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NDT Advance Access originally published online on January 24, 2006
Nephrology Dialysis Transplantation 2006 21(5):1188-1197; doi:10.1093/ndt/gfk085
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Ganoderma extract prevents albumin-induced oxidative damage and chemokines synthesis in cultured human proximal tubular epithelial cells

Kar Neng Lai, Loretta Y. Y. Chan, Sydney C. W. Tang and Joseph C. K. Leung

Division of Nephrology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong

Correspondence and offprint requests to: Professor K. N. Lai, Department of Medicine, University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong. Email: knlai{at}hkucc.hku.hk



   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. Ganoderma lucidum (Ganoderma or lingzhi) is widely used as an alternative medicine remedy to promote health and longevity. Recent studies have indicated that components extracted from Ganoderma have a wide range of pharmacological actions including suppressing inflammation and scavenging free radicals. We recently reported that tubular secretion of interleukin-8 (IL-8) induced by albumin is important in the pathogenesis of tubulointerstitial injury in the proteinuric state. In this study, we explored the protective effect of Ganoderma extract (LZ) on albumin-induced kidney epithelial injury.

Methods. Growth arrested human proximal tubular epithelial cells (PTECs) were incubated with 0.625 to 10 mg/ml human serum albumin (HSA) for up to 72 h. HSA induced DNA damage and apoptosis in PTEC in a dose- and time-dependent manner. Co-incubation of PTEC with 4–64 µg/ml LZ significantly reduced the oxidative damage and cytotoxic effect of HSA in a dose-dependent manner (P<0.001). Increased release of IL-8 and soluble intercellular adhesion molecules-1 (sICAM-1) in PTEC induced by HSA was ameliorated by co-incubation with Ganoderma (16 µg/ml). To explore the components of LZ that exhibited most protective effect in HSA-induced PTEC damages, LZ was further separated into two sub-fractions, LZF1 (MW <30 kDa) and LZF2 (MW <3 kDa), by molecular sieving using millipore membrane. PTEC were incubated with 5 mg/ml HSA in the presence of different doses of LZF1, LZF2 or unfractionated LZ.

Results. There was no difference in the degree of protection from HSA-induced cytotoxicity or oxidative DNA damage between different fractions of LZ. However, low molecular weight LZ (<3 kDa) was most effective in reducing sICAM-1 released from HSA-activated PTEC whereas the high molecular weight LZ (unfractionated LZ) was more effective in diminishing IL-8 production.

Conclusions. Our results suggest that Ganoderma significantly reduces oxidative damages and apoptosis in PTEC induced by HSA. The differential reduction of IL-8 or sICAM-1 released from HSA-activated PTEC by different components of the LZ implicates that components of Ganoderma with different molecular weights could play different roles and operate different mechanisms in preventing HSA-induced PTEC damage.

Keywords: chemokines; Ganoderma; lingzhi; proteinuria; proximal tubular epithelial cells



   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 References
 
Tubulointerstitial damage followed by scarring and progressive loss of renal function is the final common pathologic pathway in many forms of chronic proteinuric renal diseases. The severity of tubulointerstitial injury, which correlates with the amount of proteinuria, is a major determinant of the degree and rate of progression of renal failure. The putative role of urinary proteins, taken to reflect the degree of protein trafficking through the glomerular capillary, in inducing tubulointerstitial changes is supported by clinical observations as well as animal models of protein overload and experimental nephrotic syndrome [1]. Emerging evidence indicates that interstitial lesions induced by proteinuria may be mediated through tubular epithelial cell activation. One constant feature of proteinuric nephritis is the concomitant presence of tubulointerstitial inflammation, which is characterized by the infiltration of the interstitial space by mononuclear leucocytes, notably T-cells, monocytes and macrophages, which appear to play a key role in the subsequent evolution of tubulointerstitial inflammation and fibrosis. Chemokines secreted by tubular cells almost certainly play a pivotal role in leucocyte recruitment. Monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and RANTES are three C–C chemokines that have recently been shown to be upregulated by albumin in cultured proximal tubular cells [2–4].

