The glomeruli of human diabetic nephropathy patients were under oxidative stress and underwent significant pathological changes.
In total, tissues from eight normal and eight diabetic nephropathy kidneys were selected for this study. The selected clinical characteristics of patients are shown in . Pathological and immunohistochemical analyses were performed (). Compared with normal glomeruli, the glomeruli from diabetic nephropathy patients showed significant morphological changes (, compare A
). Two massive K-W nodules were formed inside the glomerulus of diabetic nephropathy patients (B
, white arrows
). It has been reported that overproduction of ROS is one of the major factors mediating tissue damage in diabetes (30
). Thus, Nrf2 is likely activated in the glomeruli of diabetic nephropathy patients. Next, Nrf2 expression in normal and diabetic nephropathy glomeruli was measured using immunohistochemical analysis. Nrf2 was hardly expressed in normal glomeruli, whereas it was upregulated in diabetic nephropathy glomeruli (, compare C
). In addition, cells with high expression of Nrf2 in the nucleus were identified as mesangial cells (D
). NQO1, a well-studied Nrf2 target gene, was also activated in glomeruli of diabetic nephropathy patients (, compare E
), indicating the activation of the Nrf2-mediated antioxidant response. Next, oxidative DNA damage was measured using immunohistochemical analysis with an anti–8-Oxo-dG antibody. Nuclear staining was detected in some cells of the glomeruli from diabetic nephropathy patients, indicting that the diabetic nephropathy kidney is under oxidative damage (, compare G
). However, the number of nuclear stained cells was low in diabetic nephropathy patients because of the massive glycogen deposition inside the glomeruli (H
). Together, these results demonstrated that the glomeruli of diabetic nephropathy patients are under severe oxidative stress and the Nrf2-mediated antioxidant response is activated.
Selected clinical characteristics of patients in the study group
FIG. 1. Significant pathological changes and activation of Nrf2 pathway in the glomeruli of human diabetic nephropathy patients. Renal biopsy samples were fixed and cut into 2-μm sections; the sections were subjected to H&E staining (A and B) (more ...)
Nrf2−/− mice suffered greater renal damage induced by STZ compared with Nrf2+/+ mice.
During the course of 16 weeks, some mice in both STZ-treated Nrf2+/+ and Nrf2−/− groups died, but the survival rate between the two groups did not show any statistical difference (A, P = 0.9477). Blood glucose levels, monitored at 3, 5, 8, 12, and 16 weeks post-injection, were significantly increased in both Nrf2+/+ and Nrf2−/− mice after STZ treatment, although no difference in glucose levels was observed between the two genotype groups (B, *P < 0.05).
FIG. 2. Nrf2−/− mice suffered greater renal damage by STZ compared with Nrf2+/+ mice. During the course of 16 weeks after STZ injection, the survival rate of mice was recorded and plotted (A). Tail-vein blood glucose levels were monitored at 3, (more ...)
At 16 weeks post-injection, mice in the STZ-treated groups had a decrease in their body weight, whereas the untreated groups gained weight (C, *P < 0.05). Interestingly, Nrf2−/− mice lost more body weight than Nrf2+/+ mice when treated with STZ (C, #P < 0.05). Next, the ratio of kidney to body weight, which indicates the enlargement of the kidney, was calculated. The ratio was significantly increased in the STZ-treated groups (D, *P < 0.05). However, there was no difference in the ratio of kidney to body weight between the two genotypes, even in the treated groups, which may be due to the greater decrease in body weight of the Nrf2−/− mice (D). As an index of renal function, UACR was measured at 0, 8, and 16 weeks after STZ injection. STZ markedly enhanced UACR at both 8 and 16 weeks post-injection (E, *P < 0.05). Although no difference was observed between Nrf2+/+ and Nrf2−/− mice at 8 weeks post-injection, the Nrf2−/− mice showed a significantly higher UACR than Nrf2+/+ mice at 16 weeks post-injection (E, #P < 0.05). All these results indicate that Nrf2−/− mice suffered greater renal damage, implicating the essential role of Nrf2 in protecting against STZ-induced diabetic nephropathy.
