MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy
MG132 protects against renal dysfunction by regulating Akt- mediated inflammation in diabetic nephropathy
OPEN Published: xx xx xxxx Diabetic nephropathy (DN), the leading cause of end-stage renal disease (ESRD). To date, mounting evidence has shown that inflammation may contribute to the pathogenesis of DN. Recent reports have shown that proteasome inhibitors display cytoprotection by reducing the phosphorylation of Akt, a serine/threonine kinase, plays a critical role in cellular survival and metabolism and can crosstalk with inflammation. Therefore, we hypothesized that MG132, specific proteasome inhibitor, could provide renoprotection by suppressing Akt-mediated inflammation in DN. In vivo, male Sprague-Dawley rats were divided into normal control group (NC), diabetic nephropathy group (DN), DN model plus MG132 treatment group (MG132), and DN model plus deguelin treatment group (Deguelin)(deguelin, a specific inhibitor of Akt). In vitro, a human glomerular mesangial cell lines (HMCs) was exposed to 5.5 mmol/L glucose (CON), 30 mmol/L glucose (HG), 30 mmol/L glucose with 0.5 umol/L MG132 (MG132) and 30 mmol/L glucose with 5 umol/L deguelin (Deguelin). Compared with NC, DN showed a significant increase in the urinary protein excretion rate and inflammatory cytokines, as well as p-Akt. Compared with CON, HMCs co-cultured with HG was notably proliferated, which is in accord with ?-smooth muscle actin (?-SMA) expression. These alterations were inhibited by administration of MG132 or deguelin. In conclusion, MG132 significantly inhibits the development of DN by regulating Akt phosphorylation-mediated inflammatory activation.
Diabetic nephropathy (DN) is one of the major causes of microvascular complications of diabetes mellitus (DM)
and the leading cause of chronic and end-stage-renal disease worldwide (CKD and ESRD, respectively)1. Based
on a study in 930 patients with type II diabetes, the Shanghai Diabetic Complications Study reported that the
prevalence of microalbuminuria and macroalbuminuria was 22.8% and 3.4%, respectively2. Major hallmarks of
DN include accumulation of extracellular matrix (ECM) proteins, such as collagens and mesangial expansion in
the kidney glomerular and tubular compartments, which contribute to renal failure in diabetes3?5. Accumulated
data have emphasized the critical role of inflammation in the pathogenesis of DN6, which acts through oxidative
stress, transcription factors, and inflammatory cytokines. However, the precise mechanisms are unknown.
Akt, a downstream target of activated phosphatidylinositol 3-kinase (PI3K)7,8, is activated by mitogens and
cytokines. Previous studies have reported the importance of the PI3K/Akt pathway, an important regulator of
growth and inflammation, in inflammation-mediated diseases, such as rheumatoid arthritis (RA)9 and psoriasis10.
In this study, we aimed to determine the effects of high glucose on the development of inflammation and
mesangial cell proliferation, as well as mesangial matrix expansion. MG132, specific proteasome inhibitor,
prevents damage by inhibiting inflammatory process by regulating Akt and exerts a marked renoprotective effect.
Material and Methods
Experimental animal. Male Sprague-Dawley rats (initial weight of 200 to 220 g; average age 8 weeks; Third
Military Medical University Animal Experiment Center) were randomly divided into two groups: normal
control rats (NC, n = 18) and diabetic nephropathy rats (DN, n = 60). NC rats were fed standard laboratory animal
food, while DN rats were fed a high-sugar and high-fat feed (laboratory animal food: Yolk: lard: Sodium
cholate: sucrose = 63.5:10:8:0.5:18) for four weeks. And then diabetic rats were subjected to right nephrectomy to
hasten the development of kidney disease. After two weeks, DN was induced by intraperitoneal injection with
streptozotocin (STZ, 40 mg/kg body weight) dissolved in citrate buffer (pH 4.5, 0.1 M). Blood glucose levels were
determined at three and seven days after STZ injection, and only rats with blood glucose levels above 16.7 mmol/L
and weakly positive urinary albumin levels were considered as the major indicators of DN, and used in this study.
