Sites Of Cellular Damage Research Articles

Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling.

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes – differentiated epithelial cells critical for filtration – are thought to lack significant capacity for regeneration. Here, we show that podocytes rapidly lose differentiation markers and enter cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression induces marked upregulation of Wnt signaling and disrupts glomerular structure resulting in a collapsing glomerulopathy resembling those in humans, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased levels of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Either silencing transgenic TERT expression or inhibition of Wnt signaling through systemic expression of the Wnt-inhibitor Dkk1 in TERT transgenic mice results in marked normalization of podocytes, including rapid cell cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected property of podocytes to reversibly enter cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt/β-catenin pathways in podocyte proliferation and disease.

ATP induced microvesicles stimulate macrophage activation (89.18)

Abstract Adenosine triphosphate (ATP) has a unique role as a damage associated molecular pattern (DAMP) as it is normally kept within cells unless there is cellular/tissue injury. As a DAMP, ATP has several known mechanisms for immune cell activation. Our lab and others have reported that exogenous ATP administration to macrophages in cell culture induces rapid shedding of microvesicles (MV). In this study, we harvested MV from ATP treated macrophages and demonstrated that MV are capable of stimulating naive macrophages to release pro-inflammatory cytokines and induce co-stimulatory surface marker upregulation. MV- induced activation is dependent on a cAMP/p38 MAPK pathway and also results in NFkappaB translocation. Activation is partially dependent on TLR4, but is not due to the TLR4 agonist HMGB1, which is present in MV. TNFalpha is also present in MV but is not required to activate macrophages since MV from TNF-deficient cells are also stimulatory. Biochemical fractionation of MV was then used to show that the stimulatory component is phospholipid in nature, and we are currently working to identify the bioactive compound. These findings suggest a novel means by which macrophages at sites of cellular damage can become sterilely activated by ATP to further propagate cellular damage via shed microvesicles.

Prospects for caspase inhibitors.

Programmed cell death, or apoptosis, is executed by a series of Cysteine Aspartyl Proteases (Caspases) that form a proteolytic cascade. Each caspase functions either to activate downstream caspases by proteolytic cleavage and/or to proteolytically cleave cellular substrates. Increased levels of apoptosis and caspase activity are frequently observed at sites of cellular damage in both acute (e.g. myocardial infarction, stroke, sepsis) and chronic (e.g. Alzheimer's, Parkinson's and Huntington's Disease) indications. Thus, inhibition of caspase activity with the aim of reducing cell death, and hence tissue damage, is predicted to be therapeutically beneficial. Herein we outline different approaches that have been taken to identify small-molecule caspase inhibitors that include both traditional (e.g. HTS, structure-based design and substrate analog approaches) and novel screening technologies (e.g. Tethering). In addition, the characterization of inhibitors emerging from these programs will also be presented. Many of these compounds demonstrate efficacy in a wide range of animal models; however, only two examples of caspase inhibitors have progressed to clinical testing. Here we will discuss issues (both compound and mechanism related) associated with developing a caspase program in the pharmaceutical industry.

Effect of repeated administration of berenil on urinary enzyme excretion with corresponding tissue pattern in rats.

The effect of repeated administration of berenil, a trypanocide, on urinary excretion of some enzyme activities in rat and their corresponding levels in the kidney and serum was investigated. Daily administration of this drug to rats resulted in increased urinary volume, excretion of protein, alkaline phosphatase and lactate dehydrogenase activities. However, the level of acid phosphatase activity was not significantly increased while muramidase activity disappeared completely during the period of drug administration. In the kidney tissue, there was a significant loss of lactate dehydrogenase activity immediately after the first dose and this trend continued until the end of drug administration. In the same tissue, there was an increase in alkaline phosphatase activity while the lysosomal enzymes were not significantly affected. In the serum, except for the increase in alkaline phosphatase activity, all other enzymes were not significantly affected. All these results indicate that there is cellular damage to rat kidney as a result of repeated berenil administration, and that the plasma membrane and the soluble portion of the cytoplasm are the primary site of injury to the cells. They also suggest that urinary enzyme excretion could be useful in determining the site of cellular damage by chemical agents in kidneys.