Ganoderma lucidium (Ganoderma or lingzhi) is a natural herbal medicinal fungus that has been used in China since 100 AD. Recently, this medicinal Chinese mushroom has been demonstrated to possess tumoricidal [5] as well as immunodulatory activities [6]. In vitro studies show Ganoderma extract activates mitogen-activated protein kinase (MAPK) and inhibits lipid peroxidation and oxidative DNA damage [7,8]. Increasing evidence has been accumulated on the medicinal application of Ganoderma in the treatment of immunological disorders [9]. In the present study, we examine the in vitro effect of Ganoderma extract on the oxidative damage and chemokines synthesis in cultured human proximal tubular epithelial cells (PTECs) induced by albumin. The modulation of tubulo-interstitial injury in this model may have potential experimental basis to support the therapeutic potential of Ganoderma.



   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 References
 
Preparation of water-soluble Ganoderma extract
In this study, Ganoderma extract (product code RLZ91; batch number 970117) was provided by Vita Green Company (Hong Kong). Briefly, to isolate water-soluble polysaccharides from Ganoderma, samples were mostly extracted with hot water (95–100°C). Then ethanol was added to the concentrated aqueous extract and the resulting precipitate (polysaccharide fraction) was collected by centrifugation. After cleaning, drying, ethanol extraction and vacuum concentration (60°C, 76 cmHg), a batch of crude extract of Ganoderma was obtained. The major components consisted of mainly polysaccharide with small amount of nucleotide, tripenoids and Ling-Zhi-8 from HPLA analysis [10,11]. A single stock of Ganoderma extract was prepared for all cell culture experiments. In one experiment, Ganoderma extract was further fractionated into three preparations (LZ, total Ganoderma extract; LZ F1: Ganoderma extract with MW <30 kDa; LZ F2 Ganoderma extract with MW <3 kDa) by passing through a series of millipore membranes with cut-off of different MW (Millipore, Bedford, MA, USA).

Reagents
Medium, reagents for cell culture, antibodies for cell characterization, and general chemicals were purchased from Sigma Chemicals (Poole, Paisley, UK). Human serum albumin (HSA) was obtained from CSL Laboratory (Parkville, Victoria, Australia). The endotoxin level in all albumin preparations were <10 EU/ml as determined using the QCL-1000 Limulus Amebocyte Lysate (LAL) kit (BioWhittaker Inc., Walkersville, MD, USA). Antibiotics, sera, agarose and DNA size markers were obtained from Gibco BRL (Paisley, UK). Enzyme immunoassay kits for detection of IL-8 and soluble intracellular adhesion molecules-1 (sICAM-1) were purchased from Bender MedSystems (Vienna, Austria). Fluorescence probe for intracellular reactive oxygen species detection was from Molecular Probes Inc. (Eugene, OR, USA). All other antibodies were from Dako A/S (Glostrup, Denmark). Other chemicals were obtained from Sigma (St Louis, MO, USA).

PTEC culture
Human PTECs were isolated according to a previously described method [4]. Briefly, renal cortical tissue was obtained from kidneys removed for circumscribed tumours. Histological examination of these kidney samples revealed no renal pathology. Cortical specimens were cut into small cubes and passed through a series of mesh sieves of diminishing pore size. PTECs were collected on the 53 µm sieve, and digested with collagenase (750 U/ml) at 3°C for 15 min. Tubular cells were isolated by centrifugation and grown in a renal epithelial basal medium (REBM) consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 10% fetal calf serum, hydrocortisone (40 ng/ml), L-glutamine (2 mM), benzyl penicillin (100 IU/ml) and streptomycin (100 µg/ml). The cells were incubated at 37°C in 5% CO2 and 95% air. They were characterized to be of proximal tubular origin by immunofluorescence and enzyme histochemistry: cells stained positively for cytokeratin, vimentin and alkaline phosphatase, but negatively for Tamm–Horsfall glycoprotein, factor VIII-related antigen and {alpha}-smooth muscle actin. Scanning electron microscopy demonstrated the presence of numerous apical microvilli of a rudimentary brush border with reassembly of tight junctions. Experiments were performed with cells up to the third passage, as it has been shown that there are no phenotypic changes up to this passage number [4]. In all experiments, there was a ‘growth arrest’ period of 48 h in serum-free medium prior to stimulation. Results were obtained from PTEC cultured from the kidney of three different donors.

