Diabetic mice are protected from normally lethal nephrotoxicity of S-1,2-dichlorovinyl-L-cysteine (DCVC): role of nephrogenic tissue repair

Streptozotocin (STZ)-induced diabetic (DB) rats are protected from nephrotoxicity of gentamicin, cisplatin and mercuric chloride, although the mechanisms remain unclear. Ninety percent of DB mice receiving a LD90 dose (75 mg/kg, ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) survived in contrast to only 10% of the nondiabetic (NDB) mice surviving the same dose. We tested the hypothesis that the mechanism of protection is upregulated tissue repair. In the NDB mice, DCVC produced steep temporal increases in blood urea nitrogen (BUN) and plasma creatinine, which were associated with proximal tubular cell (PTC) necrosis, acute renal failure (ARF), and death within 48 h. In contrast, in the DB mice, BUN and creatinine increased less steeply, declining after 36 h to completely resolve by 96 h. HPLC analysis of plasma and urine revealed that DB did not alter the toxicokinetics of DCVC. Furthermore, activity of renal cysteine conjugate beta-lyase, the enzyme that bio-activates DCVC, was unaltered in DB mice, undermining the possibility of lower bioactivation of DCVC leading to lower injury. [3H]-thymidine pulse labeling and PCNA analysis indicated an early onset and sustained nephrogenic tissue repair in DCVC-treated DB mice. BRDU immunohistochemistry revealed a fourfold increase in the number of cells in S-phase in the DB kidneys even without exposure to DCVC. Blocking the entry of cells into S-phase by antimitotic intervention using colchicine abolished stimulated nephrogenic tissue repair and nephro-protection. These findings suggest that pre-placement of S-phase cells in the kidney due to diabetes is critical in mitigating the progression of DCVC-initiated renal injury by upregulation of tissue repair, leading to survival of the DB mice by avoiding acute renal failure.     

Colchicine antimitosis causes progression of S-(1,2-dichlorovinyl)-L-cysteine-induced injury leading to acute renal failure and death in mice

Objective of the present study was to test the importance of tissue repair in the final outcome of S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced nephrotoxicity using colchicine (CLC) intervention. Male Swiss Webster (SW) mice were administered a normally nonlethal dose of DCVC (30 mg/kg, i.p.) on day 0 and CLC (2 mg/kg, i.p.) at 42 and 66 h after administration of DCVC. The mice were observed for mortality and various renal injury and repair parameters were studied during a time course of 0-14 days. Administration of 30 mg DCVC/kg led to loss of renal architecture by day 1, which sustained until day 5, and regressed thereafter to reach normal architecture by day 10 resulting in 100% survival. Renal dysfunction as assessed by increases in plasma BUN and creatinine levels was concordant during this time course. Urinary volume increased significantly between days 10 and 14 with significant increases in urinary glucose concentrations on days 1-4. Calpain leakage increased from day 1 and remained so until day 5 before declining at later time points. In contrast, CLC intervention led to marked inhibition of S-phase DNA synthesis and 100% mortality by 120 h. H&E sections of kidneys revealed loss of renal architecture on day 1 which progressively worsened from day 2 to 4. Polyuria and glycosuria were evident during the first 2 and 3 days, respectively. Calpain immunohistochemistry revealed progressive leakage of calpain in the extracellular space during 2-4 days which lead to increased renal injury as evident from significant increases in calpain specific breakdown products (CSBPs) of alpha-fodrin during the same period of time. The group of mice receiving 2 mg CLC/kg alone showed a significant increase in urinary creatinine concentration on day 5. Neither the expression nor localization of aquaporin 1 was altered in any of the treatment groups. These results show that antimitotic intervention after DCVC-initiated renal injury leads to expansion and progression of that injury, which appears to be due to proteolytic destruction of neighboring cells mediated by calpain leaking out of necrosed renal tubular epithelial cells.     

Renal injury and repair following S-1, 2 dichlorovinyl-L-cysteine administration to mice

S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of a common environmental contaminant, trichloroethylene, is a selective proximal tubular nephrotoxicant. The objective of our study was to examine the dose-response relationship of renal injury and repair following DCVC administration. Male Swiss-Webster mice were injected with DCVC [15, 30, or 75 mg/kg ip in distilled water (10 ml/kg)] and the extent of nephrotoxicity and tissue repair was assessed over a 14-day period. The renal injury due to the low and medium doses of DCVC peaked at 36 and 72 h after dosing, respectively, and then regressed over time due to a timely and adequate tissue repair response. At the highest dose tissue repair was inhibited, thereby causing progression of renal injury, which led to acute renal failure and death of the mice. The possibility that compromised tissue repair was a result of the extensive nephrotoxic injury attendant to the high dose of DCVC was investigated via an equinephrotoxicity study in which separate groups of mice received 40 (LD40) and 75 (LD90) mg DCVC/kg, respectively. Bioactivation-based renal proximal tubular injury measured in these two groups over a time course was identical but there was a marked difference in mortality due to an early and robust tissue repair in the first group relative to the second group. These results support the concept that quantitative evaluation of renal tissue repair in parallel with injury is useful in the assessment of the likely toxic outcome associated with exposure to nephrotoxic drugs and toxicants.     

Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury

Calpain activation has been implicated in the development of ischemia-reperfusion (I-R) injury. Here we investigate the effects of two inhibitors of calpain activity, PD150606 and E-64, on the renal dysfunction and injury caused by I-R of rat kidneys in vivo. Male Wistar rats were administered PD150606 or E-64 (3mg/kg i.p.) or vehicle (10%, v/v, DMSO) 30min prior to I-R. Rats were subjected to bilateral renal ischemia (45min) followed by reperfusion (6h). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (for glomerular dysfunction), fractional excretion of Na(+) (FE(Na), for tubular dysfunction) and urinary N-acetyl-beta-d-glucosaminidase (NAG, for tubular injury). Additionally, kidney tissues were used for histological analysis of renal injury, immunohistochemical analysis of intercellular adhesion molecule-1 (ICAM-1) expression and nitrotyrosine formation. Renal myeloperoxidase (MPO) activity (for polymorphonuclear leukocyte infiltration) and malondialdehyde (MDA) levels (for tissue lipid peroxidation) were determined. Both PD150606 and E-64 significantly reduced the increases in serum creatinine, FE(Na) and NAG caused by renal I-R, indicating attenuation of renal dysfunction and injury and reduced histological evidence of renal damage caused by I-R. Both PD150606 and E-64 markedly reduced the evidence of oxidative stress (ICAM-1 expression, MPO activity, MDA levels) and nitrosative stress (nitrotyrosine formation) in rat kidneys subjected to I-R. These findings provide the first evidence that calpain inhibitors can reduce the renal dysfunction and injury caused by I-R of the kidney and may be useful in enhancing the tolerance of the kidney against renal injury associated with aortovascular surgery or renal transplantation.     

Chronic toxicity of S-(trans-1,2-dichlorovinyl)-L-cysteine in mice

S-(trans-1,2-Dichlorovinyl)-L-cysteine (DCVC) exposure causes acute renal tubular cytotoxicity. To further characterize the effects of DCVC, a chronic study was undertaken. Male Swiss-Webster mice received DCVC dissolved in their drinking water at 0.01, 0.05 and 0.1 mg ml-1. At 4, 8, 21 and 37 weeks, animals were terminated. Bladders, spleens, livers, kidneys and eyes were removed for histopathological examination. At 0.05 and 0.1 mg ml-1 DCVC, growth retardation was evident by 21 weeks. By 26 weeks, all animals in the 0.1 mg ml-1 group had developed cortical cataracts. Cytomegaly, nuclear hyperchromatism and multiple nucleoli were noted in the cells of the pars recta region of the kidney by 4 weeks and correlated to time and dose. At later time points, renal tubular atrophy and early interstitial fibrosis were evident. The epithelial cytological cellular abnormalities appear to be dose-related. Minor pathological changes were noted in the spleen, while there was no effect on the liver or bladder. Chronic ingestion of DCVC results in severe kidney injury.     

Analysis of renal cell transformation following exposure to trichloroethene in vivo and its metabolite S-(dichlorovinyl)-L-cysteine in vitro

Trichloroethene (TCE) is classified as a potential human carcinogen although there is a significant debate regarding the mechanism of TCE induced renal tumor formation. This controversy stems in part from the extremely high doses of TCE required to induce renal tumors and the potential contribution of the associated nephrotoxicity to tumorigenesis. We have used Eker rats, which are uniquely susceptible to renal carcinogens, to determine if exposures to TCE in vivo or exposure to its metabolite S-(dichlorovinyl)-L-cysteine (DCVC) in vitro can transform kidney epithelial cells in the absence of cytotoxicity. Treatment with TCE (0, 100, 250, 500, 1000 mg/kg bw by gavage, 5 days a week) for 13 weeks resulted in a significant increase in cell proliferation in kidney tubule cells, but did not enhance formation of preneoplastic lesions or tumor incidence in Eker rat kidneys as compared to controls. In vitro, concentrations of DCVC, which reduced cell survival to 50%, were able to transform rat kidney epithelial cells. However, no carcinogen-specific mutations were identified in the VHL or Tsc-2 tumor suppressor genes in the transformants. Taken together, the inability of TCE to enhance formation of preneoplastic changes or neoplasia and the absence of carcinogen-specific alteration of genes accepted to be critical for renal tumor development suggest that TCE mediated carcinogenicity may occur secondary to continuous toxic injury and sustained regenerative cell proliferation.     