All experimental procedures were carried out in accordance with the recommendations of the Care and Use
Committee of the Third Military Medical University. All DN rats (n= 54) were randomly divided into three
subgroups, the untreated DN group (DN) and the DN treated with MG132 group (MG132), as well as the DN treated
with deguelin group (Deguelin) (deguelin, a specific inhibitor of Akt) which were injected intraperitoneally (i.p.)
either with an equal volume of phosphate buffer solution (PBS) alone or with MG132 10 ug/kg (Sigma, US) or
with deguelin 4.0 mg/kg (Enzo Life Sciences, Germany) beginning on the day that the DN model was established
(week 0). All rats had free access to standard chow and tap water. The 24-h urine samples were collected in
metabolic cages at weeks 4, 8 and 12 after treatment began. Rats were sacrificed at the end of weeks 4, 8 and 12 after
treatment began, and the left kidneys were harvested, weighed and transversely divided into two pieces, with one
piece fixed in 10% formalin for periodic acid-Schiff staining. Other tissues emedullated were frozen in liquid
nitrogen for detecting molecular biological expression.
Measurement of metabolic parameters. All rats were anesthetized and blood samples were drawn from
the heart and were centrifuged at 3500 rpm for five minutes. After proper dilution, the supernatant was used for
the determination of concentrations of blood glucose (Glu), triglyceride (TG) and total cholesterol (TC) using an
enzymatic method (kits from Jiu Qiang biotech company, Beijing, China). The urine protein was determined by
chemical method, and the urinary protein excretion rate of 24 h was calculated according to the formula = 24 h
total volume of urine (L) ? urinary protein levels (mg/L). Urine MCP-1 levels were measured by quantitative
sandwich ELISA using a commercial kit according to the manufacturer?s instructions (Biosource, Camarillo,
California, USA). The assay was performed in duplicate, and the intensity of the color was measured in an ELISA
reader at 450 nm.
Renal morphologic analysis. Tissue for light microscopy was fixed in 10% formalin and embedded in
paraffin. Sections were 4-? m thick and were processed for periodic acid-Schiff staining. Morphologic analyses
were performed by an experienced pathologist who was blinded to the source of the tissue. Application of a
computer image analysis system for semi-quantitative analysis of the glomerular area: that is, under a low
magnification field of vision (?100), thirty glomeruli containing the vascular pole and the urine pole were randomly
selected in each slice, and their mean areas were measured and calculated. The glomerular area (GA), defined as
the cross-sectional area containing the vascular pole and the urine pole, and the mean areas were measured and
calculated. Glomerulosclerosis was defined as index of glomerulosclerosis (IGS). The degree of sclerosis was
subjectively graded on a scale of 0 to 4: grade 0, normal; grade 1, affected glomeruli <10%; grade 2, affected glomeruli
10?25%; grade 3, affected glomeruli 25?75%; grade 4, affected glomeruli greater than 75%. IGS was calculated
using the following formula: IGS = (1 ? N1 + 2 ? N2 + 3 ? N3 + 4 ? N4)/N0 + N1 + N2 + N3 + N4. N is the
number of glomeruli in each grade of sclerosis.
Cell culture. Human glomerular mesangial cells line (HMCs) was kindly provided by Professor Ruan
Xiongzhong from Lipds Research Center of Chong Qing Medical University11. Transformed HMCs were grown
in RPMI 1640 medium (Salt Lake City, UT, USA) supplemented with 5.5 mmol/L glucose and 10% fetal bovine
serum (Sijiqing, Hangzhou, China), at 37 ?C in a humidified incubator (Heraeus, Germany) with 95% air and 5%
CO2. Cells (passages 2?3) grown to sub-confluence were used to complete all the cell based experiments. On this
basis, these cells were not passed during the 72 h and the medium was changed every 24 h. HMCs co-culture with
30 mmol/L of glucose (high glucose, HG) is defined as mimicking in vivo hyperglucemia, and HMCs were treated
with 5.5 mmol/L of glucose would be considered as control. HG with MG132 group was treated with 30 mmol/L
of glucose and 0.5 umol/L of MG132, and HG with deguelin group was treated with 30 mmol/L of glucose and
0.1 umol/L of deguelin.
