Paul A.J. Brown

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.

Agonists of Peroxisome-Proliferator Activated Receptor-Gamma Reduce Renal Ischemia/Reperfusion Injury

Background/Aims: Recent evidence indicates that peroxisome-proliferator activated receptor (PPAR) agonists protect against ischemia/reperfusion (I/R) injury. Here we investigate the effects of the PPAR-γ agonists, rosiglitazone and ciglitazone, on the renal dysfunction and injury caused by I/R of the rat kidney in vivo. Methods: Rosiglitazone or ciglitazone were administered to male Wistar rats prior to and during reperfusion. Biochemical indicators of renal dysfunction and injury were measured and histological scoring of kidney sections was used to assess renal injury. Expression of PPAR isoforms and intercellular adhesion molecule-1 during renal I/R were assessed using RT-PCR and Northern blot, respectively. Myeloperoxidase activity and activation of poly(ADP-ribose) polymerase (PARP) were used as indicators of polymorphonuclear (PMN) cell infiltration and oxidative stress, respectively. Results: Expression of PPAR-α, PPAR-β and PPAR-γ1 (but not PPAR-γ2) was observed in kidneys with down-regulation of PPAR-α expression during renal I/R. Rosiglitazone and ciglitazone significantly reduced biochemical and histological signs of renal dysfunction and injury. Renal expression of ICAM-1 caused by I/R was reduced by rosiglitazone and ciglitazone which was reflected by decreased PMN infiltration into reperfused renal tissues. Both rosiglitazone and ciglitazone reduced PARP activation indicating a reduction of oxidative stress. Conclusion: These results suggest that the PPAR-γ agonists rosiglitazone and ciglitazone reduce the renal dysfunction and injury associated with I/R of the kidney. We propose that one mechanism underlying the protective effects involves inhibition of the expression of ICAM-1, a reduction of PMN infiltration into renal tissues and subsequent reduction of oxidative stress.

Generation of endogenous hydrogen sulfide by cystathionine γ-lyase limits renal ischemia/reperfusion injury and dysfunction

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine γ-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-κB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-κB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

Inhibitors of fibrinolysis are elevated in atherosclerotic plaque.

The proteins of the fibrinolytic system have been examined in the human normal and atherosclerotic arterial wall by immunohistochemical techniques and by quantitative immunoassay of extracts. The concentration of plasminogen activator inhibitor-1 (PAI-1) increased significantly during the progression from normal vessels to fatty streaks to the developed atherosclerotic plaque. Staining for PAI-1 was strongly positive, particularly in the areas adjacent to the plaque. In these areas, PAI-1 appeared to colocalized with its binding protein vitronectin. Alpha2-antiplasmin (alpha2-AP) was present in the aorta at even higher concentrations than PAI-1; a small but significant increase was seen in some atherosclerotic compared with normal vessel walls. Tissue plasminogen activator (TPA) showed the opposite trend, being lowest in lesions with plaque. Thus, higher concentrations of the two principal inhibitors of fibrinolysis, PAI-1 and alpha2-AP, together with lower levels of TPA, are characteristic of advanced atheromatous lesions. Alteration in the balance of the fibrinolytic system, favoring its inhibition, may predispose to the development or maintenance of atherosclerotic plaque.

High density lipoprotein (HDL) reduces renal ischemia/reperfusion injury

High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) infiltrating into renal tissues during reperfusion, which was reflected by an attenuation of the increase in renal myeloperoxidase activity caused by I/R. Furthermore, HDL markedly reduced expression of the adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and P-selectin during reperfusion. The increase in renal malondialdehyde levels caused by renal I/R was also significantly reduced by HDL, suggesting attenuation of lipid peroxidation subsequent to oxidative stress. These results demonstrate that HDL significantly reduces renal I/R injury and severity of ischemic acute renal failure. It is proposed that the mechanism of protection involves reduction of the expression of adhesion molecules, resulting in reduction of PMN infiltration and oxidative stress.

Cyclin kinase inhibitor p21CIP1/WAF1 limits interstitial cell proliferation following ureteric obstruction.

