Muhammad M. Yaqoob

Bicarbonate supplementation slows progression of CKD and improves nutritional status.

Bicarbonate supplementation preserves renal function in experimental chronic kidney disease (CKD), but whether the same benefit occurs in humans is unknown. Here, we randomly assigned 134 adult patients with CKD (creatinine clearance [CrCl] 15 to 30 ml/min per 1.73 m(2)) and serum bicarbonate 16 to 20 mmol/L to either supplementation with oral sodium bicarbonate or standard care for 2 yr. The primary end points were rate of CrCl decline, the proportion of patients with rapid decline of CrCl (>3 ml/min per 1.73 m(2)/yr), and ESRD (CrCl <10 ml/min). Secondary end points were dietary protein intake, normalized protein nitrogen appearance, serum albumin, and mid-arm muscle circumference. Compared with the control group, decline in CrCl was slower with bicarbonate supplementation (5.93 versus 1.88 ml/min 1.73 m(2); P < 0.0001). Patients supplemented with bicarbonate were significantly less likely to experience rapid progression (9 versus 45%; relative risk 0.15; 95% confidence interval 0.06 to 0.40; P < 0.0001). Similarly, fewer patients supplemented with bicarbonate developed ESRD (6.5 versus 33%; relative risk 0.13; 95% confidence interval 0.04 to 0.40; P < 0.001). Nutritional parameters improved significantly with bicarbonate supplementation, which was well tolerated. This study demonstrates that bicarbonate supplementation slows the rate of progression of renal failure to ESRD and improves nutritional status among patients with CKD.

Erythropoietin Protects the Kidney against the Injury and Dysfunction Caused by Ischemia-Reperfusion

Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X(L) and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.

Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxyΔ12–14 PGJ2

Hematopoietic prostaglandin D2 synthase (hPGD2S) metabolizes cyclooxygenase (COX)-derived PGH2 to PGD2 and 15-deoxyΔ12–14 PGJ2 (15d-PGJ2). Unlike COX, the role of hPGD2S in host defense is ambiguous. PGD2 can be either pro- or antiinflammatory depending on disease etiology, whereas the existence of 15d-PGJ2 and its relevance to pathophysiology remain controversial. Herein, studies on hPGD2S KO mice reveal that 15d-PGJ2 is synthesized in a self-resolving peritonitis, detected by using liquid chromatography–tandem MS. Together with PGD2 working on its DP1 receptor, 15d-PGJ2 controls the balance of pro- vs. antiinflammatory cytokines that regulate leukocyte influx and monocyte-derived macrophage efflux from the inflamed peritoneal cavity to draining lymph nodes leading to resolution. Specifically, inflammation in hPGD2S KOs is more severe during the onset phase arising from a substantial cytokine imbalance resulting in enhanced polymorphonuclear leukocyte and monocyte trafficking. Moreover, resolution is impaired, characterized by macrophage and surprisingly lymphocyte accumulation. Data from this work place hPGD2S at the center of controlling the onset and the resolution of acute inflammation where it acts as a crucial checkpoint controller of cytokine/chemokine synthesis as well as leukocyte influx and efflux. Here, we provide definitive proof that 15d-PGJ2 is synthesized during mammalian inflammatory responses, and we highlight DP1 receptor activation as a potential antiinflammatory strategy.

High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases

Diabetic nephropathy is the leading cause of end‐stage renal disease in the Western world. Poor glycemic control contributes to the development of diabetic nephropathy, but the mechanisms underlying high glucose‐induced tissue injury are not fully understood. In the present study, the effect of high glucose on a proximal tubular epithelial cell (PTEC) line was investigated. Reactive oxygen species (ROS) were detected using the fluorescent probes dichlorofluorescein diacetate, dihydrorhodamine 123, and 2,3‐diaminonapthalene. Peroxynitrite (ONOO−) generation and nitrite concentrations were increased after 24 h of high glucose treatment (P<0.05). LLC‐PK1 cells exposed to high d‐glucose (25 mM) for up to 48 h had increased DNA fragmentation (P<0.01), caspase‐3 activity (P<0.001), and annexin‐V staining (P<0.05) as well as decreased expression of XIAP when compared with controls (5 mM d‐glucose). The ONOO− scavenger ebselen reduced DNA fragmentation and caspase‐3 activity as well as the high glucose‐induced nitrite production and DCF fluorescence. High glucose‐induced DNA fragmentation was completely prevented by an inhibitor of caspase‐3 (P<0.01) and a pan‐caspase inhibitor (P<0.001). Caspase inhibition did not affect ROS generation. This study, in a PTEC line, demonstrates that high glucose causes the generation of ONOO−, leading to caspase‐mediated apoptosis. Ebselen and a caspase‐3 inhibitor provided significant protection against high glucose‐mediated apoptosis, implicating ONOO− as a proapoptotic ROS in early diabetic nephropathy.