Cell proliferative rate assay- tetrazolium salt (MTT) colorimetric assay. The cell viability was
measured as described previously12. Briefly, HMCs were plated on M96-well plates at 1 ? 104cells/mL. After the
corresponding treatments, we incubated the cells for 4 h with 0.5 mg/mL of MTT (Amersham, LON, UK) and
then lysed the cells with dimethylsulfoxide (DMSO). Absorbance was measured at 490 nm in a microplate reader
Quantitative real-time RT-PCR analysis. Total RNA was isolated from the renal tissue using
TRIzol extraction (Invitrogen Life Technologies, Shanghai, China) and reverse-transcribed to cDNA using
ReverTra AceTM (TOYOBO, Osaka, Japan). Quantitative real-time PCR was performed with primer
pairs and probes on a Rotor-gene 6000 (Corbett Life Science, Sydney, Australia). All samples were
analyzed in triplicate, and ddH2O served as a no-template control. The relative amount of mRNA was
calculated using the comparative Ct (2???Ct) method. The primer and probe sequences were as follows: (
NF-?B (forward: 5?-AATTGCCCCGGCAT-3?; reverse: 5?-TCCCGTAACCGCGTA-3?); (
) MCP-1 (forward:
5?-CGCTTCTGGGCCTGTTGTTCC-3?; reverse: 5?-GCCGACTCATTGGGATCATC-3?); (
) TGF-?1 (forward:
Western blot analysis. Tissue samples from the renal tissue were placed in a buffer containing 20 mM
Tris-HCl, pH 6.8, 1 mM EDTA, 1% SDS, 1 mM PMSF and 1? protease inhibitor cocktail. The protein was
separated on 15% SDS-PAGE and electroblotted onto nitrocellulose (NC) membranes. The membranes were
incubated with one of the following antibodies: anti- p65 (1:1000; Cell Signaling Technology, Danvers, MA,
USA); anti-p-Akt (Ser473,1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-?-SMA(1:1000; Abcam,
USA); anti-MCP-1(1:200; Santa Cruz Biotechnology. Santa Cruz, CA, USA); anti-TGF-?1(1:500; Santa Cruz
Biotechnology. Santa Cruz, CA, USA);as the primary antibody. HRP-conjugated goat anti-rabbit IgG was used
as the secondary antibody (1:1000; Sigma, USA). All membranes were incubated with a monoclonal anti-?-actin
antibody (1:2000; Novus, USA). Immunoreactive bands were visualized with the luminescence method (Western
Blot Chemiluminescence Reagent Plus, NEN? Life Science Products Inc.). The band density was normalized to
the corresponding density of ?-actin at 42 kDa.
Data analysis. Data were compared among groups using one-way ANOVA, followed by the LSD tests or
Mann-Whitney U test. All statistical analyses were performed by the SPSS Statistical Software version 19.0. All
values are presented as mean ? S.E.M. and a value of P < 0.05 was considered statistically significant. Statistical
methods are included in the tables and figures.
Metabolic parameters. As shown in Table?1, compared with the NC group, the parameters of the kidney
weight/body weight index (KI), Scr, Glu, TG, and TC showed a noticeable increase in DN rats (all P < 0.05) at 4,
8, and 12 weeks. Compared to the DN group, treatment with MG132 or deguelin markedly lowed the increase
in KI and Glu, especially at 8 and 12 weeks (P < 0.05), but had no effect on the metabolism of Scr, TG, and TC.
UPER was kept at a low level in NC rats throughout the study period. However, UPER increased progressively
with time in DN rats and peaked at the twelfth week (Fig.?1). Treatment with MG132 or deguelin significantly
suppressed UPER at 4, 8, and 12 weeks, suggesting that MG132 could effectively prevent the increase in UPER.