Higher levels of oxidative stress and oxidative damage occurred in the glomeruli of Nrf2−/− mice than in Nrf2+/+ mice in response to STZ.
Next, STZ-induced oxidative stress were measured by Nrf2 activation and oxidative DNA damage. Nrf2−/− mice did not have any detectable levels of Nrf2 in their glomeruli, confirming the complete deletion of Nrf2 (A, c and d). Nrf2 expression was greatly enhanced in the glomeruli of the STZ-treated Nrf2+/+ group and Nrf2 nuclear staining was observed (A, compare a and b, and the insert of b). Activation of Nrf2 was confirmed by upregulation of NQO1 in response to STZ treatment in Nrf2+/+ mice, but not in Nrf2−/− mice (A, i–l). To test the role of Nrf2 in ameliorating oxidative damage under the diabetic nephropathy condition, oxidative DNA damage was compared in the glomeruli of Nrf2+/+ and Nrf2−/− mice using immunohistochemical analysis with an anti–8-Oxo-dG antibody. The results reveal that the STZ-treated Nrf2−/− mice had a greater degree of oxidative damage than the STZ-treated Nrf2+/+ mice (A, compare f and h). Intriguingly, Nrf2−/− mice displayed higher levels of oxidative damage even in the untreated condition (A, compare e and g), indicating that the basal level of Nrf2 is essential in protecting against DNA damage induced by intrinsic sources of ROS. Consistent with these results, higher levels of basal and STZ-induced oxidative DNA damage in Nrf2−/− mice were confirmed by measuring the urinary level of 8-Oxo-dG (B). These results clearly demonstrate that Nrf2−/− mice had higher production of ROS in response to STZ challenge and were more vulnerable to ROS-induced damage due to loss of Nrf2.
FIG. 3. Higher levels of oxidative stress and oxidative damage occurred in the glomeruli of Nrf2−/− mice than in Nrf2+/+ mice in response to STZ. At 16 weeks post-injection, Nrf2+/+ and Nrf2−/− mice were killed and kidneys were (more ...)
Nrf2−/− mice had more severe glomerular injury than Nrf2+/+ mice.
Kidneys were isolated and processed for pathological analysis using H&E, PAS, and Masson's trichrome staining. Glomerular lesions were detected in both Nrf2+/+
mice after STZ injection in H&E-stained tissue sections (A
, compare a
, and c
; K-W nodules are labeled with arrows in b
). Next, glycogen deposition was measured by PAS staining (A
). The severity of glomerulosclerosis was scored using a semiquantitative method, and the result is shown in B
. STZ significantly induced glomerulosclerosis in both genotype groups (A
, compare e
, and g
< 0.05). Moreover, Nrf2−/−
mice showed a higher score than Nrf2+/+
mice in response to STZ treatment (A
, compare f
< 0.05). In addition, K-W nodules were also observed in PAS-stained tissues in the STZ-treated groups (A
, compare e
, and g
, black arrows in f
). ECM deposition in glomeruli is a hallmark of many renal diseases including diabetic nephropathy (31
), Therefore, collagens and FN, the major components of ECM, were measured using Masson's trichrome staining method and immunohistochemical analysis with an anti-FN antibody, respectively. Collagen deposition was observed inside the glomeruli of the STZ-treated Nrf2−/−
mice, but not in STZ-treated Nrf2+/+
, compare j
). Slight collagen deposition in the untreated Nrf2−/−
mice was also observed (A
). In response to STZ treatment, expression of FN was increased in both genotype groups (A
, compare m
, and o
). Consistent with the observed oxidative damage in untreated Nrf2−/−
), the basal level of FN expression in Nrf2−/−
was also higher than that in Nrf2+/+
, compare m
). Collectively, these results demonstrate that Nrf2 is essential in protecting against both basal and STZ-induced glomerular injury.
