Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.

Glomerulonephritis associated with antibodies to neutrophil cytoplasm and glomerular basement membrane

The prognosis for recovery of renal function of oligoanuric patients with anti-glomerular basement membrane disease is generally regarded as poor. Five patients are reported with dialysis-dependent renal failure in whom antibodies were present simultaneously both to neutrophil cytoplasm and glomerular basement membrane all of whom responded, at least initially, to immunosuppressive therapy and plasma exchange. Two of the 5 remain in clinical and immunological remission at 25 and 51 months of follow-up. We suggest that reversal of dialysis-dependent renal failure may be possible in some patients who display this dual antibody positivity.

Intensive diabetes therapy and glomerular filtration rate in type 1 diabetes

An impaired glomerular filtration rate (GFR) leads to end-stage renal disease and increases the risks of cardiovascular disease and death. Persons with type 1 diabetes are at high risk for kidney disease, but there are no interventions that have been proved to prevent impairment of the GFR in this population.

Estimated Glomerular Filtration Rate Decline as a Predictor of Dialysis in Kidney Transplant Recipients

BACKGROUND: It is now common for individuals to require dialysis following the failure of a kidney transplant. Management of complications and preparation for dialysis are suboptimal in this group. To aid planning, it is desirable to estimate the time to dialysis requirement. The rate of decline in the estimated glomerular filtration rate (eGFR) may be used to this end.

METHODS: This study compared the rate of eGFR decline prior to dialysis commencement between individuals with failing transplants and transplant-naïve patients. The rate of eGFR decline was also compared between transplant recipients with and without graft failure. eGFR was calculated using the four-variable MDRD equation with rate of decline calculated by least squares linear regression.

RESULTS: The annual rate of eGFR decline in incident dialysis patients with graft failure exceeded that of the transplant-naïve incident dialysis patients. In the transplant cohort, the mean annual rate of eGFR decline prior to graft failure was 7.3 ml/min/1.73 m(2) compared to 4.8 ml/min/1.73 m(2) in the transplant-naïve group (p < 0.001) and 0.35 ml/min/1.73 m(2) in recipients without graft failure (p < 0.001). Factors associated with eGFR decline were recipient age, decade of transplantation, HLA mismatch and histological evidence of chronic immunological injury.

CONCLUSIONS: Individuals with graft failure have a rapid decline in eGFR prior to dialysis commencement. To improve outcomes, dialysis planning and management of chronic kidney disease complications should be initiated earlier than in the transplant-naïve population.

Estimation of glomerular filtration rate in hospitalised patients: are we overestimating renal function?

QUESTIONS UNDER STUDY AND PRINCIPLES: Estimating glomerular filtration rate (GFR) in hospitalised patients with chronic kidney disease (CKD) is important for drug prescription but it remains a difficult task. The purpose of this study was to investigate the reliability of selected algorithms based on serum creatinine, cystatin C and beta-trace protein to estimate GFR and the potential added advantage of measuring muscle mass by bioimpedance. In a prospective unselected group of patients hospitalised in a general internal medicine ward with CKD, GFR was evaluated using inulin clearance as the gold standard and the algorithms of Cockcroft, MDRD, Larsson (cystatin C), White (beta-trace) and MacDonald (creatinine and muscle mass by bioimpedance). 69 patients were included in the study. Median age (interquartile range) was 80 years (73-83); weight 74.7 kg (67.0-85.6), appendicular lean mass 19.1 kg (14.9-22.3), serum creatinine 126 μmol/l (100-149), cystatin C 1.45 mg/l (1.19-1.90), beta-trace protein 1.17 mg/l (0.99-1.53) and GFR measured by inulin 30.9 ml/min (22.0-43.3). The errors in the estimation of GFR and the area under the ROC curves (95% confidence interval) relative to inulin were respectively: Cockcroft 14.3 ml/min (5.55-23.2) and 0.68 (0.55-0.81), MDRD 16.3 ml/min (6.4-27.5) and 0.76 (0.64-0.87), Larsson 12.8 ml/min (4.50-25.3) and 0.82 (0.72-0.92), White 17.6 ml/min (11.5-31.5) and 0.75 (0.63-0.87), MacDonald 32.2 ml/min (13.9-45.4) and 0.65 (0.52-0.78). Currently used algorithms overestimate GFR in hospitalised patients with CKD. As a consequence eGFR targeted prescriptions of renal-cleared drugs, might expose patients to overdosing. The best results were obtained with the Larsson algorithm. The determination of muscle mass by bioimpedance did not provide significant contributions.