Analysis of IL-8 and ICAM-1 gene expression
Gene expression of IL-8 and ICAM-1 was determined by reverse transcription and polymerase chain reaction (RT–PCR) as previously described [4]. The oligonucleotide sequences for IL-8 were: forward-ATG ACT TCC AAG CTG GCC GTG CT, reverse-TCT CAG CCC TCT TCA AAA ACT TCT; sICAM-1: forward-GAG ACC CCG TTG CCT AAA, reverse-CCG CAG GTC CAG TTC AGT; yielding amplified products of 298 bp (IL-8) and 399 bp (ICAM-1). PCR reactions were carried out in a DNA thermal cycler (MJ Research, Watertown, MA, USA), with 28–30 cycles of amplification at an annealing temperature of 55–59°C. For semi-quantification, human glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) primers were included in every reaction as an internal control. The PCR products were separated by 1.5% wt/vol agarose gels, stained with ethidium bromide (Sigma) and the gel image was captured and analysed using the Gel Doc 1000 Densitometry System and Quantity One (Bio-Rad Laboratories Ltd, Hercules, CA, USA). The product yield was expressed as a ratio to GAPDH.

Assay of IL-8 and sICAM-1 proteins in culture supernatants
PTEC were grown to confluence in six-well cell culture plates, growth-arrested and exposed to albumin (1.25–20 mg/ml) for defined time periods (6, 24 or 48 h) at 37°C in the presence or absence of Ganoderma extract. The concentration of albumin and the duration of experiments were selected based on our previous data [4]. At the end of the experiment, cell number was counted and supernatants were collected and stored at –70°C until further use. Detection of IL-8 and sICAMI-1 levels in culture supernatants was carried out on commercially available assay kits. The detection sensitivity and intra-assay CV is 11 pg/ml±3.8% for IL-8 and 0.63 ng/ml±6.8% for sICAM-1.

Quantification of proliferation and viability of PTEC cultured with Ganoderma extract
The optimal or sub-toxic dose of Ganoderma extract was determined by cell viability measured by a commercial colorimetric kit (Chemicon International, Temecula, CA, USA) based on cleavage of the tetrazolium salt WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt]. Briefly, growth arrested PTEC were seeded into 96-well plates (0.25 x 105 cells per well) before being exposed to Ganoderma extract for 48 h. WST-1 reagent was then added and incubated at 37°C for a further 2 h. The absorbance was measured using 450 nm as the primary wavelength and 600 nm as the reference wavelength. Results were expressed as percentage changes in absorbance compared with that of the medium control.

Determination of apoptosis and oxidative damage of PTEC
The level of apoptosis was determined by a TUNEL assay carried out with the ApopTaq Fluorescein In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA) following the manufacturer's protocol. Briefly, cells were grown on Lab-Tak chamber slides (Nalgene Nunc Internatinal, Naperville, IL, USA) and fixed with 1% paraformaldehyde. The fixed cells were subsequently incubated in TdT equilibration buffer followed by TdT reaction buffer. After washing, the cells were incubated with anti-digoxigenin-fluorescein. Slides were mounted and the apoptotic cells with green fluorescence were visualized under a fluorescence microscope with a FITC filter (excitation 490 nm, emission 520 nm). For a positive control, camptothecin was added to the media (1:1000) for 24 h before analysis. In addition, activation of caspase 3 was determined using the caspase 3 activity fluorometric immunosorbent enzyme assay kit (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer's protocol.