Liver uptake of 99mTc-pyrophosphate

A 56-yr-old chronic schizophrenic with a prior history of anterior myocardial infarction was hospitalized with a 2-day history of progressive abdominal pain and shortness of breath. Physical examination revealed an agitated male with regular pulse of 100/min, blood pressure of 112/70, respiratory rate of 30/rain, and slightly elevated temperature of 100~ The jugular veins were distended to the level of the jaw and bibasilar rates were ausculted in the lungs. The liver was enlarged, firm, and mildly tender. The chest roentgenogram showed pulmonary interstitial edema and pulmonary venous congestion. On admission, creatine phosphokinase (CPK) was 2464, lactate dehydrogenase (LDH) 1110, glutamic oxalactic transaminase (SGOT) 1210, bilirubin 0.8, and alkaline phosphatase 177. The prothrombin time and partial thromboplastin time were elevated at 22.5 and 45.4 sec, respectively. Serum urea nitrogen (BUN) was 66 with creatinine level 1.9. The EKG showed right bundle branch block, old Q waves in the precordiaI leads, and new Q waves in leads I1, lI1, and AVF. Diagnosis on admission was recent inferior MI, rightand left-sided congestive heart failure, congestive hepatopathy, and acute renal failure. Subsequent CPK isoenzyme fractionation failed to support the diagnosis of acute myocardial infarction, and a 99mTc-pyrophosphate myocardial scintigram was performed on the third hospital day that showed intense labeling of the liver without demonstrable myocardial activity. (Fig. 1). It was postulated that the patient's myocardial infarction had occurred 3-5 days prior to admission, and that the persistent elevation of CPK was due to rhabdomyolysis. Right heart catheterization performed after correction of coagulation parameters with fresh frozen plasma revealed markedly elevated right-sided pressures, with right atrial pressure 23 mm Hg, RV 47/20, and pulmonary artery wedge 28. The cardiac output was 2.8 liter/min. On nitroprusside and dopamine, the hemodynamic parameters improved somewhat with diuresis and concomitant improvement in hepatic function. On the seventh hospital day, the patient developed supraventricular tachycardia, which progressed to ventricular fibrillation. Resuscitation was performed; however, a downhi|l course ensued and the patient expired 3 days later. Permission for autopsy was not granted. This case demonstrates diffuse hepatic labeling by 99mTc-pyrophosphate in a clinical setting with high likelihood of central hepatic necrosis secondary to severe congestive heart failure. Liver uptake of bone imaging agents is unusual; in a study of 250 consecutive bone scans, liver uptake occurred only in 5 cases, 2 of which were associated with metastases and the remaining 3 due to poor radiopharmaceutical quality. ''~ Diffuse hepatic uptake of 99roTe-phosphate compounds has since been shown to occur in massive liver necrosis, 2 in the presence of high serum AI +§ levels, 3 and in amyloidosis. 4 Animal studies have shown elevated liver uptake with increased phosphate salt concentration: and with combination of tin and 99mTc-reagents prior to the addition of the phosphate-chelating agent during radiopharmaceutical preparation. 6 In liver necrosis and amyloidosis, labeling occurs presumably due to phosphate localization at sites of cellular damage by mechanisms to be discussed, while in the other cases, reticuloendothelial uptake of microcolloidal particles is the postulated mechanism: ~ Focal uptake of 99roTe-phosphate compounds by hepatic metastases from adenocarcinoma of the colon, breast carcinoma, oat cell carcinoma of the lung, malignant melanoma, and esophageal squamous cell carcinoma has been reported. 7-~~ A single case of uptake by cholangiocarcinoma has also been described. 8 Tumor localization of phosphate compounds probably

Studies on the Cytotoxic Action of Silica Dusts on Macrophages in Vitro

Experiments were performed in order to investigate the site of cellular damage induced by silica psrticles in vitro to peritoneal guinea pig macrophages. Considerable amounts of cytoplasmic enzymes, lactate dehydrogenase and aldolase, were released into the medium a short time after addition of silica to the cultures, whereas lysosomal enzymes, acid phosphatase and RNAase, were detected only after more prolonged incubation and in lower quantities. Correspondingly, loss of fluorochromasia from macrophages occurred in a matter of minutes after contact with silica particles. When coarse, nonphagocytabte silica particles were used, a rapid loss of fluorochromasia and early release of cytoplasmic enzymes were also seen.