Tubulointerstitial renal injury induced by unilateral ureteric obstruction (UUO) is characterized by marked cell proliferation and apoptosis. Proliferation requires cell cycle transit that is positively regulated by cyclins and cyclin-dependent kinases (CDKs) and inhibited by the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs: p21, p27, and p57). We have shown that the absence of p27 results in markedly increased tubular epithelial cell proliferation and apoptosis following UUO (V. Ophascharoensuk, M. L. Fero, J. Hughes, J. M. Roberts, and S. J. Shankland. Nat. Med. 4: 575-580, 1998). Since p21 mRNA is upregulated following UUO, we hypothesized that p21 would also serve to limit cell proliferation and apoptosis. We performed UUO in p21 +/+ and p21 -/- mice. Cell proliferation [bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA)], apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method], interstitial myofibroblast accumulation (actin), macrophage infiltration (F4/80), and collagen I expression were quantified at days 3, 7, and 14. In contrast to p27 -/- mice, there was no difference in tubular epithelial cell proliferation or apoptosis between p21 -/- and p21 +/+ mice at any time point. However, interstitial cell proliferation at day 3 was significantly increased in p21 -/- mice [BrdU, 40.7 +/- 1.9 cells/high-power field (cells/hpf) vs. 28.8 +/- 2, P < 0.005], although, interestingly, no difference was seen in interstitial cell apoptosis. Actin/BrdU double staining demonstrated increased interstitial myofibroblast proliferation at day 3 in p21 -/- animals (10 +/- 0.12 vs. 5.8 +/- 0. 11 cells/hpf, P < 0.05), which was followed by increased myofibroblast accumulation at day 7 in p21 -/- mice. No differences were detected in interstitial macrophage infiltration, collagen I deposition or transforming growth factor-beta1 mRNA (in situ hybridization) expression. In conclusion p21, unlike p27, is not essential for the regulation of tubular epithelial cell proliferation and apoptosis following UUO, but p21 levels do serve to limit the magnitude of the early myofibroblast proliferation. This study demonstrates a differential role for the CKI p21 and p27 in this model.Tubulointerstitial renal injury induced by unilateral ureteric obstruction (UUO) is characterized by marked cell proliferation and apoptosis. Proliferation requires cell cycle transit that is positively regulated by cyclins and cyclin-dependent kinases (CDKs) and inhibited by the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs: p21, p27, and p57). We have shown that the absence of p27 results in markedly increased tubular epithelial cell proliferation and apoptosis following UUO (V. Ophascharoensuk, M. L. Fero, J. Hughes, J. M. Roberts, and S. J. Shankland. Nat. Med.4: 575-580, 1998). Since p21 mRNA is upregulated following UUO, we hypothesized that p21 would also serve to limit cell proliferation and apoptosis. We performed UUO in p21 +/+ and p21 -/- mice. Cell proliferation [bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA)], apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method], interstitial myofibroblast accumulation (actin), macrophage infiltration (F4/80), and collagen I expression were quantified at days 3, 7, and 14. In contrast to p27 -/- mice, there was no difference in tubular epithelial cell proliferation or apoptosis between p21 -/- and p21 +/+ mice at any time point. However, interstitial cell proliferation at day 3 was significantly increased in p21 -/- mice [BrdU, 40.7 ± 1.9 cells/high-power field (cells/hpf) vs. 28.8 ± 2, P< 0.005], although, interestingly, no difference was seen in interstitial cell apoptosis. Actin/BrdU double staining demonstrated increased interstitial myofibroblast proliferation at day 3 in p21 -/- animals (10 ± 0.12 vs. 5.8 ± 0.11 cells/hpf, P < 0.05), which was followed by increased myofibroblast accumulation at day 7 in p21 -/- mice. No differences were detected in interstitial macrophage infiltration, collagen I deposition or transforming growth factor-β1 mRNA (in situ hybridization) expression. In conclusion p21, unlike p27, is not essential for the regulation of tubular epithelial cell proliferation and apoptosis following UUO, but p21 levels do serve to limit the magnitude of the early myofibroblast proliferation. This study demonstrates a differential role for the CKI p21 and p27 in this model.

Upregulation and co-localization of connexin43 and cellular adhesion molecules in inflammatory renal disease

Connexin43 (Cx43) is a major component of gap junctions. These are widely distributed in the human kidney and are thought to be involved in the inflammatory response and in the regulation of cell growth. Cellular adhesion molecules (CAMs) are also thought to be important in these processes, where they possibly facilitate gap junction formation. The aims of the current study were to define for the first time the expression of Cx43 in inflammatory glomerulonephritis and to compare the localization of this connexin with that of the intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and E‐selectin. Human renal biopsies and control sections of normal human kidney were stained using the alkaline phosphatase/anti‐alkaline phosphatase immunohistochemical technique, demonstrating that Cx43 was strongly expressed on inflammatory cells, on damaged tubular cells, and on interstitial cells. This pattern of expression was paralleled closely by that of ICAM‐1 and, to a lesser extent, by that of VCAM‐1. Cx43 is therefore primarily implicated in tubulointerstitial inflammation.© 1997 John Wiley & Sons, Ltd.

Cisplatin nephrotoxicity is mediated by gamma glutamyltranspeptidase, not via a C-S lyase governed biotransformation pathway.

Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the mechanism of cisplatin nephrotoxicity in vivo and in an in vitro model system. Nephrotoxicity was induced in rats (6 mg/kg cisplatin i.p.) and mice (10 mg/kg cisplatin i.p.). Cisplatin administration significantly elevated blood urea nitrogen (BUN) and serum creatinine in male Sprague Dawley rats day 5 post-treatment (BUN Delta+28+/-5 micromol/ml; serum creatinine Delta+108+/-4 nmol/ml, P<0.05) and in male C57BL6 mice day 4 post-treatment (BUN Delta+21+/-4 micromol/ml; serum creatinine Delta+81+/-5 nmol/ml, P<0.05). Nephrotoxicity was confirmed by histological analysis that revealed significant damage to the proximal tubules of cisplatin- versus saline vehicle-treated animals. Inhibition of gamma glutamyltranspeptidase prevented cisplatin nephrotoxicity in Sprague Dawley rats (day 5 BUN Delta+1+/-2 micromol/ml; serum creatinine Delta+8+/-4 nmol/ml) and C57BL6 mice (day 4 BUN Delta+1+/-0.8 micromol/ml; serum creatinine Delta-1+/-2 nmol/ml), but not cellular toxicity in rat proximal tubular (RPT) or human proximal tubular (HPT) cultures. Inhibition of aminopeptidase N (AP-N) or renal dipeptidase (RDP) in male Sprague Dawley rats, or in RPT and HPT cell cultures, did not reduce cisplatin toxicity. In contrast to published findings inhibition of C-S lyase did not prevent the nephrotoxicity of cisplatin in vivo or cellular toxicity in vitro. These data demonstrate that the biotransformation enzymes AP-N, RDP and C-S lyase are not implicated in the metabolism of cisplatin to a nephrotoxic metabolite as has been previously hypothesised. Instead, our data demonstrate that gamma glutamyltranspeptidase is a key enzyme involved in mediating cisplatin nephrotoxicity, which potentially acts to cleave cisplatin-GSH conjugates to a toxic metabolite.

EUK-134 reduces renal dysfunction and injury caused by oxidative and nitrosative stress of the kidney

Background/Aims: Oxidative and nitrosative stress plays important roles in the pathogenesis of renal ischemia/reperfusion (I/R) injury. Here we investigate the effect of EUK-134, a synthetic superoxide dismutase and catalase mimetic, (i) on renal dysfunction and injury caused by I/R in vivo and (ii) on proximal tubular cell (PTC) injury and death caused by oxidative and nitrosative stress. Methods: Rats, subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h), were administered EUK-134 (0.3 and 3 mg/kg, i.v.) prior to and during reperfusion, after which biochemical and histological indicators of renal dysfunction and injury were measured. The expression of poly(ADP-ribose) (PAR) and inducible nitric oxide (NO) synthase (iNOS) and nitrotyrosine formation were determined immunohistochemically and used as indicators of oxidative and nitrosative stress. Primary cultures of rat PTCs, isolated and cultured from the kidney cortex, were incubated with hydrogen peroxide (H2O2; 1 mM for 2 h) in the presence of increasing concentrations of EUK-134 (1–100 µM) after which PTC injury and death were measured. The effects of EUK-134 on serum levels of NO in rats subjected to renal I/R or on NO production by PTCs incubated with interferon-γ (IFN-γ, 100 IU/ml) and bacterial lipopolysaccharide (LPS, 10 µg/ml) in combination for 24 h were also measured. Results: EUK-134 produced a significant reduction in renal dysfunction and injury caused by I/R. Specifically, serum creatinine levels, an indicator of renal dysfunction, were reduced from 227 ± 11 (n = 12, I/R only) to 146 ± 9 µM (n = 12, I/R +3 mg/kg EUK-134). Urinary N-acetyl-β-D-glucosaminidase activity, an indicator of tubular damage, was reduced from 42 ± 5 (n = 12, I/R only) to 22 ± 3 IU/l (n = 12, I/R +3 mg/kg EUK-134). EUK-134 significantly reduced renal injury caused by oxidative stress in vivo (reduction in PAR formation), and in vitro EUK-134 reduced PTC injury and death caused by H2O2. However, EUK-134 also reduced nitrosative stress caused by I/R in vivo (reduction of iNOS expression and nitrotyrosine formation), which was reflected by a significant reduction in serum NO levels in rats subjected to renal I/R. Specifically, serum NO levels were reduced from 57 ± 12 (n = 12, I/R only) to 23 ± 3 mM (n = 12, I/R +3 mg/kg EUK-134). In vitro, EUK-134 significantly reduced NO production by PTCs incubated with IFN-γ/LPS. Conclusion: We propose that EUK-134 reduces renal I/R injury not only via reduction of oxidative stress, but also by reducing nitrosative stress caused by renal I/R.

A Comparison of Peritoneal Transport in Patients with Psoriasis and Uremia

Peritoneal solute equilibration studies during prolonged intraperitoneal dwell were performed in patients with psoriasis and normal renal function, and compared to patients with renal failure undergoing peritoneal dialysis. Calcium, phosphorous, uric acid and protein equilibration were faster in uremics. No differences were observed for sodium and chloride. Mass transfer area coefficients (a measure of the mass transfer at infinite dialysate flow rates) were calculated for glucose, protein and uric acid and found to be complimentary to the results obtained by studies of solute equilibration. Possible reasons for the differences obtained are discussed.