Aldosterone Induces DNA Damage and Activation of Nrf2 Mainly in Tubuli of Mouse Kidneys

Hypertensive patients have an increased risk of developing chronic kidney disease (CKD). Many of these patients have increased levels of the blood pressure regulating mineralocorticoid aldosterone. As a protection against aldosterone-induced damage, kidney cells can upregulate key regulators of the antioxidant defense, such as nuclear factor-erythroid-2-related factor 2 (Nrf2). In the present study aldosterone-induced kidney damage and Nrf2 activation in kidney cells of mice treated with three different concentrations of aldosterone for 4 weeks was localized. Increased albumin and neutrophil gelatinase-associated lipocalin (NGAL) in urine revealed an impaired kidney function of the aldosterone-infused mice. Localization of aldosterone-induced oxidative damage (in the form of DNA lesions) in specific kidney cells showed an increase in proximal tubuli and to an even greater extend in distal tubuli. Phosphorylated Nrf2 was increased in distal tubule cells after aldosterone-infusion. Nrf2 activation in proximal tubuli or in glomeruli after aldosterone-treatment could not be observed. Nrf2 target genes and proteins analyzed, paradoxically, showed a downregulation in the whole kidney. Aldosterone-treated mice exhibited an increased kidney injury and DNA damage in distal and proximal tubuli. Nrf2 seemed only to be specifically activated in distal tubule cells, where we also detected the highest amount of oxidative damage.

P0099CALCINEURIN INHIBITION, BUT NOT DEHYDRATION, IN RATS MIMICS HUMAN RENAL HISTOPATHOLOGY OF PATIENTS WITH CHRONIC INTERSTITIAL NEPHRITIS IN AGRICULTURAL COMMUNITIES (CINAC)

Background and Aims CINAC patients present a newly discovered constellation of proximal tubular lysosomal lesions which is also observed in patients experiencing calcineurin inhibitor (CNI) nephrotoxicity, suggesting that CINAC is a toxin-induced nephropathy involving calcineurin inhibition. An alternate hypothesis advocates chronic heat stress/dehydration as the major etiological factor for CINAC. Here, we evaluated in rats to what extent heat stress/dehydration versus CNI exposure reflects proximal tubular CINAC histopathology. Method Wistar rats were divided in 3 groups. Group 1 (n=6) was given water ad libitum (control group). Group 2 (n=8) was water deprived for 10 hours per 24h, 5 days/week and were placed in an incubator (37°C) for 30 min/hr of water deprivation. Group 3 (n=8) underwent daily oral gavage with cyclosporine (50mg/kg body weight). Animals were weighed daily and urine was collected at day 3, 17 and 28. After 28 days, rats were sacrificed. Kidneys were collected for light (LM) and electron microscopic (EM) histopathological analysis as well as for cortical renal tissue proteomics. Results Cyclosporine rats developed focal cortical lesions mimicking those of CINAC patients: i.e. atrophic proximal tubuli with thickened basement membranes and associated tubulo-interstitial fibrosis, PASM staining demonstrating enlarged argyrophyllic granules in the affected proximal tubuli, LAMP1 immunofluorescent staining identifying a subset of these granules as lysosomes, and EM confirming the presence of enlarged lysosomes, some dysmorphic approaching CINAC lysosomes. In dehydrated rats, confirmed by urinary osmolality and fluctuating body weight associated with water deprivation, none of the cyclosporine features were observed. Proteomic analysis confirmed cellular toxicity by cyclosporine, whereas the dehydration group lacked any markers of such. Conclusion The histopathological analogy between CNI nephrotoxicity in rats and humans and CINAC suggests a toxicological etiology for CINAC. In rats, dehydration/heat stress alone does not lead to the constellation of proximal tubular lesions as observed in CINAC patients.

Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney

We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.