Le diabète maternel influence la morphogenèse rénale et la programmation périnatale

Le diabète maternel est un facteur de risque majeur pour le développement de malformations congénitales. Dans le syndrome de l’embryopathie diabétique, l’exposition prolongée du fœtus à de hautes concentrations ambientes de glucose induit des dommages qui peuvent affecter plusieurs organes, dont les reins. Les malformations rénales sont la cause de près de 40 pourcent des cas d’insuffisance rénale infantile. L’hyperglycémie constitue un environnement utérin adverse qui nuit à la néphrogenèse et peut causer l’agenèse, la dysplasie (aplasie) ou l’hypoplasie rénale. Les mécanismes moléculaires par lesquels les hautes concentrations ambientes de glucose mènent à la dysmorphogenèse et aux malformations demeurent toutefois mal définis. Le diabète maternel prédispose aussi la progéniture au développement d’autres problèmes à l’âge adulte, tels l’hypertension, l’obésité et le diabète de type 2. Ce phénomène appelé ‘programmation périnatale’ a suscité l’intérêt au cours des dernières décennies, mais les mécanismes responsables demeurent mal compris. Mes études doctorales visaient à élucider les mécanismes moléculaires par lesquels le diabète maternel ou un environnement in utero hyperglycémique affecte la néphrogenèse et programme par la suite la progéniture a développer de l’hypertension par des observations in vitro, ex vivo et in vivo. Nous avons utilisé les cellules MK4, des cellules embryonnaires du mésenchyme métanéphrique de souris, pour nos études in vitro et deux lignées de souris transgéniques (Tg) pour nos études ex vivo et in vivo, soient les souris HoxB7-GFP-Tg et Nephrin-CFP-Tg. Les souris HoxB7-GFP-Tg expriment la protéine fluorescente verte (GFP) dans le bourgeon urétérique (UB), sous le contrôle du promoteur HoxB7. Les souris Nephrin-CFP expriment la protéine fluorescente cyan (CFP) dans les glomérules, sous le contrôle du promoteur nephrin spécifique aux podocytes. Nos études in vitro visaient à déterminer si les hautes concentrations de glucose modulent l’expression du gène Pax2 dans les cellules MK4. Les cellules MK4 ont été traitées pendant 24h avec du milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants ou inhibiteurs de p38 MAPK, p44/42 MAPK, PKC et NF-kB. Nos résultats ont démontré que le D-glucose élevé (25mM) augmente la génération des espèces réactives de l’oxygène (ROS) dans les cellules MK4 et induit spécifiquement l’expression du gène Pax2. Des analogues du glucose tels le D-mannitol, L-glucose ou le 2-Deoxy-D-glucose n’induisent pas cette augmentation dans les cellules MK4. La stimulation de l’expression du gène Pax2 par le D-glucose dans les cellules MK4 peut être bloquée par des inhibiteurs des ROS et de NF-kB, mais pas par des inhibiteurs de p38 MAPK, p44/42 MAPK ou PKC. Ces résultats indiquent que la stimulation de l’expression du gène Pax2 par les concentrations élevées de glucose est due, au moins en partie, à la génération des ROS et l’activation de la voie de signalisation NF-kB, et non pas via les voies PKC, p38 MAPK et p44/42 MAPK. Nos études ex vivo s’intéressaient aux effets d’un milieu hyperglycémique sur la morphogenèse de la ramification du bourgeon urétérique (UB). Des explants de reins embryonnaires (E12 à E18) ont été prélevés par micro-dissection de femelles HoxB7-GFP gestantes. Les explants ont ensuite été cultivés dans un milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants, catalase ou inhibiteur de PI3K/AKT pour diverses durées. Nos résultats ont démontré que le D-glucose stimule la ramification du UB de manière spécifique, et ce via l’expression du gène Pax2. Cette augmentation de la ramification et de l’expression du gène Pax2 peut être bloquée par des inhibiteurs des ROS et de PI3K/AKT. Ces études ont démontré que les hautes concentrations de glucose altèrent la morphogenèse de la ramification du UB via l’expression de Pax2. L’effet stimulant du glucose semble s’effectuer via la génération des ROS et l’activation de la voie de signalisation Akt. Nos études in vivo visaient à déterminer le rôle fondamental du diabète maternel sur les défauts de morphogenèse rénale chez la progéniture. Dans notre modèle animal, le diabète maternel est induit par le streptozotocin (STZ) chez des femelles HoxB7-GFP gestantes (E13). Les souriceaux ont été étudiés à différents âges (naissants et âgés de une, deux ou trois semaines). Nous avons examiné leurs morphologie rénale, nombre de néphrons, expression génique et les événements apoptotiques lors de cette étude à court terme. La progéniture des mères diabétiques avait un plus faible poids, taille et poids des reins, et possédait des glomérules plus petits et moins de néphrons par rapport à la progéniture des mères contrôles. La dysmorphogenèse rénale observée est peut-être causée par l’augmentation de l’apoptose des cellules dans la région du glomérule. Nos résultats ont montré que les souriceaux nés de mères diabétiques possèdent plus de podocytes apoptotiques et plus de marquage contre la caspase-3 active dans leurs tubules rénaux que la progéniture des mères contrôles. Les souriceaux des mères diabétiques montrent une augmentation de l’expression des composants du système rénine angiotensine (RAS) intrarénal comme l’angiotensinogène et la rénine, ainsi qu’une augmentation des isoformes p50 et p65 de NF-kB. Ces résultats indiquent que le diabète maternel active le RAS intrarénal et induit l’apoptose des glomérules, menant à une altération de la morphogenèse rénale de la progéniture. En conclusion, nos études ont permis de démontrer que le glucose élevé ou l’environnement in utero diabétique altère la morphogenèse du UB, qui résulte en un retard dans la néphrogenèse et produit des reins plus petits. Cet effet est dû, au moins en partie, à la génération des ROS, à l’activation du RAS intrarénal et à la voie NF-kB. Nos études futures se concentreront sur les mécanismes par lesquels le diabète maternel induit la programmation périnatale de l’hypertension chez la progéniture adulte. Cette étude à long terme porte sur trois types de progénitures : adultes nés de mères contrôles, diabétiques ou diabétiques traitées avec insuline pendant la gestation. Nous observerons la pression systolique, la morphologie rénale et l’expression de divers gènes et protéines. Nous voulons de plus déterminer si la présence d’un système antioxydant (catalase) peut protéger la progéniture des effets néfastes des ROS causés par l’environnement in utero hyperglycémique. Les souris Catalase-Tg expriment la catalase spécifiquement dans les tubules proximaux et nous permettrons d’explorer notre hypothèse sur le rôle des ROS dans notre modèle expérimental de diabète maternel.

