Targeting superoxide dismutase to renal proximal tubule cells attenuates vancomycin-induced nephrotoxicity in rats

Vancomycin hydrochloride (VCM), a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). As it is known to induce renal dysfunction, the dose and the duration of its administration are limited. Moreover, the mechanism of VCM-induced renal dysfunction remains to be unclear. To evaluate the involvement of free radical on VCM-induced renal dysfunction, we carried out analysis with a hexamethylenediamine-conjugated superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Male Wistar rats (weighing 200-210 g) were intraperitonealy administered with 200 mg/kg of VCM twice a day for 7 days. AH-SOD 5 mg/kg/day was subcutaneously injected 5 min before every VCM injection. VCM induced renal injury dose-dependently. Biochemical analyses revealed that plasma levels of blood urea nitrogen and creatinine significantly increased in the VCM-treated group by an AH-SOD-inhibitable mechanism. VCM simultaneously elicited an increase of 8-OHdG levels and chemiluminescence intensity of free radical generation in the kidney. Histological examination revealed that VCM also elicited a marked destruction of glomeruli and necrosis of proximal tubules. AH-SOD inhibited these phenomena in the kidney. These results suggested that oxidative stress might underlie the pathogenesis of VCM-induced nephrotoxicity and targeting SOD and/or related antioxidants to renal proximal tubules might permit the administration of higher doses of VCM sufficient for eradication of MRSA without causing renal injury.     

Inhibition of vancomycin-induced nephrotoxicity by targeting superoxide dismutase to renal proximal tubule cells in the rat

Vancomycin, a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). Because vancomycin induces renal dysfunction, the dose and the duration of its administration are limited. The mechanism of vancomycin-induced renal dysfunction is not known. We recently synthesized a hexamethylenediamine-conjugated cationic superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. The present work reports the protective effects of AH-SOD against vancomycin-induced renal dysfunction. Male Wistar rats (200-210 g) were intraperitoneally administered with either 200 or 400 mg/kg of vancomycin twice a day for 7 days. Either 5 mg/kg/day AH-SOD or saline was subcutaneously injected 5 min before every vancomycin injection. Biochemical analysis revealed that plasma levels of blood urea nitrogen and creatinine increased significantly in vancomycin-treated group by an AH-SOD-inhibitable mechanism. Histological examination revealed that vancomycin also elicited a marked destruction of glomeruli and necrosis of proximal tubule by an AH-SOD inhibitable mechanism. These results suggest that oxidative stress underlies the pathogenesis of vancomycin-induced nephrotoxicity and that targeting SOD and/or related antioxidants to renal proximal tubule cells might permit the administration of higher doses of vancomycin sufficient for eradication of MRSA without causing renal injury.     

Targeting superoxide dismutase to renal proximal tubule cells inhibits mitochondrial injury and renal dysfunction inuduced by cisplatin

We recently reported the synthesis of a cationic superoxide dismutase (SOD) derivative (AH-SOD) that rapidly and selectively accumulates in and around proximal tubule cells and effectively dismutes superoxide radicals in situ. The present study revealed that administration of cis-diamminedichloroplatinum(II)-elicited oxidative stress in renal mitochondria, decreased the renal expression of Bcl-x, released cytochrome c from mitochondria to cytosol, and induced apoptosis and renal dysfunction by a mechanism that was inhibited by AH-SOD. These results suggest that targeting SOD to proximal tubule cells protects renal function and permits the administration of fairly high doses of nephrotoxic anticancer agents, such as cisplatin, without causing renal injury.     

