Toll-like receptors (TLRs) are now recognized as the major receptors for microbial pathogens on cells of the innate immune system. Recently, TLRs were also identified in many organs including the kidney. However, the cellular distribution and role of these renal TLRs remain largely unknown. In this paper, we investigated the expression of TLR4 in a cecal ligation and puncture (CLP) model of sepsis in Sprague-Dawley rats utilizing fluorescence microscopy. In sham animals, TLR4 was expressed predominantly in Tamm-Horsfall protein (THP)-positive tubules. In CLP animals, TLR4 expression increased markedly in all tubules ( proximal and distal), glomeruli, and the renal vasculature. The staining showed a strong apical distribution in all tubules. A moderately less intense cellular signal colocalized partially with the Golgi apparatus. In addition, kidneys from septic rats showed increased expression of CD14 and THP. They each colocalized strongly with TLR4, albeit in different tubular segments. We also imaged the kidneys of live septic animals with two-photon microscopy after fluorescent lipopolysaccharide (LPS) injection. Within 10 min, LPS was seen at the brush border of some proximal tubules. Within 60 min, LPS was fully cytoplasmic in proximal tubules. Conversely, distal tubules showed no LPS uptake. We conclude that TLR4, CD14, and THP have specific renal cellular and tubular expression patterns that are markedly affected by sepsis. Systemic endotoxin can freely access the tubular and cellular sites where these proteins are present. Therefore, locally expressed TLRs and other interacting proteins could potentially modulate the renal response to systemic sepsis.

Tetracyclines exhibit significant anti-inflammatory properties, inhibit matrix metalloproteinases (MMPs), and are protective in models of ischemia-reperfusion injury (IRI). Both inflammatory cascades and MMP activation have been demonstrated to modulate microvascular permeability. Because increased microvascular permeability occurs during IRI in a variety of organ systems including the kidney, we hypothesized that minocycline, a semisynthetic tetracycline, would diminish microvascular leakage during renal IRI. To test this hypothesis, we used intravital 2-photon microscopy to examine leakage of fluorescent dextrans from the vasculature in a rodent model of IRI. Minocycline significantly reduced the extent of dextran ( 500 kDa) leakage from the renal microvasculature 24 h after ischemia. Although minocycline diminished leukocyte accumulation in the kidney following ischemia, areas of leukocyte accumulation did not correlate with areas of microvascular permeability in either the saline- or minocycline-pretreated animals. Minocycline diminished the perivascular increase in MMP-2 and MMP-9, as well as the increase in MMP-2 activity 24 h after ischemia. ABT-518, a specific inhibitor of MMP-2 and MMP-9, also significantly reduced the extent of dextran ( 500 kDa) leakage from the renal microvasculature 24 h after ischemia. Our results indicate that minocycline mitigates the renal microvascular permeability defect following IRI. This effect is spatially distinct from the effect of minocycline on leukocyte accumulation and may be related to diminished activity of MMPs on the integrity of the perivascular matrix.

Tetracyclines exhibit significant anti-inflammatory properties in a variety of rheumatologic and dermatologic conditions. They have also been shown to inhibit apoptosis in certain neurodegenerative disorders. Because ischemic renal injury is characterized by both apoptosis and inflammation, we investigated the therapeutic potential of tetracyclines in a rat model of renal ischemia-reperfusion. Male Sprague-Dawley rats underwent bilateral renal artery clamp for 30 min followed by reperfusion and received either minocycline or saline for 36 h before ischemia. Minocycline reduced tubular cell apoptosis 24 h after ischemia as determined by terminal transferase-mediated dUTP nick end-labeling staining and nuclear morphology. It also decreased cytochrome c release into the cytoplasm and reduced upregulation of p53 and Bax after ischemia. The minocycline-treated group showed a significant reduction in tubular injury and cast formation. In addition, minocycline reduced the number of infiltrating leukocytes, decreased leukocyte chemotaxis both in vitro and ex vivo, and downregulated the expression of ICAM-1. Serum creatinine 24-h postischemia was significantly reduced in the minocycline-treated group. We conclude that minocycline has potent antiapoptotic and anti-inflammatory properties and protects renal function in this model of ischemia-reperfusion. Tetracyclines are among the safest and best-studied antibiotics. They are thus attractive candidates for the therapy of human ischemic acute renal failure.

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. GTP depletion was recently identified as an important inducer of apoptosis during chemical anoxia in vitro and ischemic injury in vivo. It has also been shown that GTP salvage with guanosine prevented apoptosis and protected function. This study investigates the role of p53 in mediating the apoptotic response to GTP depletion. Male Sprague-Dawley rats underwent bilateral renal artery clamp for 30 min followed by reperfusion. p53 protein levels increased significantly in the medulla over 24 h post-ischemia. The provision of guanosine inhibited the increase in p53. Pifithrin-alpha, a specific inhibitor of p53, mimicked the effects of guanosine. It had no effect on necrosis, yet it prevented apoptosis and protected renal function. Pifithrin-alpha was protective when given up to 14 h after the ischemic insult. The effects of pifithrin-alpha on p53 included inhibition of transcriptional activation of downstream p53 targets like p21 and Bax and inhibition of p53 translocation to the mitochondria. Similar results were obtained in cultured renal tubular cells. It is concluded that p53 is an important mediator of apoptosis during states of GTP depletion. Inhibitors of p53 should be considered in the treatment of ischemic renal injury.