Early Biological Indicators of S-(1,2-Dichlorovinyl)-L-Cysteine Nephrotoxicity in the Rabbit

ABSTRACT

The progression of changes in rabbit kidney function following dosing with the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC, 20–50 mg/kg) was determined. Proteinuria was observed 0.5–1 hr after administration of DCVC at doses of 20 -50 mg/kg. Blood urea nitrogen levels, glomerular filtration rates, urinary glucose excretion, and urine volume were also altered following DCVC dosing; however, these parameters were less sensitive than proteinuria as markers of early renal dysfunction. None of these latter four indicators were affected by low DCVC doses, nor were they altered by high DCVC doses until 1.5–2.5 hr after dosing. Dose-dependent morphological changes to kidney structure were also observed 5 hr after DCVC administration. Low doses caused damage restricted to brush border membranes in the pars recta, while higher doses produced a necrotic lesion encompassing all regions of the proximal tubule. This study indicates that DCVC can cause rapid renal dysfunctional changes which are first detected by elevated urinary protein excretion.     

Role of tissue repair in survival from s-(1,2-dichlorovinyl)-L-cysteine-induced acute renal tubular necrosis in the mouse.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a model nephrotoxicant in mice, causes acute tubular necrosis and death at high doses. Our earlier studies revealed that renal tissue repair was critical for survival in mice with DCVC nephrotoxicity. The objective of this study was to investigate if increasing renal tissue repair could protect mice from the lethal outcome of DCVC. Male Swiss Webster (SW) mice were administered a low dose of DCVC (15 mg/kg, ip) 72 h before injection of a normally lethal dose of DCVC (75 mg/kg, ip); this resulted in 100% protection against the lethal effect of DCVC. Because DCVC caused approximately two fold decrease in cytosolic and mitochondrial beta-lyase activity, the possibility that DCVC protection may be caused by decreased bioactivation was examined. Mercuric chloride (HgCl2, 6 mg/kg), a nephrotoxicant with no effect on beta-lyase activity, was administered 96 h before a lethal dose of DCVC. This also resulted in 100% protection from the lethal effect of DCVC. In both studies total glutathione was unchanged at any time after the lethal dose of DCVC was administered, obviating the role of glutathione in protection. In both cases the augmented and sustained tissue repair induced by priming dose and documented by 3H-thymidine pulse labeling and immunocytochemistry for proliferating cell nuclear antigen resulted in 100% survival in spite of the extensive renal injury. These findings suggest that stimulation of renal tubular repair by the priming dose, through augmented cell division, and the resistance of new cells to mechanisms of progression of injury, underlies auto- and heteroprotection against DCVC. The molecular mechanisms may have potential application in pharmacotherapeutic intervention for treatment of acute renal failure.     

Correlation of the in vivo and in vitro renal toxicity of S-(1,2-dichlorovinyl)-L-cysteine

The major site at which vinyl cysteine conjugates exert nephrotoxicity is the proximal tubule. Since this is the site of all active anion and cation transport, tubule transport integrity was used to assess nephrotoxicity. Tubules were isolated from young rabbits to study the in vivo and in vitro nephrotoxicity of the conjugate, dichlorovinyl cysteine (DCVC). In vivo exposure to DCVC caused necrosis in the pars recta region of the proximal tubules (20-100 mg/kg ip) and a dose-dependent decrease in tubular active transport. Addition of DCVC to the perfused kidney and tubule suspensions resulted in similar decreases in tubular organic ion transport. At 0.01 mM DCVC, transport was similar to controls while 1 mM DCVC completely inhibited active accumulation of the organic ions. Thus kidney tubule active transport is similarly inhibited in vivo and in vitro by DCVC indicating that bioactivation of DCVC and inhibition of active transport occur directly in the renal tubule.     

