electrolyte disturbances, 33% recovered renal function, and 56% survived. ICU admission, dialysis, sepsis and hypomagnesemia were all significantly associated with nonrecovery of renal function and with mortality. Nonrecovery of renal function was significantly associated with hypomagnesemia, as was mortality in the multivariate analysis. The risks for nonrecovery of renal function and for death were 6.94 and 6.92 times greater, respectively, for patients with hypomagnesemia. In hospitalized AIDS patients, hypomagnesemia is a risk factor for nonrecovery of renal function and for in-hospital mortality. To determine whether hypomagnesemia is a determinant or simply a marker of critical illness, further studies involving magnesium supplementation in AIDS patients are warranted.

Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively). Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

Maternal undernutrition and the offspring kidney: from fetal to adult life

Maternal dietary protein restriction during pregnancy is associated with low fetal birth weight and leads to renal morphological and physiological changes. Different mechanisms can contribute to this phenotype: exposure to fetal glucocorticoid, alterations in the components of the renin-angiotensin system, apoptosis, and DNA methylation. A low-protein diet during gestation decreases the activity of placental 11ß-hydroxysteroid dehydrogenase, exposing the fetus to glucocorticoids and resetting the hypothalamic-pituitary-adrenal axis in the offspring. The abnormal function/expression of type 1 (AT1R) or type 2 (AT2R) AngII receptors during any period of life may be the consequence or cause of renal adaptation. AT1R is up-regulated, compared with control, on the first day after birth of offspring born to low-protein diet mothers, but this protein appears to be down-regulated by 12 days of age and thereafter. In these offspring, AT2R expression differs from control at 1 day of age, but is also down-regulated thereafter, with low nephron numbers at all ages: from the fetal period, at the end of nephron formation, and during adulthood. However, during adulthood, the glomerular filtration rate is not altered, due to glomerulus and podocyte hypertrophy. Kidney tubule transporters are regulated by physiological mechanisms; Na+/K+-ATPase is inhibited by AngII and, in this model, the down-regulated AngII receptors fail to inhibit Na+/K+-ATPase, leading to increased Na+ reabsorption, contributing to the hypertensive status. We also considered the modulation of pro-apoptotic and anti-apoptotic factors during nephrogenesis, since organogenesis depends upon a tight balance between proliferation, differentiation and cell death.

Acute kidney injury in AIDS: frequency, RIFLE classification and outcome

The objective of the present study was to evaluate the characteristics of acute kidney injury (AKI) in AIDS patients and the value of RIFLE classification for predicting outcome. The study was conducted on AIDS patients admitted to an infectious diseases hospital inBrazil. The patients with AKI were classified according to the RIFLE classification: R (risk), I (injury), F (failure), L (loss), and E (end-stage renal disease). Univariate and multivariate analyses were used to evaluate the factors associated with AKI. A total of 532 patients with a mean age of 35 ± 8.5 years were included in this study. AKI was observed in 37% of the cases. Patients were classified as "R" (18%), "I" (7.7%) and "F" (11%). Independent risk factors for AKI were thrombocytopenia (OR = 2.9, 95%CI = 1.5-5.6, P < 0.001) and elevation of aspartate aminotransferase (AST) (OR = 3.5, 95%CI = 1.8-6.6, P < 0.001). General mortality was 25.7% and was higher among patients with AKI (40.2 vs17%, P < 0.001). AKI was associated with death and mortality increased according to RIFLE classification - "R" (OR 2.4), "I" (OR 3.0) and "F" (OR 5.1), P < 0.001. AKI is a frequent complication in AIDS patients, which is associated with increased mortality. RIFLE classification is an important indicator of poor outcome for AIDS patients.

Adherence to drug therapy in kidney disease

Non-adherence to drug therapy has not been extensively studied in patients with chronic kidney disease (CKD). The objective of the present study was to identify determinants of non-adherence to drug therapy in patients with CKD, not on dialysis. A prospective cohort study involving 149 patients was conducted over a period of 12 months. Adherence to drug therapy was evaluated by the self-report method at baseline and at 12 months. Patients who knew the type of drug(s) and the respective number of prescribed pills in use at the visit preceding the interview were considered to be adherent. Patients with cognitive decline were assessed by interviewing their caregivers. Mean patient age was 51 ± 16.7 years. Male patients predominated (60.4%). Univariate analysis performed at baseline showed that non-adherence was associated with older age, more pills taken per day, worse renal function, presence of coronary artery disease, and reliance on caregivers for the administration of their medications. In multivariate analysis, the factors that were significantly associated with non-adherence were daily use of more than 5 pills and drug administration by a caregiver. Longitudinal evaluation showed an increase in non-adherence over time. Medication non-adherence was lower (17.4%) at the baseline period of the study than after 1 year of the study (26.8%). Compared to the baseline period, the percentage of adherent patients who became non-adherent (22%) was lower than the percentage of non-adherent patients who became adherent (50%). In CKD patients not on dialysis, non-adherence was significantly associated with the number of pills taken per day and drug administration by third parties. Adherence is more frequent than non-adherence over time.