Gur P. Kaushal

Apoptotic mechanisms in acute renal failure

It has been generally accepted that a catastrophic breakdown of regulated cellular homeostasis, known as necrosis, is the mode of cellular injury in various forms of acute renal failure. One of the major advances in our understanding of cell death has been the recognition that the pathways traditionally associated with apoptosis as described in the landmark study by Kerr, Wyllie, and Currie in 1972 maybe very critical in the form of cell injury associated with necrosis. The pathway that is followed by the cell varies with both nature and severity of insults and may evolve from an apoptotic to a necrotic form of cell death. It is also likely that there are some common pathways that are shared and regulated in the two modes of cell death. In this review, we first describe evidence for the role of apoptotic pathways in ischemic acute renal failure, and then consider the potential mechanisms that may participate in this model of acute renal tubular injury. We then summarize the current information of apoptotic pathways related to other common causes of acute renal failure including endotoxin-induced, toxic acute renal failure and transplant rejection. A better understanding of the mechanisms of apoptosis could lead to safer and more specific therapeutic interventions for acute renal failure.

The JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide

Assessment of specific apoptosis and survival pathways implicated in anticancer drug action is important for understanding drug mechanisms and modes of resistance in order to improve the benefits of chemotherapy. In order to better examine the role of mitogen-activated protein kinases, including JNK and ERK, as well as the tumor suppressor p53, in the response of tumor cells to chemotherapy, we compared the effects on these pathways of three structurally and functionally distinct antitumor agents. Drug concentrations equal to 50 times the concentration required to reduce cell proliferation by 50% were used. Vinblastine, doxorubicin, or etoposide (VP-16) induced apoptotic cell death in KB-3 carcinoma cells, with similar kinetic profiles of PARP cleavage, caspase 3 activation, and mitochondrial cytochrome c release. All three drugs strongly activated JNK, but only vinblastine induced c-Jun phosphorylation and AP-1 activation. Inhibition of JNK by SP600125 protected cells from drug-induced cytotoxicity. Vinblastine caused inactivation of ERK whereas ERK was unaffected in cells exposed to doxorubicin or VP-16. Inhibition of ERK signaling by the MEK inhibitor, U0126, potentiated the cytotoxic effects of vinblastine and doxorubicin, but not that of VP-16. Vinblastine induced p53 downregulation, and chemical inhibition of p53 potentiated vinblastine-induced cell death, suggesting a protective effect of p53. In contrast, doxorubicin and VP-16 induced p53, and inhibition of p53 decreased drug-induced cell death, suggesting a pro-apoptotic role for p53. These results highlight the differential roles played by several key signal transduction pathways in the mechanisms of action of key antitumor agents, and suggest ways to specifically potentiate their effects in a context-dependent manner. In addition, the novel finding that JNK activation can occur without c-Jun phosphorylation or AP-1 activation has important implications for our understanding of JNK function.

Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury

In the present study, we demonstrate that rat kidney contains caspase activity that was markedly inhibited by specific peptide inhibitors of caspases but not by inhibitors of Ser, Cys, Asp, or metalloproteinases. Using primers based on the nucleotide sequence of known members of Ced-3/interleukin-1β-converting enzyme (ICE) family from human origin, we have identified by reverse-transcription (RT) polymerase chain reaction (PCR) analyses that rat kidney transcribes the genes for caspase-1 (ICE), caspase-2 (Nedd2), caspase-3 (CPP32), and caspase-6 (Mch2). RT-PCR products, when subcloned and sequenced, provided full-length cDNAs for ICE (1,209 bp) and CPP32 (786 bp) and partial cDNA products for Mch2 (561 bp) and Nedd2 (811 bp). The sequence analysis of the caspase cDNAs showed conserved catalytic site QACRG as well as Asp cleavage site. Rat kidneys subjected to ischemia-reperfusion injury revealed differential expression of caspases with marked increase in CPP32 and ICE mRNA and proteins during reperfusion, transient increase in Nedd2 mRNA and proteins during ischemia and the early period of reperfusion, and little change in Mch2 expression during the ischemia or reperfusion period. The altered expression suggests that caspases may act in concert in a cascade and may play an important role in ischemic acute renal failure.In the present study, we demonstrate that rat kidney contains caspase activity that was markedly inhibited by specific peptide inhibitors of caspases but not by inhibitors of Ser, Cys, Asp, or metalloproteinases. Using primers based on the nucleotide sequence of known members of Ced-3/interleukin-1 beta-converting enzyme (ICE) family from human origin, we have identified by reverse-transcription (RT) polymerase chain reaction (PCR) analyses that rat kidney transcribes the genes for caspase-1 (ICE), caspase-2 (Nedd2), caspase-3 (CPP32), and caspase-6 (Mch2). RT-PCR products, when subcloned and sequenced, provided full-length cDNAs for ICE (1,209 bp) and CPP32 (786 bp) and partial cDNA products for Mch2 (561 bp) and Nedd2 (811 bp). The sequence analysis of the caspase cDNAs showed conserved catalytic site QACRG as well as Asp cleavage site. Rat kidneys subjected to ischemia-reperfusion injury revealed differential expression of caspases with marked increase in CPP32 and ICE mRNA and proteins during reperfusion, transient increase in Nedd2 mRNA and proteins during ischemia and the early period of reperfusion, and little change in Mch2 expression during the ischemia or reperfusion period. The altered expression suggests that caspases may act in concert in a cascade and may play an important role in ischemic acute renal failure.