The oxidative damage of DNA in PTEC following exposure to Ganoderma extract was determined by a fluorogenic OxyDNA Assay kit (Calbiochem–Novabiochem Co., La Jolla, CA, USA). The OxyDNA Assay is an in vitro fluorescent protein binding method used for the detection of oxidative DNA in fixed and permeabilized cells. It uses 8-oxoguanine (as part of the oxidized nucleotide 8-oxoguanosine), which is a sensitive marker formed during free radical damage to DNA and is a sensitive and specific indicator of oxidative DNA damage. The assay is based on the binding of a FITC-labelled probe to the 8-oxoguanine moieties present in the DNA of the fixed and permeabilized cells. Fluorescence was visualized by fluorescence microscopy according to the protocol recommended by the manufacturer.

Measurement of intracellular reactive oxygen species (ROS)
The intracellular formation of ROS was detected by the fluorescence probe 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Molecular Probes Inc., Eugene, OR, USA). PTECs (1 x 106/ml) were cultured in the presence of different doses of HSA (4–16 mg/ml) or 16 mg/ml HSA with or without the addition of Ganoderma extract. The cells were incubated for 30 min and then loaded with 0.1 µg/ml CM-H2DCFDA. The cells were placed on ice. ROS production, expressed as mean fluorescence intensity (MFI), was immediately measured by flow cytometry using a Coulter EPICS XL analyzer (Coulter Electronic, Miami, FL, USA). Results were expressed as MFI% of control cells incubated with culture medium alone.

Statistical analysis
All data (from five experiments) were expressed as means±SD. Inter-group differences for continuous variables were assessed by the unpaired t-test. The cell viability of PTEC following exposure to different concentrations of Ganoderma extract was analyzed with multivariate ANOVA for repeated measures. Statistical analysis was performed using statistical software (Statview, SAS Intelligence, Cary, NC, USA). All P-values quoted are two-tailed and the significance is defined as P<0.05.



   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 References
 
Viability of PTEC cultured with Ganoderma
Figure 1A showed the cell viability measured by cell proliferation in PTEC following incubation of two concentrations of HSA. Incubation of PTEC with HAS at 2.5 mg/ml or 5 mg/ml reduced the cell proliferation by 68 and 73%, respectively. Ganoderma extract restored the cell viability in a dose-dependent manner (P<0.001). The cell viability improved to 85–95% of control values at a Ganoderma concentration of 16 µg/ml. It is noteworthy that cell viability fell by 11% in PTEC incubated with Ganoderma extract alone at a concentration of 64 µg/ml (without HSA). Cell viability was reduced by 18, 24, 50 and 17% when PTEC were incubated with Ganoderma extract alone at a concentration of 128, 256, 512 and 1026 µg/ml, respectively (not shown in figure). Figure 1B shows the micrographs of trypan blue stained PTEC cultured with different concentrations of Ganoderma extract upon prior incubation of 0, 2.5 or 5 mg/ml of HSA. The findings paralleled those of WST assay showing less than 20% of the cultured PTEC demonstrating uptake of the dye when incubated with HSA at 2.5 or 5 mg/ml. The dye uptake and cell morphology improved with addition of Ganoderma extract.


Figure 1
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  		Fig. 1. (A) Cell proliferation assay in PTEC incubated with HSA (for 48 h) determined by WST kit. Ganoderma extract increased the cell viability. (B) Micrographs showing reduced cell viability of PTEC following acute exposure to HSA for 48 h.

