Yuet-Ching Tay

Inhibition of NFκB activation with antioxidants is correlated with reduced cytokine transcription in PTC

We recently reported that inhibition of the transcription factor nuclear factor-κB (NFκB) with pyrrolidinedithiocarbamate (PDTC) reduced interstitial monocyte infiltration in rats with proteinuric tubulointerstitial disease, whereas N-acetylcysteine (NAC) was not effective. Here we investigate the effects of antioxidants (PDTC, NAC, and quercetin) on NFκB activation and cytokine transcription in primary cultured rat proximal tubular epithelial cells (PTC) stimulated with lipopolysaccharide. Antioxidant-mediated inhibition of NFκB activation (PDTC, 20-100 μM; NAC, 100 mM; and quercetin, 50 μM) diminished the induction of both pro- [interleukin (IL)-1β, tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, and MIP-2] and anti-inflammatory (IL-10, transforming growth factor-β1) cytokine transcription in PTC (RT-PCR analysis). PDTC and quercetin did not affect PTC viability, but NAC (100 mM) caused a threefold increase in lactate dehydrogenase leakage ( P < 0.001). We conclude that NAC is unable to suppress NFκB activation in PTC at subtoxic and physiologically relevant concentrations. Furthermore, antioxidant-mediated inhibition of NFκB is correlated with the nonselective reduction of cytokine transcription in activated tubular cells. These data might explain the protective effects of PDTC-mediated NFκB inhibition in tubulointerstitial disease in vivo.

PARTIAL DEPLETION OF MACROPHAGES BY ED7 REDUCES RENAL INJURY IN ADRIAMYCIN NEPHROPATHY

Background:  Because macrophages are considered to be possible effectors of disease in Adriamycin (ADR) nephrosis, we hypothesized that depletion of macrophages might protect against the initiation of renal injury. In the present study, a monoclonal antibody (ED7) directed against CD11b/CD18 integrin, which is expressed by macrophages, was used to investigate the pathogenetic effects of macrophages in ADR nephropathy.

Cytokine Gene Expression in Adriamycin Nephropathy: Effects of Antioxidant Nuclear Factor κB Inhibitors in Established Disease

Background/Aim: Inhibition of nuclear factor ĸB with the antioxidant pyrrolidine dithiocarbamate (PDTC) reduced tubulointerstitial injury in Adriamycin nephropathy (AN), whereas N-acetylcysteine (NAC) was ineffective. Here we hypothesize that PDTC reduces the renal cortical expression of nuclear factor ĸB dependent cytokines in AN. Methods: Male Wistar rats received a single intravenous injection of doxorubicin hydrochloride (7.5 mg/kg). NAC (150 mg/kg twice daily i.p.), PDTC (50 mg/kg twice daily i.p.), or vehicle were commenced on day 14 and continued until day 30. Results: On day 30, mRNAs of selected cytokines were increased in AN (TNF-α 3.4-fold, MCP-1 5.1-fold, IL-10 2.7-fold, TGF-β1 3.5-fold, all p < 0.05) as determined by RT-PCR. PDTC reduced IL-10 and TGF-β1 mRNAs (p < 0.05), whereas the upregulation of MCP-1 and TNF-α mRNAs was not affected. In contrast, NAC increased TNF-α and IL-10 mRNAs (p < 0.05). Nuclear protein levels of activator protein-1 were increased in AN (4.4-fold, p < 0.01) and not significantly altered by PDTC (3.0-fold, p = 0.13) or NAC (5.2-fold, p = 0.18). Conclusions: The protective effects of PDTC in AN are not associated with a local reduction in TNF-α and MCP-1 gene expression. The latter may be due to continued transactivation by activator protein-1. These data also suggest that IL-10 and TGF-β1 mRNA expressions are PDTC dependent and have a role in mediating tubulointerstitial injury.