GAMBARAN HISTOLOGIK GINJAL HEWAN COBA POSTMORTEM

Estimation of postmortem interval plays some significant roles in medicolegal investigation. This was a descriptive experimental study using one local pig as model. Samples were taken from the right and left kidneys at several time intervals: 0 minute, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 12 hours, and 24 hours postmortem. The results showed several histological changes, as follows: hydrophic degeneration in a small part of proximal tubules 30 minutes postmortem that increased after 45 minutes associated with narrowing of Bowman cavities; necrosis of glomeruli and proximal tubules associated with irregular distal tubular lumen and widening of Bowman cavities 60 minutes postmortem; necrosis of distal tubuli 12 hours postmortem; and necrosis of most kidney structures 24 hours postmortem. Conclusion: Hydrophic degeneration of proximal tubuli is the earliest histological change 30 minutes postmortem, followed by necrosis of glomeruli as well as proximal and distal tubuli that worsened after 24 hours postmortem. It is expected that this study can provide valuable contribution to medicolegal investigation, especially in early postmortem interval estimation.Keywords: postmortem interval, histological changes, postmortem, kidneyAbstrak: Penentuan saat kematian sangat penting dalam penyelidikan medikolegal. Penelitian ini merupakan penelitian deskriptif-eksperimental dengan menggunakan satu ekor babi lokal sebagai obyek penelitian. Sampel diambil dari ginjal kanan dan kiri pada beberapa interval waktu; 0 menit; 15 menit; 30 menit; 45 menit; 60 menit; 12 jam; dan 24 jam postmortem. Hasil penelitian memperlihatkan degenerasi hidropik pada sebagian kecil tubuli proksimal 30 menit postmortem yang meluas setelah 45 menit disertai penyempitan kavum Bowman; pelebaran kavum Bowman, nekrosis glomeruli dan tubuli proksimal, lumen sebagian kecil tubuli distal ireguler 60 menit postmortem; nekrosis tubuli distal 12 jam postmortem; dan nekrosis hampir seluruh struktur-struktur tersebut 24 jam postmortem. Simpulan: Degenerasi hidropik tubuli proksimal merupakan perubahan histologik yang paling dini yaitu 30 menit postmortem, disusul oleh tanda-tanda nekrosis pada sebagian kecil glomeruli dan tubuli proksimal serta nekrosis tubuli distal yang meluas setelah 24 jam postmortem. Penelitian ini diharapkan dapat memberikan kontribusi yang bermakna untuk kepentingan medikolegal, terutama dalam perkiraan saat kematian dini.Kata kunci: saat kematian, perubahan histologik, ginjal

IMMUNOHISTOCHEMICAL LOCALIZATION OF CATALASE IN MAMMALIAN TISSUES

The distribution of catalase was investigated in bovine tissues using fluorescent antibody techniques. The immunochemical properties of liver catalase were also examined. Two distinct components of liver catalase in immune system were found. Both of them possessed common antigenicity with erythrocyte catalase. No cross-reactivity was observed by immunodiffusion between catalase and the other hemoproteins such as lactoperoxidase, cytochrome c and hemoglobins. Bovine liver, pancreas, kidney, spleen and peripheral blood were examined. Catalase was located mainly in the cytoplasm of hepatic cells, acinar cells of the pancreas, epithelia of proximal tubuli, splenic cells scattered in the red pulp and some leukocytes. It was not found in any nucleus. Intracorpuscular catalase could be revealed in the erythrocytes treated with surface-active agents but not in frozen sections.

QUANTITATION OF TISSUE-BOUND RENAL AMINOPEPTIDASE BY A MICRODENSITOMETRIC TECHNIQUE

Relationships between biochemical and histochemical assay systems can be evaluated by appropriate techniques. With rat kidney as the test object the enzyme localized in the tissue section hydrolyzing l-leucyl-β-naphthylamide (LNA) was found to be identical with a purified particle-bound, cobalt-activated aminopolypeptidase previously characterized biochemically. A soluble sulfhydryl-dependent aminopeptidase, present in the rat kidney and also known to hydrolyze LNA, was not demonstrated in the histochemical system, although the diazonium salt employed incidentally caused the insolubilization in the tissue section of a significant proportion of previously extractable, particle-bound enzyme. Linearity between the enzymic activity present and the measurable enzymic activity was demonstrated to exist at the substrate concentration, pH level, section thickness and diazonium salt concentrations used in the assay procedure, but did not exist when these factors were varied beyond certain well defined limits. In addition, the effect of diazonium salt on both the inhibition and insolubilization of enzyme in the tissue section could be quantitated. Based on this evaluation of the enzymes hydrolyzing LNA in the rat kidney, a microdensitometric assay method employing a constant flow incubating chamber was developed to characterize and quantitate the LNA-hydrolyzing enzyme in the proximal tubuli. This study defines many of the parameters necessary for the future investigation by quantitative and qualitative methods of histochemical systems capable of providing resolutions of a higher order of magnitude and the retention of a greater proportion of extractable enzyme within the tissue section.