 
Effect of Ganoderma extract on apoptosis and oxidative damage of PTEC induced by albumin
Following exposure to HSA at either 2.5 or 5 mg/ml, PTEC exhibited significant apoptotic changes demonstrated by TUNEL (Figure 2A). The apoptotic changes were abolished with addition of Ganoderma extract at a concentration of 16 µg/ml. Activation of caspase 3 was observed in PTEC incubated with HSA for 24 h. The caspase 3 activation was significantly reduced in the presence of Ganoderma extract at concentrations of 8 and 16 µg/ml (P<0.001 vs PTEC incubated without Ganoderma) (Figure 2B). Similarly, Ganoderma extract at a concentration of 16 µg/ml reduced the oxidative damage of DNA in PTEC incubated with two cytotoxic concentrations of HSA (Figure 2C). The cytoprotective effect of Ganoderma extract on the oxidative damage of DNA in PTEC induced by HSA was dose-dependent (Figure 3A) while incubation of PTEC with Ganoderma extract alone (at a concentration between 4–16 µg/ml) showed no oxidative damage of DNA (Figure 3B) or induction of intracellular ROS generation in PTEC (Figure 4).


Figure 2
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  		Fig. 2. (A) Apoptosis assay by TUNEL showing increased apoptotic changes in PTEC incubated with HSA (for 24 h). The apoptotic changes were minimized with Ganoderma extract (LZ). (B) Activation of caspase 3 was observed in PTEC incubated with HSA (for 24 h) and this caspase 3 activation was significantly reduced (*P<0.001 vs PTEC incubated without Ganoderma) in the presence of 8 and 16 µg/ml of Ganoderma extract. (C) Oxidative damage of DNA in PTEC incubated with HSA (for 24 h). The oxidative DNA damage was minimized with Ganoderma extract.

 

Figure 3
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  		Fig. 3. (A) Representative cytometric results of ROS generation as determined by flow cytometry. The cytoprotective effect of Ganoderma extract (LZ) on the ROS released in PTEC induced by HSA (24 h incubation) was dose-dependent. (B) Incubation of PTEC with Ganoderma extract alone (at a concentration between 4–16 µg/ml) showed no induction of ROS generation.

 

Figure 4
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  		Fig. 4. ROS generation as determined by flow cytometry. The results represent mean±SD of five separate experiments. *P<0.001 when compared with PTEC incubated with HSA (for 24 h) in the absence of Ganoderma.

 
Effect of Ganoderma extract on IL-8 and sICAM-1 expression from PTEC induced by albumin
There was a significant increase of ICAM-1 expression (Figure 5A) with a 7-fold increase in sICAM-1 release from PTEC following exposure to HSA at 5 mg/ml (Figure 5B). Ganoderma extract reduced the ICAM-1 mRNA expression and sICAM-1 release from PTEC induced by HSA in a dose-dependent manner up to a concentration of 16 µg/ml, above which no further reduction was observed. Incubation of PTEC with Ganoderma extract alone (at a concentration between 2–16 µg/ml) showed no significant induction of ICAM-1 mRNA expression or sICAM-1 release from PTEC. Two-fold and 3.5-fold increases in sICAM-1 release, but not ICAM-1 mRNA expression, were detected with Ganoderma extract at a concentration of 32 or 64 µg/ml, respectively. Upon exposure to HSA at 5 mg/ml, there was a significant increase in IL-8 mRNA expression and an 18-fold increase in IL-8 release from PTEC (Figure 6B). Again, Ganoderma extract reduced the IL-8 mRNA expression and IL-8 release from PTEC induced by HSA in a dose-dependent manner up to a concentration of 32 µg/ml, above which no further reduction was observed. Incubation of PTEC with Ganoderma extract alone (at a concentration between 2–16 µg/ml) showed no induction of IL-8 mRNA expression or IL-8 release from PTEC although a 2-fold increase was detected with Ganoderma extract at a concentration of 32 µg/ml.