Synthesis of a superoxide dismutase derivative that circulates bound to albumin and accumulates in tissues whose pH is decreased

Protection of tissues from oxidative stress is one of the major prerequisites for aerobic life. Since intravenously injected Cu2+/Zn2+-type superoxide dismutase (SOD) disappears from the circulation with a short half-life of 5 min, its clinical use as a scavenger for superoxide radical is limited. We synthesized a human erythrocyte type SOD derivative (SM-SOD) by linking 2 mol of hydrophobic organic anion, alpha-4-[( 6-(N-maleimido)hexanoyloxymethyl]cumyl]half-butyl-esterified poly(styrene-co-maleic acid) (SM), to the cysteinyl residues of the dimeric enzyme without decreasing enzymic activity. SM-SOD, but not SOD, bound to an albumin-Sepharose column; the bound SM-SOD was eluted by a buffer solution containing 0.5% sodium dodecyl sulfate or 10 mM warfarin, suggesting that SM-SOD reversibly binds to the warfarin site on albumin. Due to the amphipathic nature of the SMI moiety, SM-SOD bound also to cell membranes particularly when the pH was decreased. In vivo analysis in the rat revealed that intravenously injected SM-SOD circulated bound to albumin with a half-life of 6 h. Postischemic reperfusion arrhythmias were almost completely prevented by a single dose of SM-SOD, but not SOD. Thus, the prolonged half-life of SM-SOD in the circulation and its preferential accumulation in an injured site with decreased pH appeared to be responsible for preventing myocardial injury. These results suggest that superoxide radical and/or its metabolite(s) would play an important role in the pathogenesis of postischemic reperfusion arrhythmias and that SM-SOD may be useful for decreasing tissue injury in ischemic heart disease.     

Overexpression of manganese superoxide dismutase protects against ATP depletion-mediated cell death of proximal tubule cells

We have previously shown that in vivo renal ischemia/reperfusion results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. Current studies were designed to compare the effect of ATP depletion (Antimycin A treatment) on cell death pathways using renal proximal tubular cells and identical cells that overexpress MnSOD. ATP depletion in wild-type cells induced an apoptotic cascade that involved caspase 9 activation; MnSOD overexpressing cells afforded protection against apoptosis. This protection did not appear to involve a cytochrome c-related mechanism, but may be related to altered levels of nitric oxide within the cell. Further studies suggested that nitric oxide was required to protect the renal cells from caspase-mediated cell death. Interestingly, treatment of renal cell extracts with reductants (DTT and ascorbate) enhanced caspase activation. Taken together, these results suggest that cysteine nitrosylation may be playing a role in caspase dysfunction in cells overexpressing MnSOD following ATP depletion.     

Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells.

Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells. Epinephrine has known to be a very important factor in the regulation of renal sodium excretion. However, the effect of epinephrine on Na+/glucose cotransporter was not fully elucidated. Thus, we examined effect of epinephrine on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signal pathways in the primary cultured rabbit renal proximal tubule cells (PTCs). Epinephrine inhibited alpha-MG uptake in a time- and dose-dependent manner and also decreased SGLT1 and SGLT2 protein level. Both phentolamine and propranolol completely prevented epinephrine-induced inhibition of alpha-MG uptake. The epinephrine-induced inhibition of alpha-MG uptake was blocked by SQ-22536 or myristoylated PKA inhibitor amide 14-22 and epinephrine increased the intracellular cAMP content. In western blotting analysis, epinephrine increases phosphorylation of p44/42 and p38 MAPKs and PD 98059 or SB 203580 blocked the effect of epinephrine. In addition, epinephrine increased AA release and PGE2 production and effects of epinephrine on alpha-MG uptake and AA release were blocked by staurosporine and bisindolylmaleimide I or mepacrine and AACOCF3. Indeed, epinephrine translocated PKC or cPLA2 from cytosol to membrane fraction. In conclusion, epinephrine partially inhibits the alpha-MG uptake through PKA, PKC, p44/42, p38 MAPK, and cPLA2 pathways in the PTCs.     

Inhibition of carrageenin-induced paw edema by a superoxide dismutase derivative that circulates bound to albumin.