Inflammation, oxidative stress, glomerular filtration rate, and albuminuria in elderly men : a cross-sectional study

BACKGROUND: The role of inflammation and oxidative stress in mild renal impairment in the elderly is not well studied. Accordingly, we aimed at investigating the associations between estimated glomerular filtration rate (eGFR), albumin/creatinine ratio (ACR), and markers of different inflammatory pathways and oxidative stress in a community based cohort of elderly men. FINDINGS: Cystatin C-based GFR, ACR, and biomarkers of cytokine-mediated inflammation (interleukin-6, high-sensitivity C-reactive protein[CRP], serum amyloid A[SAA]), cyclooxygenase-mediated inflammation (urinary prostaglandin F2alpha [PGF2alpha]), and oxidative stress (urinary F2 isoprostanes) were assessed in the Uppsala Longitudinal Study of Adult Men(n = 647, mean age 77 years). RESULTS: In linear regression models adjusting for age, BMI, smoking, blood pressure, LDL-cholesterol, HDL-cholesterol, triglycerides, and treatment with statins, ACE-inhibitors, ASA, and anti-inflammatory agents, eGFR was inversely associated with CRP, interleukin-6, and SAA (beta-coefficient -0.13 to -0.19, p < 0.001 for all), and positively associated with urinary F2-isoprostanes (beta-coefficient 0.09, p = 0.02). In line with this, ACR was positively associated with CRP, interleukin-6, and SAA (beta- coefficient 0.09-0.12, p < 0.02 for all), and negatively associated with urinary F2-isoprostanes (beta-coefficient -0.12, p = 0.002). The associations were similar but with lower regression coefficients in a sub-sample with normal eGFR (>60 ml/min/1.73 m2, n = 514), with the exception that F2-isoprostane and SAA were no longer associated with eGFR. CONCLUSION: Our data indicate that cytokine-mediated inflammation is involved in the early stages of impaired kidney function in the elderly, but that cyclooxygenase-mediated inflammation does not play a role at this stage. The unexpected association between higher eGFR/lower albuminuria and increased F2-isoprostanes in urine merits further studies.