Therefore, these results indicate that both MG132 and deguelin could markedly prevent renal hypertrophy and
Effect of MG132 on renal histopathologic changes. In this study, we found typical glomerular damage
in the kidneys of DN rats, including mesangial cell proliferation, mesangial matrix accumulation and
expansion(?), compared with NC (Fig.?2A). Treatment with MG132 and deguelin prevented these changes (Fig.?2C,D).
As Fig.?2E shows, for DN, the mean GA was approximately 1.5-fold that of NC at 12 weeks. However,
administration of MG132 or deguelin decreased GA by approximately 18% and 20%.
MG132 suppresses high glucose-induced HMC proliferation. To investigate the effect of MG132 on
the proliferation of HMCs, HMCs proliferation was detected by the MTT assay. As shown in Fig.?3A, compared
with the CON group, high glucose facilitates HMCs proliferation along with the temporal elongation. However,
incubation with MG132 or deguelin inhibited high glucose-induced HMCs proliferation.
MG132 suppresses high glucose-induced expression of ?-SMA. ECM accumulation plays crucial
roles in early renal hypertrophy and late glomerular sclerosis in diabetic nephropathy; ?-SMA as one of the
important indicators of fibrosis; therefore, we evaluated the effect of MG132 on the expression of ?-SMA. As
shown in Fig.?4, ?-SMA was significantly higher in the HG group. After treatment with MG132 or deguelin for
24, 48, and 72 h, ?-SMA was significantly decreased at all timing points.
Effect of MG132 on the renal sclerotic degree. IGS is index for evaluating the sclerotic degree of
glomerulosclerosis. As Fig.?5 shows, IGS in the DN group was prominent at 12 weeks, but MG132 and deguelin
inhibited the sclerotic degree by approximately 65% and 70%, respectively.
Effect of MG132 on Akt phosphorylation. Akt is a well-established protein that regulates cell growth,
survival and anti-apoptotic mechanisms. Akt activation is regulated through phosphorylation. Renal tissue
western blotting (Fig.?6A) demonstrated that p-Akt(Ser473) protein expression was increased in the DN group;
however, compared with the DN group (P < 0.05), p-Ak(Ser473) augmentation in the DN group was partially reversed
by MG132. There was no significant difference between the MG132 and deguelin groups, indicating that the
proteasome inhibitor MG132 partially reversed the p-Akt(Ser473) increase in DN. In addition, similar to the in
vivo experiment (Fig.?6B), the relative expression of p-Akt(Ser473) increased with time in the HG group; the most
significant changes were observed after 72h. After MG132 or deguelin intervention, p-Akt (Ser473) expression was
significantly decreased. These data suggest that high glucose led to p-Akt(Ser473) expression; however, elevated
p-Akt(Ser473) expression was significantly decreased by the addition of MG132.
Effect of MG132 on the expression of NF-?B. NF-?B is a pleiotropic transcription factor that is mainly
represented by the p65/p50 heterodimeric complex, which is found in multiple cell types. It regulates the
transcription of multiple genes and is involved in the inflammatory response, cell proliferation, and apoptosis13. To
further elucidate the protective mechanisms of MG132 on the diabetic kidney, we measured the mRNA of NF-?B
and protein level of p65. As Fig.?7A shows, significantly elevated of NF-?B expression was detected in the DN
group compared to the NC group (P < 0.05). Treatment with MG132 reduced the extent of the change. Similarly,
deguelin remarkably decreased the expression level of NF-?B after 8 and 12 weeks (P < 0.05). We also found that
the change trend of NF-KB is consistent with p65 (Fig.?7B); the DN group demonstrated a significant elevation
compared with the NC group. However, MG132 and deguelin efficiently inhibited the expression of p65.