Purification and properties of glucosidase I from mung bean seedlings

The microsomal enzyme fraction from mung bean seedlings was found to contain glucosidase activity capable of releasing [3H]glucose from the glucose-labeled Glc3Man9GlcNAc. The enzymatic activity could be released in a soluble form by treating the microsomal particles with 1.5% Triton X-100. When the solubilized enzyme fraction was chromatographed on DE-52, it was possible to resolve glucosidase I activity (measured by the release of [3H]glucose from Glc3Man9GlcNAc) from glucosidase II (measured by release of [3H]glucose from Glc2Man9GlcNAc). The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose, and concanavalin A-Sepharose. The purified enzyme was free of glucosidase II and aryl-glucosidase activities. Only a single glucose residue could be released from the Glc3Man9GlcNAc by this purified enzyme and the other product was the Glc2Man9GlcNAc. Furthermore, this enzyme was inhibited in a dose-dependent manner by kojibiose, an alpha-1,2-linked glucose disaccharide, but not by other alpha-linked glucose disaccharides. These data indicate that this glucosidase is a specific alpha-1,2-glucosidase. The pH optimum for the glucosidase I was about 6.3 to 6.5, and no requirements for divalent cations were observed. The enzyme was inhibited strongly by the glucosidase processing inhibitors, castanospermine and deoxynojirimycin, and less strongly by the plant pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. However, the enzyme was not inhibited by the mannosidase processing inhibitors, swainsonine, deoxymannojirimycin or 1,4-dideoxy-1,4-imino-D-mannitol. The stability of the enzyme under various conditions and other properties of the enzyme were determined.

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation, and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity. Addedum to: Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy and apoptosis are associated in cisplatin injury to renal tubular epithelial cell injury. Am J Physiol Renal Physiol 2008; 294:F777-87.

The new kids on the block: ADAMTSs, potentially multifunctional metalloproteinases of the ADAM family

Cell-cell and cell-matrix interactions are of vital importance not only for proper cellular homeostasis during embryogenesis and development of an organism, but also in pathological states in diseases ranging from tumor metastasis to AIDS. Tissues owe their dynamic structure both to changes in expression of adhesive proteins and their receptors and to the regulated action of secreted proteinases, particularly members of the metalloproteinase family. Many of these secreted and cell surface proteins and metalloproteinases are found at critical locations that facilitate their involvement in cell-cell and cell-matrix interactions. Metalloproteinases belong to a superfamily of zinc-dependent proteases known as metzincins. Based on sequence and structural similarities, metzincins

Transcriptional activation of caspase-6 and -7 genes by cisplatin-induced p53 and its functional significance in cisplatin nephrotoxicity.