Figure 5
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  		Fig. 5. ICAM-1 mRNA expression at 6 h (A) and sICAM-1 protein synthesis (B) at 24 h in supernatant from PTEC cultured with HSA in the absence or presence of different concentrations of Ganoderma. The results represent mean±SD of five separate experiments. *P<0.001 when compared with PTEC incubated with HSA in the absence of Ganoderma. #P<0.05 when compared with PTEC incubated with HSA in the absence of Ganoderma.

 

Figure 6
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  		Fig. 6. IL-8 mRNA expression at 6 h (A) and IL-8 protein synthesis (B) at 24 h in supernatant from PTEC cultured with HSA in the absence or presence of different concentrations of Ganoderma. The results represent mean±SD of five separate experiments. *P<0.001 when compared with PTEC incubated with HSA in the absence of Ganoderma.

 
Pathophysiological actions of different fractions of Ganoderma separated by molecular weight
We then fractionated the Ganoderma extract into three preparations (LZ, total Ganoderma extract; LZ F1, Ganoderma extract with MW <30 kDa; LZ F2, Ganoderma extract with MW <3 kDa) (Figure 7A). All three fractions of Ganoderma extract restored the cell viability in a dose-dependent manner with cell viability improved to 77–88% of control values at a concentration of 16 µg/ml (Figure 7B). Similarly, all three fractions at identical concentration exhibited comparable reduction of ROS generation in PTEC induced by preincubation with HSA (Figure 8). Incubation with all three fractions of Ganoderma extract at either 16 or 32 µg/ml significantly reduced the sICAM-1 release from PTEC induced by HSA (Figure 9A). The reduction with either LZ F1 or LZ F2 fractions was lower than that of unfractionated LZ. In contrast, reduction of IL-8 release was only observed in PTEC that were incubated with unfractionated Ganoderma extract at a dose-dependent manner (Figure 9B). However, similar reduction of IL-8 release from PTEC was not observed with either LZ F1 or LZ F2 fractions.


Figure 7
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  		Fig. 7. (A) Fractions of Ganoderma extract separated by molecular weight. (B) The three fractions expressed similar protective effect on albumin-induced cytotoxicity on PTEC.

 

Figure 8
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  		Fig. 8. Fractions of Ganoderma extract separated by molecular weight expressed similar protective effect on albumin-induced ROS generation (48 h incubation) in PTEC. The results represent mean±SD of five separate experiments. *P<0.001 when compared with PTEC incubated with HSA in the absence of Ganoderma.

 

Figure 9
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  		Fig. 9. (A) sICAM-1 protein synthesis at 24 h in supernatant from PTEC cultured with HSA in the absence or presence of different fractions of Ganoderma. The results represent mean±SD of five separate experiments. *P<0.001 when compared with PTEC incubated with HSA in the absence of Ganoderma extract. #P<0.05 when compared with PTEC incubated with HSA in the presence of the same concentration of unfractionated Ganoderma extract (LZ). (B) IL-8 protein synthesis at 24 h in supernatant from PTEC cultured with HSA in the absence or presence of different fractions of Ganoderma. The results represent mean±SD of five separate experiments. *P<0.001 when compared with PTEC incubated with HSA in the absence of Ganoderma extract. #P<0.05 when compared with PTEC incubated with HSA in the presence of the same concentration of unfractionated Ganoderma extract (LZ).

 


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
References
 
The fungus Ganoderma lucidum—also known as ‘Lingzhi’ in Chinese, ‘Reishi’ in Japanese, and ‘Youngzhi’ in Korean—is a member of the genus Ganoderma and has been traditionally used as a popular herbal medicine for the promotion of health in the Orient. The genus Ganoderma, however, was established in the west by a Finnish botanist, Karsten, in 1881 [5], and more than 120 species have been reported in the world since then. In addition to G. lucidum (red), other members of the genus Ganoderma that are commonly known to possess medicinal/nutritional values include G. applanatum (brown), G. tsugae (red-brown), G. sinense (dark purple to black) and G. capense (dark red). In the present study, we utilize a primary PTEC culture to demonstrate the presence of reno-protective compounds in the Chinese medicinal mushroom, G. lucidum. Incubation with Ganoderma extract results in improved cell viability and reverses the cytotoxicity induced by HSA. More importantly, the Ganoderma extract also protects the PTEC from apoptosis and DNA damage induced by exposure to HSA. Furthermore, the ability of Ganoderma extract to reduce the release of sICAM-1 and IL-8 from PTEC upon exposure to HSA suggests that Ganoderma extract comprises a rich source of immunomodulatory compounds that possess anti-inflammatory actions.