Effect of MG132 on inflammatory cytokine expression. MCP-1, a member of the CC chemokine
family of proinflammatory cytokines14,15, plays an important role in the propagation of focal inflammation and
macrophage infiltration16. As Fig.?8A,B shows, the level of MCP-1 was significantly increased compared to the
NC group (P < 0.05), but MG132 and deguelin effectively suppressed this increase (P < 0.05). TGF-?1, another
proinflammatory cytokine, is a pivotal mediator of matrix accumulation that results in the development of
glomerulosclerosis17?19. In this study, we found that the expression level of TGF-?1 was elevated in the DN group
compared with the NC group (all P < 0.05). However, treatment with MG132 and deguelin decreased the level of
TGF-?1 (all P < 0.05). Moreover, the urinary MCP-1 concentration was in accordance with the MCP-1 level of the
tissues, and the MCP-1concentration was decreased by treatment with MG132 and deguelin (Fig.?8E, P < 0.05).
This study demonstrated that the proteasome inhibitor MG132 had a preventative effect on impaired renal
function induced by persistent high glucose. Several factors support this concept. First, HMCs co-cultured with
high glucose noticeably proliferated, while there was a depressant effect when MG132 was added. MG132 also
decreased blood glucose, the urinary protein excretion rates, and glomerulosclerosis in DN rats. Second, high
glucose increased the expression of ?-SMA and inflammatory transcripts; however, these expression levels were
markedly reduced by MG132. Third, p-Akt(Ser473) was elevated by hyperglycemia and was significantly
attenuated by the administration of MG132. More importantly, the effect of MG132 was in parallel with deguelin, a
specific inhibitor of Akt. These results provide the first evidence that MG132 effectively prevents the progression
triggers a negative feedback loop on the PI3K/Akt pathway, leading to suppression of Akt33,34. In this study, we
found that HMCs incubated with high glucose demonstrated increased proliferation, which is consistent with
the expression of p-Akt(Ser473). However, deguelin effectively decreased the level of proliferation. These results
suggest that Akt plays a significantly role in the pathology of chronic renal injury.
The most important finding in this study is that we first demonstrated that MG132 has an equivalent effect on
alleviating renal deterioration induced by high glucose as deguelin, as evidenced by in vitro and in vivo studies.
In vivo research showed that MG132 effectively reduced mesangial cell proliferation, mesangial matrix
accumulation, and urine protein excretion for the indicted time in diabetic nephropathy rats. In vitro studies also
revealed that most mesangial cell phenotypic transformation markers induced by high glucose were suppressed
by MG132, including decreased mesangial cell proliferation and the expression of ?-SMA. These findings are in
line with Sternesjo35, who implicated the proteasome in interleukin-1??mediated suppression of islet function.
Interesting, we also found that MG132 supressed the expression of p-Akt(Ser473). In particular, Tang36
demonstrated that proteasome inhibitors, clasto-lactacystin blactone (LA) or epoxomicin (Epo) reduced p-Akt and
activation of autophagy in ARPE-19 cells, possibly through inhibition of PI3K/Akt/mTOR signalling. Therefore, we
speculated that MG132, a proteasome inhibitor, would be a drug of practical value for the treatment of diabetic
nephropathy through inhibition of the Akt signalling pathway.
Recently, it is believed that DN is one kind of chronic inflammation. Persistent and enhanced inflammation,
and finally leads to excessive fibronectin production and extracellular matrix accumulation resulting in
acceleration of the pathogenesis of glomerular sclerosis and tubulointerstitial fibrosis. The ubiquitin-proteasome system
(UPS) is related to inflammatory signal transmission, such as NF-?B and its downstream signalling cascade.
NF-?B is mainly represented by the p65/p50 heterodimeric complex and this complex is retained in the
cytoplasm in an inactive form bound to an additional inhibitory subunit ? I?B?37. During activation, the
inhibitory subunit I?B? is rapidly phosphorylated at Ser32 and Ser36 by IKK?/? and subsequently ubiquitinated and
degraded by the 26S proteasome complex. Once released, free NF-?B translocates to the nucleus and activates
the transcription of various inflammatory gene products. MG132 plays a pivotal role in blocking the degradation
of ubiquitinconjugated proteins and permeable strains of yeast by the 26S complex. It inhibits NF-?B activation
by reducing the degradation of I?B?. We found that expression of NF-?B and p65 were significantly higher in
the DN treated group, as compared with the NC group. In MG132-treated rats the expression of NF-?B and p65
were down-regulated, as compared with DN rats. These results indicated that MG132, inhibited activation of
NF-?B. Similar to UPS, Akt appears to require IKK to efficiently stimulate the transactivation domain of the p65
subunit of NF-?B38. Deguelin, a specific Akt inhibitor, it suppressed NF-?B, suggesting specificity toward NF-?B.