Although the possible involvement of superoxide radical and its metabolite(s) in the pathogenesis of various types of edema have been suggested, direct evidence supporting this concept is lacking. Since intravenously administered Cu2+Zn2(+)-type superoxide dismutase (SOD) rapidly disappeared from the circulation with a half-life of 4 min, the enzyme could not be used to test whether superoxide radicals played a critical role in the modulation of vascular permeability. We previously synthesized a SOD derivative (SM-SOD) by linking poly(styrene co-maleic acid butyl ester) (SM) to the enzyme (Ogino, T., Inoue, M., Ando, Y., Awai, M., Maeda, H. and Morino Y. (1988) Int. J. Pept. Protein Res. 32, 1583-1588); SM-SOD circulates bound to albumin with a half-life of 6 h. To test whether superoxide radicals play an important role in the regulation of vascular permeability, the effect of SM-SOD on experimental paw edema was studied in the rat. Subcutaneous injections of carrageenin to the paw rapidly induced local edema by increasing vascular permeability. Intravenous administration of SM-SOD markedly inhibited the carrageenin-induced increase in vascular permeability and suppressed the development of paw edema. In contrast, the same dose of SOD showed no such inhibitory effect. These results suggest that superoxide radical and/or its metabolite(s) might play a critical role in the pathogenesis of carrageenin-induced vasogenic edema.     

Streptomycin toxicity in primary cultures of flounder renal proximal tubule cells

Summary The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 μg/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-dcultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated culture were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity. This investigation was supported by the University of Connecticut Research Foundation and by grant PCM-8003452 from the National Science Foundation, Washington, DC.     

Heterogeneity of pH in the aqueous cytoplasm of renal proximal tubule cells

Heterogeneity of cytosolic pH was studied with compounds that distribute between the cytosol and mitochondrial matrix in fundamentally different ways, i.e., according to the extent of ionization or according to the function of H+-coupled transport systems. Results show that the average cytosolic pH is considerably more alkaline than the region to which mitochondria are exposed. Because mitochondria are localized predominantly in the basal region, the results are consistent with a transcellular pH gradient within the cytosol of proximal tubule cells. Experiments analyzing the effects of inhibiting efflux of HCO3- at the basal surface and Na+-H+ exchange at the apical surface support the interpretation that the function of these systems contributes to the transcellular pH gradient. The existence of a heterogeneity in pH within the cytosol has important implications concerning the function and regulation of numerous cell processes.     

Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

An mRNA that specifically accumulates in maize roots delineates a novel subset of developing cortical cells

A near full-length cDNA clone (pZRP3) corresponding to an mRNA that accumulates specifically in roots of maize was isolated. The ZRP3 mRNA is ca. 600 nucleotides in length. The amino acid sequence of the predicted polypeptide is rich in leucine (16%), proline (11%), and cysteine (8.5%). The zrp3 gene appears to be expressed exclusively in roots, whereas other ZRP3-related genes are expressed in additional organs of the maize plant. In situ hybridization shows that ZRP3 mRNA accumulation is largely confined to the cells of the cortical ground meristem. Furthermore, accumulation of this mRNA occurs within a distinct subset of cortical cells, the inner three to four cell layers.Journal paper number J-14572 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project Number 2997.     

Sodium-alanine cotransport in renal proximal tubule cells investigated by whole-cell current recording

Sodium-alanine cotransport was investigated in single isolated proximal tubule cells from rabbit kidney with the whole-cell current recording technique. Addition of L-alanine at the extracellular side induced an inward-directed sodium current and a cell depolarization. The sodium-alanine cotransport current was stereospecific and sodium dependent. Competition experiments suggested a common cotransport system for L-alanine and L-phenylalanine. Sodium-alanine cotransport current followed simple Michaelis-Menten kinetics, with an apparent Km of 6.6 mM alanine and 11.6 mM sodium and a maximal cotransport current of 0.98 pA/pF at -60 mV clamp potential. Hill plots of cotransport current suggested a potential-independent coupling ratio of one sodium and one alanine. The apparent Km for sodium and the maximal cotransport current were potential dependent, whereas the apparent Km for L-alanine was not affected by transmembrane potential. The increase in Km for alanine with decreasing inward-directed sodium gradients suggested a simultaneous transport mechanism. These results are consistent with a cotransport model with potential-dependent binding or unbinding of sodium (high-field access channel) and a potential-dependent translocation step.     

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.     

Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells

The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.     

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.