EARLY ADMINISTRATION OF PDTC IN ADRIAMYCIN NEPHROPATHY: EFFECT ON PROTEINURIA, CORTICAL TUBULOINTERSTITIAL INJURY, AND NF- B ACTIVATION

The persistence of NF-κB independent inflammatory signals in the cortical tubulointerstitium may explain the incomplete suppression of interstitial monocyte accumulation by the antioxidant NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), in nephrotic rats with established Adriamycin nephropathy (AN). Because PDTC is known to have anti-proteinuric effects, in this study we investigated whether earlier commencement, during the pre-nephrotic phase of AN, would be more effective in reducing interstitial monocyte accumulation. Male Wistar rats with AN received either vehicle or PDTC (50 mg/kg bd i.p.i.) from d7 until d30 (n = 8 per group). On d30, PDTC reduced renal cortical lipid peroxidation (43%), wet kidney weight and tubulointerstitial injury in AN, but did not decrease proteinuria. Accordingly, inhibition of interstitial ED-1 accumulation remained incomplete (52%). Interestingly, the early administration of PDTC in AN, induced polyuria and renal cortical NF-κB DNA-binding activity was reduced by only 35%. These results suggest that: (i) the combination of an anti-proteinuric agent with PDTC may be required to completely suppress interstitial monocyte cell accumulation in AN and, (ii) the timing and duration of PDTC therapy are an important determinant of its efficacy to reduce NF-κB activation, in vivo.

Significance of CD25 positive cells and macrophages in noncrescentic IgA nephropathy.

The aim of this study was to determine whether infiltration by CD25 positive cells, macrophages, and activated macrophages in the kidney is predictive of chronic histological injury and renal prognosis in adults with noncrescentic IgA nephropathy. Renal biopsies of 36 patients with noncrescentic IgA nephropathy were examined by immunohistochemistry for glomerular and interstitial CD4, CD8, and CD25 positive cells, monocytes/macrophage (Mac387), and activated macrophages (27E10). Renal injury (glomerulosclerosis, mesangial cell hypercellularity, tubular atrophy, and interstitial fibrosis) at the time of biopsy and renal prognosis (follow-up creatinine and creatinine clearance) were assessed. The mean follow-up period was 22.5 ± 16.5 months. The number of interstitial CD8 positive cells was the best predictor of renal injury at the time of biopsy, and was positively correlated with glomerulosclerosis (p = 0.04), tubular atrophy (p = 0.04), and interstitial fibrosis (p = 0.01) but not with mesangial cell hypercellularity. The number of interstitial Mac387 and 27E10 positive cells were the best predictors of renal prognosis (r2 = 0.33 and 0.34 respectively, both p < 0.01). These data suggest the presence of CD8 cells and macrophages in the kidney at the time of biopsy could potentially serve as pathological markers to identify patients with IgA nephropathy, which may warrant more aggressive medical therapy.

Mild gentamicin nephrotoxicity reduces the progression of chronic adriamycin nephrosis

The effect of mild acute tubular injury on the progression of tubulointerstitial fibrosis was studied in pair‐fed uninephrectomized male Wistar rats with established adriamycin nephrosis (n=34). Rats were stratified into three groups according to endogenous creatinine clearance (CrCl), proteinuria (Upr) and body weight (BW): (i) group 1 (Fe, n=12) received a single intraperitoneal injection of ferric nitrilotriacetate (5 mg Fe/kg BW); (ii) group 2 (G, n=10) three daily subcutaneous injections of gentamicin (60 mg/kg BW) and; (iii) group 3 (C, n=12) saline injections. Serial CrCl (day 2, day 5, weeks 2, 4, 6 and 8) and renal histology (week 8) were examined following administration of nephrotoxin. CrCl was reduced on d2 (Fe: 0.78±0.23 mL/min; mean±SD) and day 5 (G: 0.91±0.36 mL/min) as compared with C (1.22±0.12 mL/min; P<0.05). There was no change in the serum creatinine and functional recovery occurred by d5 (Fe) and week 2 (G). Upr decreased transiently in G at week 2 (G: 482±208 mg/day vs C: 716±233; P=0.05) despite similar food intake, baseline Upr and CrCl. At week 8, CrCl in Fe (0.84±0.40 mL/min) was similar to C (0.84±0.58 mL/min), whereas in G it remained stable (1.27±0.39 mL/min; P<0.05). By morphometric analysis, mean relative interstitial volume (RIV) and glomerulosclerosis (GS) in Fe (RIV: 28.5±13.4%; GS: 10.3±12.3%) was no different to C (RIV: 24.5±12.5%; GS: 20.9±20.0%), whereas both parameters were reduced in G (RIV: 14.1±8.1%; GS: 4.0±4.8%; P<0.05). Mild gentamicin nephrotoxicity therefore reduced the progression of adriamycin nephrosis. The mechanism of this finding is unclear, but it may relate to altered glomerular and tubular cell handling of protein.