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

 Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate β-lyase. However, intravenous administration of the α-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate β-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme β-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloro ethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate β-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by β-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmacokinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves. Received: 16 October 1995/Accepted: 9 January 1996     

Disrupted G1 to S phase clearance via cyclin signaling impairs liver tissue repair in thioacetamide-treated type 1 diabetic rats

Previously we reported that a nonlethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats because of irreversible acute liver injury owing to inhibited hepatic tissue repair, primarily due to blockage of G(0) to S phase progression of cell division cycle. On the other hand, DB rats receiving 30 mg TA/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic (NDB) rats receiving 300 mg TA/kg, resulting in a delay in recovery from liver injury and survival. The objective of the present study was to test the hypothesis that impaired cyclin-regulated progression of G(1) to S phase of the cell cycle may explain inhibited liver tissue repair, hepatic failure, and death, contrasted with delayed liver tissue repair but survival observed in the DB rats receiving 300 in contrast to 30 mg TA/kg. In the TA-treated NDB rats sustained MAPKs and cyclin expression resulted in higher phosphorylation of retinoblastoma (pRb), explaining prompt tissue repair and survival. In contrast, DB rats receiving the same dose of TA (300 mg/kg) exhibited suppressed MAPKs and cyclin expression that led to inhibition of pRb, inhibited tissue repair, and death. On the other hand, DB rats receiving 30 mg TA/kg exhibited delayed up regulation of MAPK signaling that delayed the expression of CD1 and pRb, explaining delayed stimulation of tissue repair observed in this group. In conclusion, the hepatotoxicant TA has a dose-dependent adverse effect on cyclin-regulated pRb signaling: the lower dose causes a recoverable delay, whereas the higher dose inhibits it with corresponding effect on the ultimate outcomes on hepatic tissue repair; this dose-dependent adverse effect is substantially shifted to the left of the dose response curve in diabetes.     

Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.     

Early biological indicators of S-(1,2-dichlorovinyl)-L-cysteine nephrotoxicity in the rabbit

The progression of changes in rabbit kidney function following dosing with the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC, 20-50 mg/kg) was determined. Proteinuria was observed 0.5-1 hr after administration of DCVC at doses of 20-50 mg/kg. Blood urea nitrogen levels, glomerular filtration rates, urinary glucose excretion, and urine volume were also altered following DCVC dosing; however, these parameters were less sensitive than proteinuria as markers of early renal dysfunction. None of these latter four indicators were affected by low DCVC doses, nor were they altered by high DCVC doses until 1.5-2.5 hr after dosing. Dose-dependent morphological changes to kidney structure were also observed 5 hr after DCVC administration. Low doses caused damage restricted to brush border membranes in the pars recta, while higher doses produced a necrotic lesion encompassing all regions of the proximal tubule. This study indicates that DCVC can cause rapid renal dysfunctional changes which are first detected by elevated urinary protein excretion.     

Production of DNA single strand breaks in rabbit renal tissue after exposure to 1,2-dichlorovinylcysteine

S-(trans-1,2-dichlorovinyl)-L-cysteine (DCVC) is a recognized nephrotoxin. To investigate the genotoxic effects of DCVC on the kidney, DNA strand breaks were measured as an indicator of DCVC induced damage. To ascertain if bioactivation of DCVC occurred in the kidney, 3 experimental systems were used: in vivo; isolated perfused kidneys; and isolated proximal tubules of albino male rabbits. A dose-dependent increase in strand breaks in the kidney tubular DNA occurred after in vivo dosing with 5-100 mg/kg DCVC and after in vitro exposure to 10(-5)-10(-2) M DCVC. These results demonstrate the genotoxic effect of this compound on renal tissue.     

Mechanism of S-(1,2-dichlorovinyl)-L-cysteine- and S-(1,2-dichlorovinyl)-L-homocysteine-induced renal mitochondrial toxicity

The mechanism by which the nephrotoxic S-conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) produce toxicity in rat kidney mitochondria was studied by examining their effects on mitochondrial function, structural integrity, and metabolism. Both S-conjugates inhibited succinate-linked state 3 respiration and impaired the ability of mitochondria to retain Ca2+ and to generate a membrane potential; 30-60 min were required for maximal expression of these functional changes. Mitochondrial structure was damaged, as indicated by enhanced polyethylene glycol-induced shrinkage of matrix volume and by leakage of protein and malic dehydrogenase from the matrix; 60-120 min were required for maximal expression of these structural changes. Much shorter incubation times (15-30 min) were required for DCVC and DCVHC to decrease ATP concentrations, to alter the concentrations of several citric acid cycle intermediates, and to inhibit succinate:cytochrome c oxidoreductase and isocitrate dehydrogenase activities. Lipid peroxidation and oxidation of glutathione to glutathione disulfide also occurred. The relative time courses of these pathological changes indicate that the initial effects of DCVC and DCVHC in renal mitochondria are the inhibition of energy metabolism and the oxidation of glutathione. These changes then lead to alterations in mitochondrial function and ultimately to irreversible damage to mitochondrial structure.     

