Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus.

The Coral Triangle is widely considered the most important centre of marine biodiversity in Asia while areas on its periphery such as the South China Sea, have received much less interest. Here, we demonstrate that a small population of the knobbly sea star Protoreaster nodosus in Singapore has similarly high levels of genetic diversity as comparable Indonesian populations from the Coral Triangle. The high genetic diversity of this population is remarkable because it is maintained despite decades of continued anthropogenic disturbance. We postulate that it is probably due to broadcast spawning which is likely to maintain high levels of population connectivity. To test this, we analysed 6140 genome-wide single nucleotide polymorphism (SNP) loci for Singapore's populations and demonstrate a pattern of near panmixia. We here document a second case of high genetic diversity and low genetic structure for a broadcast spawner in Singapore, which suggests that such species have high resilience against anthropogenic disturbances. The study demonstrates the feasibility and power of using genome-wide SNPs for connectivity studies of marine invertebrates without a sequenced genome.

Leiomyoma of the oesophagus managed by thoracoscopic enucleation.

The authors document two patients with oesophageal leiomyoma. In the first patient, a 41-year-old man, enucleation of the oesophageal leiomyoma was initially attempted by a thoracoscopic approach, but because of adherence of the tumour to the oesophageal mucosa, enucleation was completed by thoracotomy. Thoracoscopic enucleation was successfully performed in the second patient, a 62-year-old man. This paper includes a literature review on the pathology, diagnosis and surgical approach in the management of oesophageal leiomyoma. In conclusion, prudent use of thoracoscopic approach in the enucleation of oesophageal leiomyoma could potentially result in shorter hospital stay, decreased postoperative pain and reduced requirement for postoperative analgesia.

NK cells do not mediate renal injury in murine adriamycin nephropathy.

In adriamycin nephropathy (AN), a model of chronic proteinuric renal injury, the absence of functional B and T cells with residual natural killer (NK) cells, and macrophages in severe combined immunodeficient (SCID) mice results in more severe disease than in immunocompetent mice. We have recently shown expression of the stimulatory NK cell molecule NKG2D and its ligand RAE-1 in the adriamycin (ADR) kidney. Therefore, we sought to determine the role of NK cells in AN. We used anti-asialo GM1 NK cell depletion in immunocompetent BALB/c mice with AN, and also compared AN in immunodeficient SCID mice and immunodeficient nonobese diabetic (NOD)-SCID mice (that have impaired NK cell function). The number of NK cells was increased in AN in BALB/c mice compared with normal controls. NK cell depletion or reduction of NK function in NOD-SCID mice did not affect the severity of disease. In both wild type and immunodeficient models, ADR upregulated RAE-1 in the kidney. High levels of Class I major histocompatibility complex molecules were found in both models of AN. In conclusion, NK cells do not play a significant role in AN.

Divide-and-conquer approach for the exemplar breakpoint distance.

MOTIVATION: A one-to-one correspondence between the sets of genes in the two genomes being compared is necessary for the notions of breakpoint and reversal distances. To compare genomes where there are paralogous genes, Sankoff formulated the exemplar distance problem as a general version of the genome rearrangement problem. Unfortunately, the problem is NP-hard even for the breakpoint distance. RESULTS: This paper proposes a divide-and-conquer approach for calculating the exemplar breakpoint distance between two genomes with multiple gene families. The combination of our approach and Sankoff's branch-and-bound technique leads to a practical program to answer this question. Tests with both simulated and real datasets show that our program is much more efficient than the existing program that is based only on the branch-and-bound technique. AVAILABILITY: Code for the program is available from the authors.

Role of CD8(+) cells in the progression of murine adriamycin nephropathy.