Many biological effects of Ganoderma have recently been reported in scientific literature. Many of these suggest that Ganoderma has both immunoreactive and immunomodulatory effects. It exhibits anti-allergic properties to inhibit histamine release from mast cells [12]. Ganoderma also reduces production of IgG antibodies and suppresses IL-2 and ICAM-1 release from T cells [13,14]. On the other hand, Ganoderma enhances splenic natural killer cell activity and serum interferon production in mice [15], and induces neuronal differentiation of PC12 cells through activation of MAP kinases [7]. The bidirectional effect of Ganoderma in autoimmunity is also supported by our findings. While Ganoderma suppresses the inflammatory and apoptotic activities in PTEC induced by HSA within a dose range, incubation with Ganoderma alone at a higher concentration induces cytotoxicity and release of inflammatory mediators. These findings strongly indicate the necessity to determine a therapeutic dose for specific anti-inflammatory action.

During the past two decades, modern research has revealed that Ganoderma contains a variety of chemical ingredients, including triterpenes, polysaccharides, nucleosides, steroids, fatty acids, alkaloids, proteins, peptides, amino acids, and inorganic elements [11]. Among these ingredients, triterpenes and polysaccharides have attracted considerable attention as they have been shown to possess diverse and potentially significant pharmacological activities. Our water-soluble preparation of Ganoderma extract contains many bioactive ingredients (mainly polysaccharides and some triterpenes). Three fractions of Ganoderma extract were prepared according to the molecular weight of the ingredients. All three fractions restored the cell viability in a dose-dependent manner and exhibited comparable reduction of the oxidative damage of DNA in PTEC induced by preincubation with HSA at identical concentrations of Ganoderma extract. Incubation with all three fractions of Ganoderma extract at either 16 or 32 µg/ml significantly reduced the sICAM-1 release from PTEC induced by HSA. However, the reduction with either LZ F1 or LZ F2 fractions was significantly lower than that of unfractionated LZ. These findings suggest the bioactivities against cytotoxicity or oxidative damage of DNA and the anti-inflammatory effect on sICAM-1 release reside in ingredients with low molecular weight <3 kDa. Ganoderma extract contains different low molecular weight components including monosaccharides, oligosaccharide, triterpenes, vitamins and selenium. Of particular interest, the triterpenes have been reported to possess significant anti-oxidative activity [16]. In contrast, the bioactivity against the neutrophil chemoattractant effect of IL-8 lies in ingredients with molecular weight higher than 30 kDa. Our findings in PTEC illustrate the diverse and potentially significant pharmacological activities of the extensive array of chemical ingredients in Ganoderma. Peak profiling (fingerprint) of different saccharides using the high-performance thin-layer chromatography and/or automated multiple development are likely the next step forward for screening the biological activities of different ingredients in Ganoderma [17].

Our present data show that Ganoderma extract at concentrations of 32 and 64 µg/ml slightly upregulate the sICAM-1 release by PTEC. The increased release of sICAM-1 was not due to the upregulation of ICAM-1 mRNA expression. This increased release of sICAM-1 could be due to the increased cleavage of cell surface ICAM-1. However, the precise mechanism of this increased cleavage remains unknown. It has been reported that Ganoderma extract activates the MAPK in pheochromocytoma [7]. The extracellular signal-regulated kinase (ERK1/2) has been implicated in the cleavage of groups of transmembrane proteins [18]. It could be possible that high concentrations of Ganoderma extract increase the surface ICAM-1 cleavage through activation of the MAPK.