FIG. 4. Nrf2−/− mice had more severe glomerular injury than Nrf2+/+ mice. Kidney tissue sections were subject to H&E (Fig. A, a–d), PAS (A, e–h), and trichrome staining (A, i–L), as well as immunohistochemical analysis (more ...)
Nrf2−/− mice had higher TGF-β1 transcription and FN expression.
Next, the molecular mechanism by which Nrf2 protects against STZ-induced glomerular injury was explored. First, activation of the Nrf2 pathway by STZ-induced ROS was tested. Data shown in A represent the average reading of eight mice in each group. As expected, there was no Nrf2 mRNA detected in Nrf2−/− mice (A, Nrf2 panel #P < 0.05). In addition, STZ treatment did not induce Nrf2 mRNA expression in Nrf2+/+ mice (A, Nrf2 panel), which is consistent with the previous findings that upregulation of Nrf2 is primarily regulated at the level of Nrf2 protein stability. However, the downstream genes of Nrf2, NQO1, and GST were transcriptionally activated in response to STZ in Nrf2+/+ mice and only slightly in Nrf2−/− mice (, NQO1 and GST panels, *P < 0.05), indicating the activation of the Nrf2-mediated antioxidant response. Intriguingly, although both basal and STZ-induced levels of NQO1 in Nrf2−/− mice were lower than that in Nrf2+/+ mice, the basal level of GST was similar between these two genotype groups (A, NQO1 panel, #P < 0.05, and GST panel).
Next, mRNA expression of TGF-β1, FN, and collagen IV were measured. As shown in B, the basal level of TGF-β1 in Nrf2−/− mice is higher than Nrf2+/+ mice (B, TGF-β1 panel, #P < 0.05). STZ treatment induced transcription of TGF-β1 in both Nrf2+/+ and Nrf2−/− mice, and the highest transcription was observed in the STZ-treated Nrf2−/− mice (B, TGF-β1 panel, *P < 0.05, #P < 0.05). In agreement with the notion that TGF-β1 positively regulates expression of FN, the mRNA expression pattern of FN is similar to that of TGF-β1 (B, FN panel, *P < 0.05, #P < 0.05), demonstrating that FN was overexpressed, especially in the STZ-treated Nrf2−/− mice. To our surprise, although the basal level of collagen IV mRNA expression in Nrf2−/− mice was higher, compared with Nrf2+/+ mice (B, collagen IV panel, #P < 0.05), there was no difference in collagen IV mRNA expression between the control and STZ-treated groups. It is likely that the observed collagen deposition in the glomeruli of the STZ-treated Nrf2−/− mice in the Masson's trichrome stained tissues (, l) came from other types of collagen, rather than collagen IV. In another set of experiments, NQO1 and FN were chosen as representative Nrf2 and TGF-β1 downstream genes, respectively, and their protein levels were measured by immunoblot analysis. NQO1 was induced by STZ treatment in Nrf2+/+ mice, while there was no detectable level of NQO1 in Nrf2−/− mice (C, NQO1 panel). Quantification data showed nearly fourfold induction of NQO1 in response to STZ in Nrf2+/+ mice (C, lower left panel, *P < 0.05). The protein level of FN was induced more than three- to fourfold by STZ treatment, both in Nrf2+/+ and Nrf2−/− mice (C, FN panel, and lower right panel, *P < 0.05). Furthermore, Nrf2−/− mice had both higher basal and STZ-induced levels of FN than Nrf2+/+ mice, with the highest FN expression detected in the STZ-treated Nrf2−/− mice (C, FN panel, and lower right panel, #P < 0.05). Taken together, these data demonstrate that STZ is able to activate the Nrf2-mediated antioxidant response, which in turn negatively regulates TGF-β1–mediated ECM production, especially FN.