BACKGROUND: Many studies have shown that interstitial inflammation in human and experimental renal disease is characterized by T-cell infiltration, but published data on the involvement of inflammatory cell subsets in progressive tubulointerstitial lesions are often conflicting. A previous study suggested a role for cytotoxic T lymphocytes in the damaging effect of CD4(+) T-cell depletion in murine adriamycin (ADR) nephropathy, a model of focal segmental glomerulosclerosis (FSGS), and tubulointerstitial inflammation. The aim of this study was to investigate the role of CD8(+) cells in this model. METHODS: Male BALB/c mice were treated with five intraperitoneal injections of anti-CD8 monoclonal antibody (mAb), beginning from five days after ADR treatment, when overt proteinuria was established. Seven mice in each of groups A (ADR + mAb), B (ADR only), and C (saline treated, age matched) were sacrificed at week 6. Changes in renal function and histopathological features were assessed. Tubulointerstitial inflammation and glomerular inflammation were examined immunohistochemically. RESULTS: mAb treatment reduced CD8(+) cell levels to <2% of normal in spleen. Proteinuria in group A was no different from that in group B at week 6, but was markedly higher than in group C. Creatinine clearance was significantly ameliorated by anti-CD8 treatment (71.8 +/- 4.9 microL/min vs. 29.2 +/- 2.8 in group B and 81.9 +/- 3.7 in group C). Morphometric analysis showed less FSGS in group A compared with group B (6.5 +/- 1.9 vs. 13.0 +/- 2.8, P < 0.001), as well as less tubular atrophy (indicated by increased ratio of tubule cell height to tubular diameter, 0.25 +/- 0.24 in group A vs. 0.04 +/- 0.02 in group B, P < 0.05). CD8 depletion also reduced interstitial expansion (6.3 +/- 2.2% vs. 16.4 +/- 3.1 in group B, P < 0.001) and fibrosis (P < 0.01). Macrophage infiltration in tubulointerstitium was less in group A than in group B (P = 0.052). The number of interstitial CD4(+) cells appeared to increase after anti-CD8 treatment, but was not statistically different between groups A and B. CONCLUSION: Anti-CD8 treatment protects against renal functional and structural injury in this murine model of chronic proteinuric renal disease.

Depletion of CD4(+) T cells aggravates glomerular and interstitial injury in murine adriamycin nephropathy.

BACKGROUND: CD4(+) T cells play an important role in various types of immunologic renal disease, including lupus nephritis, IgA nephropathy, and crescentic glomerulonephritis. CD4(+) T cells are also major infiltrating lymphocytes in chronic tubulointerstitial inflammation associated with nonimmunological renal diseases. We suspected that CD4(+) T cells might contribute to disease progression and loss of renal function in chronic proteinuric renal disease (CPRD). To investigate this possibility, the effect of monoclonal antibody against CD4(+) lymphocytes (anti-CD4) was studied in a murine model (adriamycin nephropathy) of CPRD. METHODS: Adriamycin nephropathy was produced in male BALB/c mice by a single intravenous injection of adriamycin (11 mg/kg). Anti-CD4 was given by intraperitoneal injection following the development of proteinuria at days 5, 6, 7, 21, and 37 after adriamycin. After six weeks, renal function and histology were studied by histomorphometry, immunohistochemistry, and flow cytometry. RESULTS: Flow cytometric analysis showed a marked decrease in the number of CD4(+) T cells in blood and spleen of the antibody-treated animals (N = 7, P < 0.01). Adriamycin plus CD4(+) depletion mice had significantly greater mesangial expansion, glomerular sclerosis, and interstitial expansion than the mice on adriamycin alone. Interstitial infiltration with macrophages and CD8(+) cells was significantly increased in adriamycin plus CD4(+) depletion mice. Creatinine clearance (17.5 +/- 0.54 vs. 29.2 +/- 0.89 microL/min, P < 0.001) was significantly worse in the adriamycin plus CD4(+) depletion mice than in adriamycin alone mice and correlated with histologic change in glomeruli and interstitium. CONCLUSIONS: Depletion of CD4(+) T cells promotes glomerular and interstitial injury in mice with established adriamycin nephropathy. These findings suggest that CD4(+) T cells have a protective role against the progression of adriamycin nephropathy.

