Generation of a novel decay accelerating factor (DAF) knock-out rat model using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9), genome editing

Decay accelerating factor (DAF), a key complement activation control protein, is a 70 kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf^?/?) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf^+/+ rats. Renal histology in Daf^?/? rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf^+/+ rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf^?/? rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.

     

Caspase-3 immunohistochemical expression is a marker of apoptosis,increased grade and early recurrence in intracranial meningiomas

Caspase-3 is the ultimate executioner caspase that is essential for the nuclear changes associated with apoptosis. We investigated caspase-3 immunohistochemical expression in 58 primary intracranial meningiomas, using one monoclonal antibody detecting both precursor and cleaved caspase-3 (CPP32) and a second recognizing only the cleaved activated form (ASP175). Caspase-3 expression was analyzed in relation to baseline apoptosis—as illustrated by the expression of anti-single stranded DNA (ss-DNA), the antiapoptotic protein bcl-2, proliferation indices (Ki-67, PCNA, topoisomerase IIa, mitosin C), hormonal status (estrogen, progesterone, androgen receptors), standard clinicopathological parameters and patients’ disease-free survival. Caspase-3 immunostaining was observed in 62% of cases for CPP32 and in 24% for ASP175. In both instances, the labeling index (LI) was significantly correlated with ss-DNA LI (p=0.038 and p=0.018). CPP32 but not ASP175 LI positively correlated with the mitotic index (p=0.001) and PCNA LI (p=0.004). Both CPP32 and ASP175 LIs were increased in nonbenign meningiomas (p<0.0001 and p=0.0035 respectively). In univariate and multivariate survival analyses, caspase-3 predicted meningioma recurrence, independently affecting disease-free survival (p=0.011 and p=0.047 respectively for CPP32; p<0.0001 and p=0.012 respectively for ASP175). Caspase-3 may prove to be a useful predictor of early recurrence in a group of neoplasms characterized by the frequent discordance between histology and clinical behavior.     

Phenotypic characterization of a novel HO-1 depletion model in the rat

Although the protective role of HO-1 induction in various forms of kidney disease is well established, mechanisms other than heme catabolism to biliverdin, bilirubin and carbon monoxide have recently been identified. Unraveling these mechanisms requires the generation of appropriate animal models. The present study describes the generation of a HO-1 deficient Hmox1 ^?/? rat model and characterizes its renal and extrarenal phenotype. Hmox1 ^?/? rats had growth retardation and splenomegaly compared to their Hmox1 ^+/+ littermates. Focal segmental glomerulosclerosis-type lesions and interstitial inflammatory infiltrates were prominent morphologic findings and were associated with increased blood urea nitrogen, serum creatinine and albuminuria. There was no increase in iron deposition in glomeruli, tubules or interstitium. Iron deposition in spleen and liver was reduced. Electron microscopic examination of glomeruli revealed edematous podocytes with scant areas of foot process effacement but otherwise well preserved processes and slit-diaphragms. Of the filtration barrier proteins examined, β-catenin expression was markedly reduced both in glomeruli and extrarenal tissues. Since the rat is the preferred laboratory animal in experimental physiology and pathophysiology, the rat model of HO-1 deficiency may provide a novel tool for investigation of the role of this enzyme in renal function and disease.     

Expression of Ser729 Phosphorylated PKC Epsilon in Experimental Crescentic Glomerulonephritis: An Immunohistochemical Study

PKCε, a DAG-dependent, Ca²⁺- independent kinase attenuates extent of fibrosis following tissue injury, suppresses apoptosis and promotes cell quiescence. In crescentic glomerulonephritis (CGN), glomerular epithelial cells (GEC) contribute to fibro-cellular crescent formation while they also transdifferentiate to a mesenchymal phenotype. The aim of this study was to assess PKCε expression in CGN. Using an antibody against PKC-ε phosphorylated at Ser⁷²⁹, we assessed its localization in rat model of immune-mediated rapidly progressive CGN. In glomeruli of control animals, pPKCε was undetectable. In animals with CGN, pPKCε was expressed exclusively in glomerular epithelial cells (GEC) and in GEC comprising fibrocellular crescents that had acquired a myofibroblasttype phenotype. In non-immune GEC injury induced by puromycin aminonucleoside and resulting in proteinuria of similar magnitude as in CGN, pPKCε expression was absent. There was constitutive pPKCε expression in distal convoluted tubules, collecting ducts and thick segments of Henley’s loops in both control and experimental animals. We propose that pPKC_ε expression occurring in GEC and in fibrocellular crescentic lesions in CGN may facilitate PKCε dependent pathologic processes.