Nrf2 was activated by high glucose–induced ROS production in HRMCs.
To further confirm that the activation of Nrf2 by STZ in vivo is due to high glucose–induced ROS production, in vitro experiments were carried out. Because mesangial cells play a crucial role in the initiation and progression of many renal diseases, including diabetic nephropathy (31
), HRMCs were used for this in vitro study. An enhanced nuclear protein level of Nrf2 was detected in cells growing under high glucose medium compared with low glucose medium (A
< 0.05). Consistent with this result, high glucose induced the mRNA level of NQO1, HO-1, and GST, without affecting Nrf2 mRNA levels (B
< 0.05), indicating the activation of the Nrf2 pathway. This in vitro study recapitulates the observed Nrf2 activation in STZ-treated Nrf2+/+
mice and in human renal tissues from a diabetic nephropathy patient. It is conceivable that high glucose induced Nrf2 activity through ROS production. Thus, ROS levels were measured. Indeed, the cells growing in high glucose medium had substantially higher levels of ROS (C
< 0.05). To further confirm the notion that Nrf2 activation by high glucose is through generation of ROS, N
-acetylcysteine (NAC), a ROS scavenger, was included in the medium. As shown in D
, NAC inhibited the activation of high glucose–induced Nrf2 and NQO1 (D
< 0.05, #P
< 0.05). Collectively, these results indicate that hyperglycemia is able to activate the Nrf2 pathway through generation of ROS.
FIG. 6. Nrf2 was activated by high glucose–induced ROS production in HRMCs. Before exposure to low-glucose (LG) (1 g/l) or high-glucose (HG) (5.4 g/l) DMEM, cells were starved for 24 h with low-glucose DMEM containing 0.5% FBS. Cells were then incubated (more ...)
Nrf2 negatively regulated TGF-β1 and FN in HRMCs.
To confirm the negative effects of Nrf2 on TGF-β1, as observed in the in vivo study shown in B, regulation of the TGF-β1 promoter activity by Nrf2 was studied using luciferase reporter gene analysis. Overexpression of Nrf2 inhibited the promoter activity of TGF-β1 in a dose-dependent manner (A, upper panel, *P < 0.05). Overexpression of Nrf2 was confirmed by immunoblot analysis with an anti-HA antibody (A, lower anti-HA panel). Consistent with the result obtained from overexpressed Nrf2, induction of endogenous Nrf2 by tert-butylhydroquinone inhibited the promoter activity of TGF-β1 in a dose-dependent manner (B, upper panel, *P < 0.05, and lower anti-Nrf2 panel). Consistent with the Nrf2-mediated negative regulation of the TGF-β1 promoter, Nrf2 also negatively regulated the protein level of FN, a TGF-β1–downstream gene (A and B, FN panel). In addition, changes in TGF-β1 mRNA in response to reduced expression of Nrf2 by Nrf2-siRNA were measured in cells growing in low- and high-glucose medium. As expected, cells growing in high glucose medium had a higher level of TGF-β1 expression (C, *P < 0.05). Knockdown of Nrf2 significantly enhanced the mRNA level of TGF-β1 in both conditions (C, #P < 0.05), again demonstrating the negative effect of Nrf2 on TGF-β1 expression. Next, another parallel set of samples was used for immunoblot analysis. High glucose induced Nrf2, aldose reductase, γGCS, and NQO1, as well as FN (D, compare lane 1 and 3, *P < 0.05). Nrf2-siRNA reduced the protein levels of Nrf2, aldose reductase, γGCS, and NQO1, while it enhanced FN in both low- and high-glucose medium (D, compare lane 1 and 2, and lane 3 and 4, #P < 0.05).
FIG. 7. Nrf2 negatively regulated TGF-β1 and FN in HRMCs. HRMCs growing were transfected with plasmids for TGF-β1–promoter-firefly luciferase and renilla luciferase (internal control), along with different amounts of an expression vector (more ...)