Early administration of PDTC in adriamycin nephropathy: effect on proteinuria, cortical tubulointerstitial injury, and NF-kappaB activation.

The persistence of NF-kappaB independent inflammatory signals in the cortical tubulointerstitium may explain the incomplete suppression of interstitial monocyte accumulation by the antioxidant NF-kappaB 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-kappaB 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-kappaB activation, in vivo.

Cytokine gene expression in Adriamycin nephropathy: effects of antioxidant nuclear factor kappaB inhibitors in established disease.

BACKGROUND/AIM: Inhibition of nuclear factor kappaB 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 kappaB 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-alpha 3.4-fold, MCP-1 5.1-fold, IL-10 2.7-fold, TGF-beta1 3.5-fold, all p < 0.05) as determined by RT-PCR. PDTC reduced IL-10 and TGF-beta1 mRNAs (p < 0.05), whereas the upregulation of MCP-1 and TNF-alpha mRNAs was not affected. In contrast, NAC increased TNF-alpha 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-alpha 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-beta1 mRNA expressions are PDTC dependent and have a role in mediating tubulointerstitial injury.

Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.

BACKGROUND: As an experimental analogue of human focal glomerular sclerosis (FGS), adriamycin (ADR)-induced nephropathy is well-characterized in rats. Previously, this model has not been fully established in mice. The extension of this model to the mouse would be useful in the application of genetic and monoclonal antibody technology to characterize mechanisms of progressive renal disease. Herein, we describe a stable and reproducible murine model of chronic proteinuria induced by ADR. METHODS: Male BALB/c mice were intravenously injected with a single dose of ADR (10 to 11 mg/kg). Seven mice were sacrificed at weeks 1, 2, 4, and 6. Renal function, quantitative morphometric analysis, and electron microscopic studies were performed. Peripheral CD4+ and CD8+ T cells were evaluated using flow cytometric analysis of splenocytes. The leukocytic inflammatory pattern was analyzed by immunohistochemical examination. RESULTS: Overt proteinuria was observed from day 5 and remained significantly elevated throughout the study period. A focal increase in reabsorption droplets in tubular cells appeared at weeks 1 and 2, and numerous intraluminal casts were present after two weeks. Glomerular vacuolation and mild FGS appeared at week 4. At week 6, extensive focal and even global glomerular sclerosis, associated with moderate interstitial expansion and severe inflammation, were observed. A prominent macrophage infiltration appeared within both interstitium and glomeruli at week 2, followed by accumulation of both CD4+ and CD8+ T cells in interstitium but not glomeruli. There were almost no B lymphocytes seen at any time. CONCLUSION: This model should be useful in unraveling the pathogenesis of glomerular and interstitial inflammation and fibrosis in chronic proteinuric renal disease.

Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kappaB dependent.

BACKGROUND: Endotoxin is an important factor in the development of acute renal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the most potent triggers for renal cells to produce monocyte chemoattractant protein-1 (MCP-1), a key cytokine involved in immune cell recruitment into the renal interstitium in acute and chronic renal diseases. Knowledge about the transcriptional regulation of MCP-1 in renal tubular epithelial cells in response to LPS is incomplete. METHODS: Transcriptional regulation of MCP-1 was investigated in rat proximal tubule cells (PTCs) in primary culture and was exposed to LPS using electromobility shift assay and supershift analysis for nuclear factor-kappaB (NF-kappaB) and Western blot for the NF-kappaB inhibitory protein IkappaB. To prove the role for NF-kappaB, activator protein (AP-1), and sequence-specific transcription factor (Sp1), mutation and deletion analysis was performed using a 3.5 kb fragment of rat MCP-1 5'-flanking region inserted into a luciferase reporter construct transfected into tubular epithelial cell line (NRK-52E). RESULTS: LPS increased NF-kappaB in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. IkappaBalpha decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. IkappaBbeta levels were depressed within one hour and remained low throughout the culture period after LPS stimulation. The activity of the transfected 5'-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-kappaB binding sites, located in the enhancer region of the 5'-flanking region, resulted in a total loss of LPS-induced increase in luciferase activity. Mutation of putative AP-1 and Sp1 sites, located in the proximal promoter region of MCP-1, reduced basal luciferase activity in unstimulated cells, but had no effect on LPS-stimulated luciferase activity. CONCLUSIONS: These studies prove that NF-kappaB is critical for LPS-induced MCP-1 transcription, and AP-1 and Sp1 are essential for basal expression of MCP-1 in rat tubule cells. The species-specific nature of transcriptional regulation of MCP-1 has important implications for the delineation of treatment to prevent endotoxin-mediated renal injury.