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. Recently, we linked anoxia-induced apoptosis in renal cell cultures specifically to the depletion of GTP. We therefore hypothesized that enhancing GTP repletion in vivo might protect function by reducing apoptosis in postischemic tubules. Male C57 black mice (the "I" group of animals) underwent bilateral renal artery clamp for 32 minutes to induce ischemia and then received either normal saline ("NS") or guanosine ("G"). After 1 hour of reperfusion, renal GTP levels in NS/I were reduced to nearly half of those in sham operated mice, whereas these levels were nearly unchanged in G/I mice. Morphologic examination of tubular injury revealed no significant differences between the two groups. However, there was a significant reduction in the number of apoptotic tubular cells in the medulla in the G/I group as compared with the NS/I group. At 24 hours, creatinine was significantly elevated in the NS/I group, compared to the G/I group. We conclude that guanosine protects against renal ischemic injury by replenishing GTP stores and preventing tubular apoptosis.

Guanine nucleosides are toxic to some forms of cancer. This toxicity is pronounced in cancers with upregulated guanine nucleotide synthesis, but the mechanisms are poorly understood. We investigated this toxicity by measuring the effects of guanine nucleosides on nucleotides in Jurkat cells using HPLC. We also measured proliferation and cell death with microscopy and fluorescence-activated cell sorting. Guanosine increased GTP to 600% and reduced ATP to 40% of control. This resulted in cell death with a predominance of necrosis. Deoxyguanosine caused similar increases in GTP but at earlier time points. Cell death was severe with a predominance of apoptosis. Deoxyguanosine but not guanosine increased dGTP to 800% of control. Adenosine inhibited the effects of guanosine, in part by competing for uptake. In stimulated leukocytes, guanosine and deoxyguanosine altered the nucleotide pools in a way qualitatively similar to that observed in Jurkat cells. However, proliferation was enhanced rather than impaired. In conclusion, guanosine and deoxyguanosine are toxic to Jurkat cells through two mechanisms: ATP depletion, causing necrosis, and the accumulation of dGTP, resulting in apoptosis.

OBJECTIVE:To investigate the effects of human interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination, on the viability of human muscle cells in culture.METHODS:Cultures of human muscle cells were treated with various concentrations of recombinant IFN-gamma and TNF-alpha alone and in combination, and the cytotoxic effects of the cytokines on muscle cells were assessed by measuring lactic dehydrogenase (LDH) release in supernatants and by observation of the cells for morphologic changes under phase microscopy.RESULTS:Exposure of muscle cells to 100 U/ml of either IFN-gamma or TNF-alpha for 9 days caused no cytotoxic effects, as assessed by LDH release in supernatants of muscle cell cultures and by microscopic observation of the cell cultures. However, when IFN-gamma and TNF-alpha were added together in the muscle cell cultures, they caused significant cytotoxic effects. Thus, in combination, IFN-gamma and TNF-alpha at 100 U/ml each caused significant release of LDH (3rd day 9%, 4th day 28.5%, 7th day 55.5%, 9th day 74%) in the supernatants of treated cultures compared to controls. Moreover, inspection by phase microscopy showed clear damage of muscle cells; from Days 3 to 4 progressive vacuolation, detachment of cells, and finally disintegration of the muscle cells by the 8th to 10th day was observed. The synergistic cytotoxic effect of IFN-gamma and TNF-alpha occurred at concentrations as low as 1 U/ml and 10 U/ml, respectively.CONCLUSION:Our study demonstrates for the first time a direct synergistic cytotoxic effect of IFN-gamma and TNF-alpha on human muscle cells in culture. Given that T cells and macrophages are prominent in the chronic inflammatory cell infiltrates of the affected muscles in patients with myositis, our findings suggest that IFN-gamma and TNF-alpha may play an important role in the pathogenesis of muscle destruction of this disorder.

OBJECTIVE:To investigate the effects of human interferon-gamma (IFN-gamma) on cultured human skeletal muscle cells.METHODS:Muscle cell cultures were treated with various concentrations of recombinant human IFN-gamma, and muscle cell proliferation, creatine kinase synthesis and muscle cell cytotoxicity were analyzed.RESULTS:Treatment of muscle cell cultures with IFN-gamma resulted in significant inhibition of myoblasts proliferation, growth, and fusion into multinucleated myotubes. IFN-gamma inhibited creatine kinase synthesis if applied before, but not after, the myoblasts begin to differentiate into myotubes. The effect of IFN-gamma was dose dependent and observed at a concentration of IFN-gamma as low as 10 U/ml. Despite these cytostatic effects, IFN-gamma was not cytotoxic to cultured muscle cells even with very high (10,000 U/ml) IFN-gamma doses.CONCLUSION:IFN-gamma inhibits muscle cell proliferation and differentiation in vitro. These findings suggest that IFN-gamma, a T cell lymphokine, may inhibit muscle regeneration and the repair of injured muscle fibers in myositis.

We examined the proliferative responses of peripheral blood mononuclear cells (PBMC) to autologous and homologous muscle homogenates in 21 patients with early, active, untreated polymyositis/dermatomyositis (PM/DM), 8 patients with chronic PM/DM, 10 patients with myopathies other than PM/DM, 7 patients with connective tissue diseases without myositis, and 12 healthy individuals. PBMC from patients with PM/DM and from control subjects were incubated with various dilutions of autologous and homologous muscle homogenates. PBMC from patients with active PM/DM underwent significant proliferation on exposure to both the autologous muscle and the homologous muscle homogenates. In contrast, PBMC from patients with chronic PM/DM, other myopathies, connective tissue diseases without myositis, and from healthy individuals did not respond to either autologous or homologous muscle. Our findings demonstrate that the PBMC of patients with PM/DM are sensitized to muscle.