Correlation of the in vivo and in vitro Renal Toxicity of S-(1,2-Dichlorovinyl)-L-Cysteine

ABSTRACT

The major site at which vinyl cysteine conjugates exert nephrotoxicity is the proximal tubule. Since this is the site of all active anion and cation transport, tubule transport integrity was used to assess nephrotoxicity. Tubules were isolated from young rabbits to study the in vivo and in vitro nephrotoxicity of the conjugate, dichlorovinyl cysteine (DCVC). In vivo exposure to DCVC caused necrosis in the pars recta region of the proximal tubules (20–100 mg/kg ip) and a dose-dependent decrease in tubular active transport. Addition of DCVC to the perfused kidney and tubule suspensions resulted in similar decreases in tubular organic ion transport. At 0.01 mM DCVC, transport was similar to controls while 1 mM DCVC completely inhibited active accumulation of the organic ions. Thus kidney tubule active transport is similarly inhibited in vivo and in vitro by DCVC indicating that bioactivation of DCVC and inhibition of active transport occur directly in the renal tubule.     

Age- and sex-dependent dichlorovinyl cysteine (DCVC) accumulation and toxicity in the mouse kidney: relation to development of organic anion transport and beta-lyase activity

The age- and sex-dependent changes in mouse kidney accumulation and toxicity of S-1,2-dichlorovinyl cysteine (DCVC) was investigated. The results were compared to developmental changes in the basal activities of organic anion transport in vitro (PAH uptake) and of cysteine conjugate beta-lyase (substrate: benzothiazolyl cysteine). Following 14C-DCVC (5 mg/kg body wt. orally), the renal 14C-accumulation increased with age, whereas the degree of tubular DCVC lesions was about the same at all time points. Regarding the sex differentiation in adult mice, both the kidney 14C-accumulation levels and the kidney lesion (5 mg/kg DCVC) were most accentuated in the female mouse. However, at a higher dose (25 mg/kg), the male kidney was most affected. Changes in the anion transport and beta-lyase activities did not directly mirror the age-dependent increase in kidney radioactivity. Sex differences in anion transport and beta-lyase activities were also seen, the former activity being highest in the male mouse and the latter in the female. The conflicting results of 14C-accumulation and histopathology in developing mice, may be explained by the ongoing development of the kidney; increase in the number of functionally active nephrons may result in an increased 14C-accumulation (in d.p.m./mg wet wt.) but still the same degree of lesion, when estimated per nephron. In the adult mice, the higher susceptibility of the female may be correlated to the higher beta-lyase activity in the same sex. Regarding the inversed results at a higher dose, rate limitations of transport and bioactivation systems may play a role.     

CSF-1R inhibition attenuates ischemia-induced renal injury and fibrosis by reducing Ly6C+ M2-like macrophage infiltration

Acute kidney injury (AKI) to chronic kidney disease (CKD) progression has become a life-threatening disease. However, an effective therapeutic strategy is still needed. The pathophysiology of AKI-to-CKD progression involves chronic inflammation and renal fibrosis driven by macrophage activation, which is physiologically dependent on colony-stimulating factor-1 receptor (CSF-1R) signaling. In this study, we modulated macrophage infiltration through oral administration of the CSF-1R inhibitor GW2580 in an ischemia–reperfusion (I/R)-induced AKI model to evaluate its therapeutic effects on preventing the progression of AKI to CKD. We found that GW2580 induced a significant reduction in the number of macrophages in I/R-injured kidneys and attenuated I/R-induced renal injury and subsequent interstitial fibrosis. By flow cytometry, we observed that the reduced macrophages were primarily Ly6C⁺ inflammatory macrophages in the GW2580-treated kidneys, while there was no significant difference in the number and percentage of Ly6C⁻CX3CR1⁺ macrophages. We further found that these reduced macrophages also demonstrated some characteristics of M2-like macrophages, which have been generally regarded as profibrotic subtypes in chronic inflammation. These results indicate the existence of phenotypic and functional crossover between Ly6C⁺ and M2-like macrophages in I/R kidneys, which induces AKI worsening to CKD. In conclusion, therapeutic GW2580 treatment alleviates acute renal injury and subsequent fibrosis by reducing Ly6C⁺ M2-like macrophage infiltration in ischemia-induced AKI.     

Cytotoxicity of trichloroethylene and S-(1, 2-dichlorovinyl)-L-cysteine in primary cultures of rat renal proximal tubular and distal tubular cells

Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2-10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1-1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.