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

We recently reported that inhibition of the transcription factor nuclear factor-kappaB (NFkappaB) 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 NFkappaB activation and cytokine transcription in primary cultured rat proximal tubular epithelial cells (PTC) stimulated with lipopolysaccharide. Antioxidant-mediated inhibition of NFkappaB activation (PDTC, 20-100 microM; NAC, 100 mM; and quercetin, 50 microM) diminished the induction of both pro- [interleukin (IL)-1beta, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-2] and anti-inflammatory (IL-10, transforming growth factor-beta1) 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 NFkappaB activation in PTC at subtoxic and physiologically relevant concentrations. Furthermore, antioxidant-mediated inhibition of NFkappaB is correlated with the nonselective reduction of cytokine transcription in activated tubular cells. These data might explain the protective effects of PDTC-mediated NFkappaB inhibition in tubulointerstitial disease in vivo.

Inhibition of nuclear factor-kappaB activation reduces cortical tubulointerstitial injury in proteinuric rats.

BACKGROUND: Protein-induced chemokine expression in proximal tubular cells is mediated by the transcription factor nuclear factor-kappa B (NF-kappaB). We hypothesized that in vivo inhibition of renal NF-kappaB activation would reduce interstitial monocyte infiltration in a rat model of nonimmune proteinuric tubulointerstitial inflammation. METHODS: Male Wistar rats received a single intravenous injection of doxorubicin hydrochloride [adriamycin (ADR), 7.5 mg/kg] and were studied 7, 14, 21, and 28 days later. In a second study, inhibitors of NF-kappaB [N-acetylcysteine (NAC; 150 mg/kg, b.i.d., i.p.), pyrrolidine dithiocarbamate (PDTC, 50 mg/kg, b. i.d., i.p.)] or vehicle were commenced on day 14 after the onset of proteinuria and were continued until day 30. RESULTS: Rats injected with ADR had increased proteinuria (UpV, day 28, 474 +/- 57; control, 18 +/- 2 mg/day; P < 0.01) and cortical tubulointerstitial injury [tubule cell atrophy, interstitial volume, and monocyte/macrophage (ED-1) infiltration]. Electrophoretic mobility shift assay of nuclear extracts from whole cortex of ADR rats demonstrated that NF-kappaB activation (p50/65, p50/c-Rel) increased from day 7 (4.7 +/- 0.2 fold-increase above control; P < 0.01) was maximal at day 28 (6.2 +/- 0.7; P < 0.01) and correlated with UpV (r = 0.63; P < 0.05) and interstitial ED-1 infiltration (r = 0.67; P < 0.01). Chronic treatment of ADR rats with PDTC suppressed NF-kappaB activation (by 73%; P < 0.05) without any effect on UpV. NF-kappaB inhibition with PDTC was accompanied by a reduction in tubule cell atrophy (59%; P < 0.01), interstitial volume (49%; P < 0.05) and ED-1 infiltration (48%; P < 0.01), and cortical lipid peroxidation (41%; P < 0.05) compared with vehicle-treated ADR rats. In contrast NAC had no effect on NF-kappaB activation, tubulointerstitial injury, or UpV in ADR rats. CONCLUSION: The activation of NF-kappaB may have an important role in mediating cortical interstitial monocyte infiltration and tubular injury in nonimmune proteinuric tubulointerstitial inflammation.

Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor kappaB in proximal tubule cells.

The transcription and translation of monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, are increased in proximal tubule epithelial cells (PTC) stimulated with pathophysiologically relevant concentrations of albumin. The purpose of this study was to investigate whether nuclear factor kappaB (NFkappaB)/Rel proteins play a role in albumin-induced MCP-1 transcription. Confluent monolayers of rat PTC in primary culture were stimulated with delipidated bovine serum albumin. NFkappaB, the NFkappaB inhibitory protein (IkappaB), and MCP-1 transcription were assessed using electrophoretic mobility shift assays, Western immunoblotting, semiquantitative reverse transcription-PCR, and ribonuclease protection assays. Activation of NFkappaB by delipidated bovine serum albumin (15 mg/ml) was detectable within 2 h, maximal after 8 h, and maintained for at least 16 h of continuous exposure. Supershift analysis showed that the activated proteins were composed of p50/p50, p50/p65, and p50/c-Rel dimers. dimers. Cytoplasmic IkappaBalpha levels were decreased 30 min after stimulation and returned to unstimulated levels by 4 to 8 h. IkappaBbeta levels were decreased at 2 h and there was no recovery until 8 h. Inhibition of NFkappaB with pharmacologic agents (N-tosyl-phenylalanine chloromethyl ketone and dexamethasone) and an antisense oligonucleotide to the rat p65 subunit of NFkappaB significantly reduced MCP-1 transcription. The 3.6-kb 5' flanking region of the rat MCP-1 gene was cloned and sequenced, and two putative kappaB binding sites were identified within the enhancer region. Therefore, albumin increased NFkappaB and reduced IkappaB levels in PTC, and MCP-1 expression was dependent on NFkappaB activation. It is concluded that the activation of NFkappaB/Rel proteins modulates chemokine production in PTC in response to albumin and is likely to have an important role in the mediation of tubulointerstitial injury in proteinuric renal disease.

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. 2. State 4 (resting) respiration was increased in adriamycin nephrosis in comparison with control (51 +/- 2 vs 43 +/- 2 ng atoms oxygen (O)/min per mg protein, respectively; P < 0.02). 3. Mitochondrial iron concentration was increased in nephrotic rats compared with control (9.52 +/- 0.70 vs 5.97 +/- 0.26 nmol Fe/mg protein, respectively; P < 0.001) and rates of state 3, state 4 and uncoupled respiration and the severity of proteinuria correlated with mitochondrial iron concentration. 4. To further define the relationship between mitochondrial iron accumulation and altered respiratory function, rats were loaded with iron. 5. In comparison with control, acute iron loading of normal rats impaired creatinine clearance (1.48 +/- 0.02 vs 0.40 +/- 0.29 mL/min), increased kidney weight (1.33 +/- 0.07 vs 1.74 +/- 0.14 g) and impaired mitochondrial enzyme activity (e.g. cytochrome oxidase 185.0 +/- 46.6 vs 362.0 +/- 32.8 delta log [cytochrome C]/min per mg protein; P < 0.05), but had no significant effect on rates of mitochondrial respiration or on mitochondrial fragility. 6. Mitochondrial iron concentration was not increased by iron loading, despite a similar increment in cytoplasmic iron to that seen in rats with adriamycin nephrosis. 7. In summary, resting mitochondrial respiration is increased in nephrotic rats in proportion to mitochondrial iron accumulation. Changes in mitochondrial oxygen consumption do not appear to be a primary event in the tubular cell injury of iron loading.

Proteinuria and tubulointerstitial injury.

These studies have demonstrated pathways whereby one urinary protein, holotransferrin, may alter proximal tubular cell function and cause tubular cytotoxicity, and at least two urinary proteins, albumin and transferrin, may mediate the development of interstitial inflammation in proteinuric renal disease.

Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein.