Asha in vitro kinase assays showed that deguelin is not a direct inhibitor of IKK, but this agent seems to block
the activation of IKK by interfering with upstream regulatory kinases39. Other evidence indicated that IKK is a
downstream target of Akt. Bhandari provided indirect evidence that renal cortical matrix accumulation in Type 2
DM is, at least in part, attributable to Akt effects40. Another new finding of the study is that, deguelin inhibited the
high expression of NF-?B and p65 in DN group. In our previous research have shown that renal 26S proteasome
activity and concentration, the indicators of UPS, were significantly higher in DN rats than in NC rats at the end
of 4, 8 and 12 weeks; these increase reflects the activation of UPS in kidney of DN rats41. Therefore, it is reasonable
to assume that administration of MG132 and deguelin may constitute a new molecular basis for the inhibition of
inflammatory activation in rats with diabetic nephropathy by interruption of activated Akt.
Increasing evidence suggests that inflammation due to proinflammatory cytokines and chemokines secreted
by renal cells and macrophages infiltrating the kidney can substantially contribute to DN. In this study, it is
interesting to note that there was a significant increase in NF-?B in the DN group compared with the NC group.
Furthermore, the results also showed that MCP-1 was significantly elevated in the kidneys of the DN group.
Meanwhile, the data in this study demonstrated that UPER was increased in line with urinary MCP-1. More
importantly, we found that MG132 not only reduced NF-?B but also reduced the expression of MCP-1 in DN
group kidney tissue and decreased urine excretion. NF-?B, the major inflammatory transcription factor that
triggers the transcription of several inflammation mediators, such as endothelin-1 (ET-1), VCAM-1,
intercellular adhesion molecule-1 (ICAM-1), IL-6, and TNF-?42, is expressed in mesangial cells43, renal tubule cells, and
podocytes in individuals with DM44. MCP-1, which is a member of the CC chemokine family of
proinflammatory cytokines, plays an important role in the propagation of focal inflammation and macrophage infiltration.
Several recent studies have indicated that MCP-1 null mice are protected against DN and blockade of the MCP-1
receptor, C-C chemokine receptor type 2 (CCR-2), using propagermanium-ameliorated diabetic
glomerulosclerosis. However, expression of urinary MCP-1 and the secretory volume of UPER were decreased with MG132
administration. Previous studies have shown that urinary excretion of MCP-1 is correlated with diabetic
glomerular injury45, as well as an increased risk of death and cardiovascular events46,47. These results are supported by
Banba45, whose study indicated that increases in MCP-1 expression and interstitial macrophage infiltration
coincide with the development of hyperglycemia and precede a rise in albuminuria in type 1 DN in mice. Bondar48
and Wolkow49 documented that urinary excretion of proinflammatory factors in patients with DN correlated with
the excretion of urine albumin. In a model of STZ-induced type 1 DN, mice genetically deficient in MCP-1 were
found to have reduced renal injury compared with wild-type mice with equivalent hyperglycemia. Therefore,
MCP-1 plays a critical role in diabetic kidney impairment caused by inflammation, and the proteasome
inhibitor MG132 inhibited inflammation and reduced the excretion of urine protein in DN rats. Major hallmarks of
DN include the accumulation of ECM proteins, such as collagens (leading to fibrosis), and mesangial expansion
(leading to hypertrophy) in the kidney glomerular and tubular compartments, which contribute to renal failure
in diabetes. However, the molecular mechanism of this phenomenon has not been established. To verify this
hypothesis, we incubated HMCs with high glucose and determined the expression of ?-SMA; we found that
the level of protein expression was remarkably increased. TGF-?1, the most abundant TGF-? family member
isoform, is a pleiotropic cytokine that has been established as a central mediator of kidney inflammation and
fibrosis; TGF-?1 is involved in inflammatory responses associated with the NF-?B pathway and binds to latent
TGF-?-binding protein (LTBP) and initiates downstream signals50. In the present research, we provided evidence
that increased expression of TGF-?1 was significantly inhibited by treatment with MG132. These results were
supported by the work of Ma51, who found that MG132 significantly attenuated hypertension-induced cardiac
remodelling and dysfunction via downregulation of TGF-?1. These results were also supported by Sakairi52, who
confirms rat renal fibroblasts NRK-49F cells and tubular epithelial cells, NRK-52E, were treated with TGF-? in
the presence or absence of a proteasome inhibitor, MG132 or lactacystin. Proteasome inhibitors attenuate TGF-?