Ganoderma extract at concentration of ≥8 µg/ml significantly reduces ROS generation by PTEC exposed to HSA (5 mg/ml). However, higher concentration (≥16 µg/ml) is needed to reduce the sICAM-1 or IL-8 release as well as for maintaining the viability of PTEC cultured with HSA. Our data suggest that the protection of Ganoderma extract in PTEC against albumin-induced injury is not totally dependent on suppression of ROS generation. We and others have previously demonstrated that activation of NF-{kappa}B in PTEC is an important downstream event mediated by albumin [3,4]. We speculate that other ROS-independent signalling mediators could also participate in albumin-induced NF-{kappa}B activation. Higher concentration of Ganoderma extract is needed to suppress this ROS-independent NF-{kappa}B activation. Further experiments are needed to confirm this hypothesis.

There has been increasing evidence linking excessive protein trafficking through the glomerulus and progressive renal tubulointerstitial inflammation leading to chronic renal failure. A hallmark of inflammation is the invasion into injured tissue by activated leucocytes. This step is critically dependent on the rapid expression of chemokines. Data are already available to show that overexposure to filtered proteins upregulates tubular expression of pro-inflammatory mediators such as IL-8 [4] and macrophage migration inhibitory factor [19]. Apparently, the involvement of infiltrating inflammatory cells in the glomeruli and renal interstitium is important in mediating tubular injury and renal fibrosis. As a consequence, resident renal cells can become activated. In recent years, much attention has focused on the role of PTEC in the orchestration of inflammatory cell infiltration and interstitial fibrosis. Mediators released by infiltrating cells are directly responsible for the activation of proximal tubular cells, which in turn may intensify the inflammatory process via local production of various mediators which attract more inflammatory cells. The outcome of this chain reaction is the generation of a positive feedback loop of activation, leading to the overproduction of extracellular matrix components resulting in fibrosis and ultimately loss of tissue integrity and renal failure. Treatment options for tubulointerstitial injury related to proteinuric state remain limited other than blockade of the renin–angiotensin system aiming to reduce intra-renal pressure and proteinuria. As most available treatment is only targeted at preventing the development of renal failure rather than towards the primary etiology, long-term therapy is frequently required. Treatment with few side-effects is preferable. Ingredients purified from medicinal herbs are distinctly attractive with the notion that side-effects are fewer with natural compounds. Prevention of chronic renal failure had previously been reported with compounds purified from medicinal herbs [20]. Our present in-vitro study provides data suggesting the therapeutic potential of Ganoderma to ameliorate the tubulointerstitial injury related to proteinuric state.

In summary, this study is the first to show that Ganoderma extract can reduce the cytotoxicity, apoptosis, DNA damage and release of chemokines in PTEC stimulated by human albumin in an experimental setting mimicking proteinuric state. The reno-protective effect appears to be dose-related and a higher dose may induce pro-inflammatory effect. Currently, therapeutic interventions are primarily aimed at reducing glomerular protein ultrafiltration and controlling systemic hypertension. It is conceivable that future therapeutic strategies may be targeted at the pathobiologic processes that lie further downstream, namely the activation of tubular epithelial cells and their interaction with infiltrating leucocytes. Understanding the therapeutic potential of Ganoderma extract and its bioactivities is likely to yield novel insight into the development of new anti-inflammatory agents.



   Acknowledgments
 
The study was partly supported by the Research Grant Committee (Hong Kong SAR) [HKU7424/03M]. J.C.K. Leung is supported by the L&T Charitable Fund and INDOCAFE. Previously presented in abstract form at the American Society of Nephrology Annual Scientific Meeting, St Louis, MO, 29 October–21 November 2004.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 9.05
Accepted in revised form: 29.12.05


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