Cytokines play a pivotal role in synthesis and deposition of extracellular matrix in chronic renal failure (CRF). The proinflammatory properties of monocyte chemoattractant protein (MCP)-1 make it an ideal candidate cytokine for the production of interstitial inflammation in CRF. To investigate the possible role of proteinuria in inducing proximal tubular (PT) MCP-1, MCP-1 mRNA levels were measured by Northern blot and reverse transcription PCR in confluent monolayers of PT cells in primary culture in media containing a variety of proteins. PT cells produced MCP-1 mRNA in response to bovine serum albumin (BSA), delipidated BSA (dBSA; 0.5 to 30 mg/ml), holotransferrin, and apotransferrin (1 to 8 mg/ml). Unstimulated PT cells expressed very low levels of MCP-1 mRNA, detectable by reverse transcription PCR but not by Northern blot. The expression of MCP-1 mRNA reached a peak (sixfold greater than control) within 4 h of exposure to dBSA and was maintained for at least 24 h with continued exposure. Removal of dBSA from the media led to a rapid decline in MCP-1 mRNA expression. dBSA-induced MCP-1 expression was inhibited by lysine, an inhibitor of protein uptake, and reproduced by dBSA purified by gel and size-selective filtration. dBSA influenced MCP-1 expression at the level of transcription and probably translation, as evidenced by abrogation of MCP-1 by actinomycin D and superinduction with the protein synthesis inhibitor cycloheximide. The concentration of MCP-1 protein in response to dBSA added to the apical surface of PT cells was 2.4-fold greater in basolateral than in apical media, indicating basolateral secretion of MCP-1 protein. In summary, PT cell MCP-1 mRNA and protein expression are upregulated by albumin and transferrin, in concentrations similar to those of proteinuric urine. This effect could explain the link between proteinuria and interstitial inflammation in CRF.

Mechanisms of iron-induced proximal tubule injury in rat remnant kidney.

The proposition that proximal tubule (PT) iron accumulation may cause PT injury by lysosomal destabilization or reactive oxygen species generation in human and animal chronic renal disease was examined in partially nephrectomized [remnant kidney (RK)] and sham-operated (SO) Wistar rats. Electron microscopic histochemistry with horseradish peroxidase indicated iron uptake into and release from lysosomes. PT cytoplasmic iron was seen in RK but not in SO by energy-dispersive X-ray spectrometry. Total (9.66 +/- 1.89 vs. 3.30 +/- 0.31 nmol/mg protein; P < 0.01), low-molecular-weight (1.39 +/- 0.09 vs. 0.91 +/- 0.07; P < 0.001), and catalytic iron (1.88 +/- 0.27 vs. 1.28 +/- 0.09; P = 0.05) were higher in RK cytoplasm than in SO. Lysosomal enzyme activity was greater in RK than in SO [e.g., N-acetyl-beta-D-glucosaminidase (NAG): 0.75 +/- 0.05 vs. 0.57 +/- 0.06 mumol p-nitrophenol.h-1.mg protein-1; P < 0.05] and was increased further by chronic iron loading (e.g., RK and NAG: 0.84 +/- 0.04 vs. 0.60 +/- 0.07; P < 0.05). There was no enzymatic evidence of lysosomal fragility, and chronic iron loading of RK decreased fragility as assessed by NAG release (1.36 +/- 0.14 vs. 2.17 +/- 0.14; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary protein alters tubular iron accumulation after partial nephrectomy.