MITOCHONDRIAL FUNCTION IN RAT RENAL CORTEX IN RESPONSE TO PROTEINURIA AND IRON

1. Proximal tubular cell dysfunction in chronic glomerular disease (CGD) has been ascribed, in part, to reabsorption of transferrin‐iron from tubular fluid and subsequent cytosolic peroxidative injury. To investigate a possible role for altered mitochondrial function in tubular cell injury in CGD, renal cortical mitochondrial respiratory function was examined in rats with adriamycin nephrosis.

Effect of nephrotoxins on tubulointerstitial injury and NF-κB activation in adriamycin nephropathy

In a previous study we found that an episode of acute subclinical nephrotoxicity with gentamicin (G) (but not that induced by another proximal tubular cell nephrotoxin: ferric nitrilotriacetate, FeNTA), paradoxically reduced the progression of renal function and injury in uninephrectomized rats with nephrotic glomerular disease due to Adriamycin nephropathy (AN). Here, we hypothesized that subclinical exposure to G reduces early renal cortical tubulointerstitial inflammation and NF-κB activation in AN. To test this hypothesis, male Wistar rats with established AN received either G (10, 40, or 80 mg/kg by daily s.c.i. for 3 days), FeNTA (1.25, 5, or 10 mg/kg by a single i.p.i.), or vehicle (n = 8 per group), 13 to 15 days after disease induction. Although G and FeNTA caused acute tubular necrosis in a dose-dependant manner (day 17), only the highest doses (10 mg/kg and 80 mg/kg) produced an acute elevation in the serum creatinine. On day 33, chronic tubulointerstitial inflammation (tubular atrophy, interstitial ED-1 + /CD8 + cell accumulation) and NF-κB activation were exacerbated only in the groups that caused functional nephrotoxicity. These data suggest that: 1) the protective effect of subclinical G nephrotoxicity in chronic AN does not involve early changes in interstitial inflammation or NF-κB activation; and 2) a single episode of G exposure must be accompanied by clinically apparent nephrotoxicity in order to accelerate progression in a nonuremic model of chronic glomerular disease.

Mitochondrial function in partially nephrectomized rats

Summary: Previous demonstrations of increased oxygen consumption and renal iron accumulation in partially nephrectomized (RK) rats on the one hand, and acute mitochondrial toxicity of iron in normal rats on the other suggest a complex relationship between mitochondrial function and iron. Renal cortical mitochondrial respiratory function was therefore investigated in RK and iron‐loaded rats. State 4 (resting) respiration was increased in RK rats (49±1 vs 43±1 natom 0/min, P. <0.001) in comparison to control. Mitochondrial enzyme activity was lower in RK than control rats (e.g. succinate dehydrogenase 158.2 ± 13.6 vs 237.2±17.4 Δlog[cyt C]/min/mg/prot, P<0.01). Acute iron‐loading impaired creatinine clearance (1.39 ± 0.07 vs 0.40 ± 0.29 mL/min) and mitochondrial enzyme activity (e.g. cytochrome oxidase 362.0 ± 32.8 vs 185.0 ± 46.6 Δlog[cyt C]/min/mg/prot, P<0.05) in control rats, but RK rats were more resistant to injury and there was no change in mitochondrial enzyme activity. Mitochondrial fragility was similar in RK and control rats, and in both was unaffected by iron‐loading. In conclusion, rates of resting mitochondrial respiration are increased in partially nephrectomized rats, despite reduced intrinsic activity of two iron‐dependent mictchondrial enzymes. Mitochondrial function is more resistant to acute injury with iron in partially nephrectomized rats.