signalling by blocking Smad signal transduction in vitro. As mentioned above, MG132 effectively inhibited renal
inflammation and fibrosis through attenuation of NF-?B in DN rats. Similarly, administration of deguelin greatly
diminished the expression of NF-?B and MCP-1, as well as TGF-?1, suggested that MG132 inhibition of
inflammation is in line with deguelin and is associated with NF-?B. It is notable that deguelin alleviates inflammation;
whether this is a direct action on NF-?B needs to be investigated. In this study, deguelin suppressed NF-?B
activation through a variety of stimuli, suggesting that it must act at a step common to all of these activators.
It is worth noting that MG132 decreased blood glucose compared with DN, which is supported by Zhou53,
who found that glucose-dependent insulinotropic polypeptide receptor (GIP-R) was rescued by treating isolated
islets with the proteasomal inhibitors lactacystin and MG132. After inhibition, the islets were once again capable
of increasing the intracellular cAMP levels in response to increase insulin secretion and subsequent effects on
glucose metabolism54?56. Hofmeister suggested that glucokinase aggregation due to proteasome blocking with
MG132, bortezomib, epoxomicin or lactacystin could be detected in MIN6 cells57. Similarly, deguelin could also
decrease blood glucose, which is related with relieving insulin resistance58?60. In recent years, more and more
evidences (clinical and animal experiment) suggest DN can?t be prevented by simply lowering blood glucose
owing to the ?metabolic memory?, supported by Kowluru61,who found in diabetic rats, poor glucose control
led to hyperglycemia-induced changes in retinal cell apoptotic marker expression, which were sustained for as
long as several months following glucose normalization. Not only diabetic nephropathy itself is associated with
inflammation, but also we have confirmed that MG132 and deguelin can reduce transcription factor and its
expression of inflammatory factors, so as to reduce proteinuria. Whatever the mechanism, these findings indicate
that MG132 treatment effectively protected the kidneys of rats against the complications of DM.
In summary, we showed that MG132 is a proteasome inhibitor that can effectively provide renoprotection in
DN rats via inhibition of the PI3K/Akt pathway-related inflammatory response. Although the precise mechanism
should be explored in future studies, and one must be cautious in applying animal models to human disease, these
studies provide a theoretical basis for further study of the clinical prevention and treatment of DN.
This work was supported by the two National Nature Science Foundation Grants of China (81370820 and
81400737) to Dr Feng. We are grateful to Prof Shi-Wen?Zhou, Li-Xia?Guang, and Yin?Xu for giving technical and
experimental condition support.
Wei?Zeng and Bing?Feng designed the experiment scheme and wrote the paper. Wei?Qi and Jian-Ying?Tang
prepared figures 1?2. Jiao?Mu prepared figure 3. Yi?Wei, Li-Ling?Yang and Qian?Zhang prepared figures 4?6.
Qiong?Wu did data analysis prepared figures 7?8 and Table 1.
Competing Interests: The authors declare no competing interests.
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