This study examined the role of cisplatin-induced p53 activation in regulation of caspases and cellular injury during cisplatin nephrotoxicity. The executioner caspase-6 and -7 but not caspase-3 were identified as transcriptional targets of p53 in cisplatin injury as revealed by chromatin immunoprecipitation, a reporter gene and electrophoretic mobility shift assays, and real-time PCR following overexpression and inhibition of p53. DNA binding by p53 involved the first introns of the human and mouse caspase-7 gene and the mouse caspase-6 gene. Studies in human kidney, breast, ovary, colon, and prostate tumor cell lines also validated these findings. Treatment of p53 (−/−) cells with cisplatin did not induce caspase-6 and -7 expression and subsequent activation. In caspase-3 (−/−) cells, inhibition of caspase-6 and -7 activations markedly prevented cisplatin-induced cell death. In an in vivo model of cisplatin nephrotoxicity inhibition of p53 activation by a p53 inhibitor suppressed transactivation of the caspase-6 and -7 genes and prevented renal failure. p53 (−/−) mice were resistant to cisplatin nephrotoxicity as assessed by renal function and histology. These studies provide first evidence for p53-dependent transcriptional control of the caspase-6 and -7 genes and its functional significance in cisplatin injury to renal cells and functional implication of cisplatin-induced p53 induction in vitro and in vivo in cisplatin nephrotoxicity.

Apoptotic Pathways of Oxidative Damage to Renal Tubular Epithelial Cells

Toxic renal failure induced by gentamicin, glycerol, or cisplatin, as well as ischemic renal failure in vivo and hypoxia/reoxygenation of tubular epithelial cells in vitro, induces the production of reactive oxygen metabolites (ROM). Generation of ROM is responsible for the induction of tubular epithelial cell death, which is mediated by caspases and/or endonucleases. Scavenging of ROM protects tubular epithelium from caspase and endonuclease activation and from cell death. Thus, the inhibition of ROM production combined with the pharmacological control of caspase and endonuclease pathways may provide future modalities in the prevention or treatment of acute renal failure in humans.

Thalidomide protects endothelial cells from doxorubicin‐induced apoptosis but alters cell morphology

Summary.  Antiangiogenesis agents are now being used in clinical trials to reduce the risk of recurrence of cancer. Several of these agents, however, are associated with thrombosis, especially when used in combination with chemotherapy. Antiangiogenesis and thrombosis are both endothelial‐related activities, and we therefore evaluated one presumed antiangiogenesis agent (thalidomide) on intact cultured endothelial cells, and on cultured endothelial cells injured by preincubation with doxorubicin. We evaluated cell viability, caspase‐3 activation, morphology of cells using light microscopy, and protease activated receptor‐1 (PAR‐l) expression. In our experiments, doxorubicin induced a dose‐ and incubation time‐dependent and caspase‐3‐mediated apoptosis of endothelial cells. Thalidomide alone caused no changes in intact endothelial cells in terms of morphology, cell viability or activation of caspase‐3. In contrast, when thalidomide was added to doxorubicin‐injured endothelial cells, there was protection from cell death, increase in viability of endothelial cells, induction of differentiation and formation of neotubules. Doxorubicin reduced the expression of thrombin receptor, PAR‐1, as evaluated by immunostaining and flow cytometry. Thalidomide did not alter PAR‐1 expression in untreated cells but restored its expression reduced by doxorubicin. These findings suggest that thalidomide may be procoagulant, not by enhancing doxorubicin‐mediated endothelial cell injury, but by altering the expression of PAR‐1 on injured endothelium and resulting in endothelial dysfunction, which may explain hypercoagulability in patients treated with chemotherapy followed by thalidomide.

Autophagy in acute kidney injury

Autophagy is a conserved multistep pathway that degrades and recycles damaged organelles and macromolecules to maintain intracellular homeostasis. The autophagy pathway is upregulated under stress conditions including cell starvation, hypoxia, nutrient and growth-factor deprivation, endoplasmic reticulum stress, and oxidant injury, most of which are involved in the pathogenesis of acute kidney injury (AKI). Recent studies demonstrate that basal autophagy in the kidney is vital for the normal homeostasis of the proximal tubules. Deletion of key autophagy proteins impaired renal function and increased p62 levels and oxidative stress. In models of AKI, autophagy deletion in proximal tubules worsened tubular injury and renal function, highlighting that autophagy is renoprotective in models of AKI. In addition to nonselective sequestration of autophagic cargo, autophagy can facilitate selective degradation of damaged organelles, particularly mitochondrial degradation through the process of mitophagy. Damaged mitochondria accumulate in autophagy-deficient kidneys of mice subjected to ischemia-reperfusion injury, but the precise mechanisms of regulation of mitophagy in AKI are not yet elucidated. Recent progress in identifying the interplay of autophagy, apoptosis, and regulated necrosis has revived interest in examining shared pathways/molecules in this crosstalk during the pathogenesis of AKI. Autophagy and its associated pathways pose potentially unique targets for therapeutic interventions in AKI.