Estimating individual glomerular volume in the human kidney : clinical perspectives

Background. Measurement of individual glomerular volumes (IGV) has allowed the identification of drivers of glomerular hypertrophy in subjects without overt renal pathology. This study aims to highlight the relevance of IGV measurements with possible clinical implications and determine how many profiles must be measured in order to achieve stable size distribution estimates.

Methods. We re-analysed 2250 IGV estimates obtained using the disector/Cavalieri method in 41 African and 34 Caucasian Americans. Pooled IGV analysis of mean and variance was conducted. Monte-Carlo (Jackknife) simulations determined the effect of the number of sampled glomeruli on mean IGV. Lin’s concordance coefficient (RC), coefficient of variation (CV) and coefficient of error (CE) measured reliability.

Results. IGV mean and variance increased with overweight and hypertensive status. Superficial glomeruli were significantly smaller than juxtamedullary glomeruli in all subjects (P < 0.01), by race (P < 0.05) and in obese individuals (P < 0.01). Subjects with multiple chronic kidney disease (CKD) comorbidities showed significant increases in IGV mean and variability. Overall, mean IGV was particularly reliable with nine or more sampled glomeruli (RC > 0.95, <5% difference in CV and CE). These observations were not affected by a reduced sample size and did not disrupt the inverse linear correlation between mean IGV and estimated total glomerular number.

Conclusions. Multiple comorbidities for CKD are associated with increased IGV mean and variance within subjects, including overweight, obesity and hypertension. Zonal selection and the number of sampled glomeruli do not represent drawbacks for future longitudinal biopsy-based studies of glomerular size and distribution.

A design-based method for estimating glomerular number in the developing kidney

Low glomerular (nephron) endowment has been associated with an increased risk of cardiovascular and renal disease in adulthood. Nephron endowment in humans is determined by 36 wk of gestation, while in rats and mice nephrogenesis ends several days after birth. Specific genes and environmental perturbations have been shown to regulate nephron endowment. Until now, design-based method for estimating nephron number in developing kidneys was unavailable. This was due in part to the difficulty associated with unambiguously identifying developing glomeruli in histological sections. Here, we describe a method that uses lectin histochemistry to identify developing glomeruli and the physical disector/fractionator principle to provide unbiased estimates of total glomerular number (Nglom). We have characterized Nglom throughout development in kidneys from 76 rats and model this development with a 5-parameter logistic equation to predict Nglom from embryonic day 17.25 to adulthood (r2 = 0.98). This approach represents the first design-based method with which to estimate Nglom in the developing kidney.

Glomerular hypertrophy in subjects with low nephron number: contributions of sex, body size and race

Background. We have shown that low nephron number (Nglom) is a strong determinant of individual glomerular volume (IGV) in male Americans. However, whether the same pattern is present in female Americans remains unclear. The contributions of body surface area (BSA) and race to IGV in the context of Nglom also require further evaluation. Methods. Kidneys without overt renal disease were collected at autopsy in Mississippi, USA. The extremes of female Nglom were used to define high and low N glom for both sexes. Nglom and IGV were estimated by design-based stereology. A total of 24 African and Caucasian American females (n = 12 per race; 6 per Nglom extreme) were included. These subjects were subsequently matched to 24 comparable males by age and Nglom and to 18 additional males by age, Nglom and BSA. Results. IGV average and variance were very similar in female African and Caucasian Americans with high and low Nglom. Males with low Nglom from both races showed greater IGV average and variance than comparable females matched by age and Nglom. These differences in IGV between sexes were not observed in Caucasian Americans with low Nglom that were matched by age, Nglom and BSA. In contrast, glomeruli from African Americans were larger than those from Caucasian Americans, especially in subjects with high Nglom. Conclusions. While female Americans with low Nglom did not show glomerular hypertrophy, comparable males with low Nglom showed marked glomerular hypertrophy that was closely associated with high BSA. Glomerular size in African Americans may be confounded by multiple additional factors. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.