Reactive oxygen species (ROS) have been implicated in progression of disease in the rat remnant kidney (RK) model of chronic renal failure. Substantial amounts of iron accumulate in proximal tubular lysosomes of RK and could damage tubules by ROS generation. The effect of dietary protein intake on ROS, tubular damage and iron accumulation assessed by energy dispersive analysis was determined in RK (5/6 nephrectomy, N = 12) and sham-operated kidneys (SO, N = 10). In RK, mean lysosomal iron concentration, urinary iron and protein excretion and morphological damage were increased and GFR decreased. Dietary protein loading (40% vs. 12%) increased the number of iron-containing lysosomes (P < 0.05) and the mean lysosomal iron (P < 0.02) in proximal tubular cells after four weeks. In RK, high protein diet increased renal weight (P < 0.01), numerical density of iron-containing lysosomes and tubular damage (both P < 0.05). ROS generation, assessed by tissue and plasma malondialdehyde (MDA), was also increased (both P < 0.05). Plasma MDA correlated with tubular iron accumulation (r = 0.75). In RK fed a high protein diet (N = 18) treatment with the iron-chelator desferrioxamine reduced serum iron, urinary volume, and tubular iron accumulation and damage compared to controls (P < 0.01). In summary, in RK dietary protein manipulation altered urinary iron and protein excretion, proximal tubular iron accumulation, renal cortical ROS generation and ultrastructural damage. Desferrioxamine treatment reduced tubular lysosomal iron and ultrastructural damage. These results suggest a role for tubular iron as a determinant of tubular injury associated with dietary protein loading in rats with partial nephrectomy.

Lysosomal iron accumulation in diabetic nephropathy.

Iron accumulates within proximal tubular lysosomes in several models of renal disease and may play a role in the progression of proteinuric chronic renal disease by the generation of reactive oxygen species. In this study, tubular iron was examined at an ultrastructural level by energy dispersive x-ray spectrometry in streptozotocin (STZ) and BB diabetic rats, and in humans with diabetic nephropathy, and compared to their respective nondiabetic controls. Substantial amounts of iron accumulated in the secondary lysosomes of proximal tubules in STZ diabetic rats (4.16 +/- 0.47 iron-containing lysosomes/microns 2 x 10(-3) tubular area vs. 0.90 +/- 0.29 in controls, p < 0.001). Proximal tubular iron was related independently with urinary protein and transferrin excretion, suggesting increased cellular uptake of iron from the tubular fluid. Lysosomal iron accumulation was also associated with tubular damage (r = 0.55, p < 0.001). Minimal amounts of tubular iron were observed in BB diabetic and nondiabetic littermates. In humans with diabetic nephropathy, increased proximal tubular lysosomal iron concentration (35.6 +/- 13.0 mg% Fe vs. 9.5 +/- 2.7, p < 0.05) and numbers of iron-containing lysosomes were observed compared to nondiabetic controls, and the latter correlated with elevation of serum creatinine (r = 0.94, p = 0.016). These results suggest that filtered iron enters proximal tubular lysosomes across the brush-border membrane and are consistent with a role for iron in causing the tubular damage of diabetic nephropathy.

Hepatotoxicity induced by diethylnitrosamine causes no significant disturbances of systemic glucose homeostasis in rats.

In rats, a moderately hepatotoxic single dose of diethylnitrosamine (DEN) 100 mg/kg causing depletion of liver glycogen, elevation of aspartate aminotransferase and decreased liver uptake of 3-O-methylglucose, resulted in substantial changes in insulin and glucagon balance. Two days after DEN, insulin binding to liver membranes and insulin removal by the liver were sharply reduced whereas its binding to muscle and adipocyte membranes remained unaltered. Serum insulin (random and after an overnight fast) remained normal. Intravenous (I.V.) insulin (10 U/kg) caused the usual degree of hypoglycemia that, however, lasted longer than in the control animals. Removal of glucagon by liver was also depressed in spite of its normal binding to hepatocytes, and peripheral serum glucagon was increased three-fold. I.V. glucagon (40 micrograms/kg) resulted in a blunted response of plasma glucose. I.V. glucose tolerance test (1 g/kg) remained normal in spite of the insulin increase to a level twice as high as in the controls, and in spite of nonsuppressed glucagon. These changes were still present after 1-3 months, but disappeared by 6 months. The results demonstrate remarkable ability of homeostatic mechanisms to preserve normal plasma glucose and glucose tolerance in spite of dramatic changes in insulin and glucagon.