Erythropoietin attenuates the tissue injury associated with hemorrhagic shock and myocardial ischemia.

Here we investigate the effects of erythropoietin (EPO) on the tissue/organ injury caused by hemorrhagic shock (HS), endotoxic shock, and regional myocardial ischemia and reperfusion in anesthetized rats. Male Wistar rats were anesthetized with thiopental sodium (85 mg/kg i.p.) and subjected to hemorrhagic shock (HS; i.e., mean arterial blood pressure reduced to 45 mmHg for 90 min, followed by resuscitation with shed blood for 4 h), endotoxemia (for 6 h), or left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h). HS and endotoxemia resulted in renal dysfunction and liver injury. Administration of EPO (300 IU/kg i.v., n = 10) before resuscitation abolished the renal dysfunction and liver injury in hemorrhagic, but not endotoxic, shock. HS also resulted in significant increases in the kidney of the activities of caspases 3, 8, and 9. This increase in caspase activity was not seen in HS rats treated with EPO. In cultured human proximal tubule cells, EPO concentration-dependently reduced the cell death and increase in caspase-3 activity caused by either ATP depletion (simulated ischemia) or hydrogen peroxide (oxidative stress). In the heart, administration of EPO (300 IU/kg i.v., n = 10) before reperfusion also caused a significant reduction in infarct size. In cultured rat cardiac myoblasts (H9C2 cells), EPO also reduced the increase in DNA fragmentation caused by either serum deprivation (simulated ischemia) or hydrogen peroxide (oxidative stress). We propose that the acute administration of EPO on reperfusion and/or resuscitation will reduce the tissue injury caused by ischemia-reperfusion of the heart (and other organs) and hemorrhagic shock.

Gsk-3β inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat

Objective:Serine-threonine protein kinase glycogen synthase kinase (GSK)-3 is involved in regulation of many cell functions, but its role in regulation of inflammatory response is unknown. Here we investigate the effects of GSK-3β inhibition on organ injury/dysfunction caused by lipopolysaccharide or coadministration of lipopolysaccharide and peptidoglycan in the rat. Design:Prospective, randomized study. Setting:University-based research laboratory. Subjects:Ninety-nine anesthetized male Wistar rats. Interventions:Study 1: Rats received either intravenous Escherichia coli lipopolysaccharide (6 mg/kg) or vehicle (1 mL/kg; saline). Study 2: Rats received either intravenous E. coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle. The potent and selective GSK-3β inhibitors TDZD-8 (1 mg/kg intravenously), SB216763 (0.6 mg/kg intravenously), and SB415286 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered 30 mins before lipopolysaccharide or lipopolysaccharide and peptidoglycan. Measurements and Main Results:Endotoxemia resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), aspartate aminotransferase, alanine aminotransferase (markers for hepatocellular injury), lipase (indicator of pancreatic injury), and creatine kinase (indicator of neuromuscular injury). Coadministration of lipopolysaccharide and peptidoglycan resulted in hepatocellular injury and renal dysfunction. All GSK-3β inhibitors attenuated the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan. GSK-3β inhibition reduced the Ser536 phosphorylation of nuclear factor-&kgr;B subunit p65 and the messenger RNA expression of nuclear factor-&kgr;B-dependent proinflammatory mediators but had no effect on the nuclear factor-&kgr;B/DNA binding activity in the lung. GSK-3β inhibition reduced the increase in nuclear factor-&kgr;B p65 activity caused by interleukin-1 in human embryonic kidney cells in vitro. Conclusions:The potent and selective GSK-3β inhibitors TDZD-8, SB216763, and SB415286 reduced the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan in the rat. We propose that GSK-3β inhibition may be useful in the therapy of the organ injury/dysfunction associated with sepsis, shock, and other diseases associated with local or systemic inflammation.

Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis:

Calpain and caspase are families of cysteine proteases that have important roles in the initiation, regulation and execution of cell death. The function of both groups of proteases in the progression of apoptotic and necrotic pathways is presented here in the context of a concise overview of regulated cell death. Many of the morphological differences between apoptotic and necrotic processes are thought to be as a consequence of the action of cysteine proteases. Recent studies suggest that caspase and calpain cascades are tightly interrelated and an appreciation of how these proteases cross-talk should enable a greater understanding of how the boundaries between apoptotic and necrotic cell death have become blurred. Furthermore, an assessment of the contribution that caspase and calpain make to human physiology and pathology is provided, with a description of how these proteases can be detected and quantified. Lastly, an evaluation is made of how caspase and calpain activation might be exploited diagnostically.

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF‐κB (kidney), expression of iNOS and COX‐2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF‐κB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF‐κB to DNA and the degradation of IκBα, IKBβ, and IκBε. Selective inhibition of iNOS activity with L‐NIL reduced the circulatory failure and liver injury, while selective inhibition of COX‐2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF‐κB and thus prevention of the expression of NFκB‐dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX‐2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.—McDonald, M. C., Mota‐Filipe, H., Paul, A., Cuzzocrea, S., Abdelrahman, M., Harwood, S., Plevin, R., Chatterjee, P. K., Yaqoob, M. M., Thiemermann, C. Calpain inhibitor I reduces the activation of nuclear factor‐κB and organ injury/dysfunction in hemorrhagic shock. FASEB J. 15, 171–186 (2001)

Recombinant human erythropoietin protects the liver from hepatic ischemia-reperfusion injury in the rat

Recently, erythropoietin was shown to have both hematopoietic as well as tissue‐protective properties. Erythropoietin (EPO) had a protective effect in animal models of cerebral ischemia, mechanical trauma of the nervous system, myocardial infarction, and ischemia‐reperfusion (I/R) injury of the kidney. It is not known whether EPO protects the liver against I/R injury. Using a rat model of liver I/R injury, we aimed to determine the effect of the administration of human recombinant erythropoietin (rhEPO) on liver injury. Rats were subjected to 30 min of liver ischemia followed by 2 h of reperfusion. When compared with the sham‐operated rats, I/R resulted in significant rises in the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, gamma‐glutamyl transferase, tissue lipid peroxidation, caspase‐3 activity and altered histology. Administration of rhEPO 5 min before ischemia was able to reduce the biochemical evidence of liver injury; however, this protection was not evident when rhEPO was administered 5 min before reperfusion. Mechanistically, early administration of rhEPO was able to reduce the oxidative stress and caspase‐3 activation, suggesting the subsequent reduction of apoptosis. This study provides the first evidence that rhEPO causes a substantial reduction of the liver injury induced by I/R in the rat.

Dexamethasone Ameliorates Renal Ischemia-Reperfusion Injury

In the setting of renal ischemia-reperfusion injury (IRI), the effect and mechanism of action of glucocorticoids are not well understood. In rat renal IRI, a single dose of dexamethasone administered before ischemia, or at the onset of reperfusion, ameliorated biochemical and histologic acute kidney injury after 24 h. Dexamethasone upregulated Bcl-xL, downregulated ischemia-induced Bax, inhibited caspase-9 and caspase-3 activation, and reduced apoptosis and necrosis of proximal tubular cells. In addition, dexamethasone decreased the number of infiltrating neutrophils and ICAM-1. We observed the protective effect of dexamethasone in neutrophil-depleted mice, suggesting a neutrophil-independent mechanism. In vitro, dexamethasone protected human kidney proximal tubular (HK-2) cells during serum starvation and IRI-induced apoptosis, but inhibition of MEK 1/2 abolished its anti-apoptotic effects in these conditions. Dexamethasone stimulated rapid and transient phosphorylation of ERK 1/2, which required the presence of the glucocorticoid receptor and was independent of transcriptional activity. In summary, in the setting of renal ischemia-reperfusion injury, dexamethasone directly protects against kidney injury by a receptor-dependent, nongenomic mechanism.