Angptl3 knockout plays a certain protective role in the occurrence and progression of mice ADR nephropathy
Angptl3 knockout has previously been shown to play an important role in the early stage of ADR nephropathy [19]. To observe whether the Angptl3 knockout plays a similar renal protective role in the end stage of nephropathy, we compared differences in proteinuria, hypoproteinemia, renal function, general condition (weight, fur quality, activity and appetite) and survival rates among different experimental groups at different time points up to 12 weeks in our ADR nephropathy mouse model. Angptl3 knockout (Angptl3−/−) mice have phenotypes similar to those of wild-type (Angptl3+/+) mice, except for the presence of hypolipidemia [19]. Therefore, we used average data from Angptl3+/+ and Angptl3−/− mice treated with or without saline injection as controls.
Proteinuria was seen as early as 1 week following ADR injection in Angptl3+/+ mice and persisted throughout the observation period (until week 12). The urine albumin: creatinine ratio showed a significant increase in week 1 (P < 0.05), peaked around week 8 (P < 0.001) and decreased slightly in week 12 (P < 0.01). A similar trend was observed in Angptl3−/− mice injected with ADR. However, the urine albumin: creatinine ratio were significantly lower at every time point (P < 0.05) in the Angptl3−/− mice compared to the similarly injected Angptl3+/+ mice (Fig. 1a).
The serum albumin levels in both the Angptl3−/− and Angptl3+/+ mice injected with ADR showed decreasing trends: started to decrease in week 1, and remained decreased significantly throughout the observation period. However, at 4 weeks post ADR injection, the serum albumin levels of the Angptl3+/+ mice were significantly lower than those of the Angptl3−/− mice (P < 0.05), and remained significantly lower through week12 (P < 0.05) (Fig. 1a).
Renal function, measured as the blood urea nitrogen (BUN) and serum creatinine (Scr) levels, remained stable in the ADR-injected Angptl3+/+ and Angptl3−/− mice during the first 2 weeks but started to decline in week 4. The difference between the treated and control mice became statistically significant in week 8 and continued to be significant for the remainder of the experiment. The BUN and Scr levels were significantly lower in the ADR-injected Angptl3−/− mice compared to than in the ADR-injected Angptl3+/+ mice following ADR injection at 8 and 12 weeks (all P < 0.05) (Fig. 1a).
At 3 days post ADR injection, the Angptl3+/+ mice started to look sicker than the Angptl3−/− mice. The Angptl3+/+ mice showed increased hair loss, and their hair appeared dull compared with that of the Angptl3−/− mice. The Angptl3+/+ mice demonstrated reduced activity; compared with the Angptl3−/− mice, the Angptl3+/+ mice maintained a curled body position (data not shown but available from the authors). A higher proportion of the Angptl3+/+ than Angptl3−/− mice died throughout the observation period (Fig. 1b).
Histological examination by light microscopy revealed no evidence of renal injury during the first 2 weeks in either the Angptl3+/+ or Angptl3−/− mice injected with ADR. The Angptl3+/+ mice receiving ADR injection showed podocytes vacuolization in week 4, podocytes vacuolization and balloon adhesions appeared in week 8. Two-thirds (4/6) of the Angptl3+/+ mice manifested typical glomerulosclerosis formation in week 12. In contrast, the ADR-injected Angptl3−/− mice showed occasional podocyte vacuolization in week 8, and glomerulosclerosis was absent in all of the ADR-injected Angptl3−/− mice in week 12 (Fig. 2).
Examination with a TEM showed significant differences in the podocyte lesions, including foot process effacement, microvillus transformation and focal detachment from the basement membrane between the Angptl3+/+ and Angptl3−/− mice with receiving ADR injection from week 1 to week 12. The podocyte lesions progressed more rapidly and were more severe in the Angptl3+/+ mice receiving ADR injection than in the Angptl3−/− mice receiving ADR injection. As observed in week 1, podocyte foot process effacement manifested in Angptl3+/+ mice, while an almost normal podocyte structure was observed in the Angptl3−/− mice. Multifocal podocyte foot process effacement and significant podocyte microvillus transformation was observed in the Angptl3+/+ mice, while fewer podocyte foot process effacement and no microvillus transformation were detected in the Angptl3−/− mice in week 4; Apart from multifocal podocyte foot process effacement and significant podocyte microvillus transformation, focal podocyte foot process detachment from the basement membrane appeared earlier in the Angptl3+/+ mice week8, while, the same pathological lesions were seen in Angptl3−/− mice until week 12 (Fig. 3).
Angptl3 knockout delays glomerulosclerosis formation in mice with ADR nephropathy by attenuating podocyte loss
Podocyte injury and the subsequent cell loss promote glomerulosclerosis in various glomerular diseases [25, 26]. If cells are lost, then it should be possible to visualize this process by structural analysis. In this partial study, histological sections stained with PAS were examined under a light microscope for detached cells (detached from the glomerular basement membrane [GBM]), and a TEM was used to show rounded cells in Bowman’s capsule. The number of detached cells in Bowman’s capsule was calculated, and the average number of detached cells per randomly chosen glomerular section from the Angptl3+/+ mice injected with ADR for 12 weeks was significantly greater than that of the Angptl3−/− mice undergoing a similar injection protocol (P < 0.01, n = 30). Analysis with a TEM showed that the average number of detached, rounded cells per randomly chosen glomerular section in the Angptl3+/+ mice injected with ADR for 12 weeks was markedly higher than that of the Angptl3−/− mice undergoing a similar injection protocol (P < 0.01, n = 6) (Fig. 4a).
Transferase-mediated dUTP nick-end labeling (TUNEL) staining for identifying apoptotic cells in the renal tissue was performed with the different groups. TUNEL-positive cells were not observed in the glomeruli of saline-injected mice. However, after ADR injection for 12 weeks, TUNEL-positive cells were present in the glomeruli of both Angptl3+/+ and Angptl3−/− mice, and the number of TUNEL-positive cells per glomerulus in the Angptl3+/+ mice was greater than that in the Angptl3−/− mice (P < 0.05, n = 30) (Fig. 4b).
Most of the detached cells present in Bowman’s capsule appeared to be podocytes. To confirm podocyte loss in mice with ADR nephropathy, two podocyte-specific markers, nephrin and podocin, were assessed by immunofluorescence staining and confocal microscopy to detect the podocyte density in glomeruli. The fluorescence intensities of nephrin and podocin were both reduced significantly in glomeruli receiving ADR injection for 12 weeks compared with control glomeruli. Furthermore, the fluorescence intensities of both markers in Angptl3+/+ mice were weaker than those in Angptl3−/− mice (Fig. 5a).
Urine podocyte mRNAs mark the progression of glomerular disease [27]. To examine the hypothesis that podocytes are lost from glomeruli to the urine after ADR injection, positive cells in the urine were analyzed for evidence of podocyte loss using real-time PCR. Primers for nephrin and podocin were used. Nephrin and podocin mRNAs were not detected in mice without ADR injection. Additionally, nephrin and podocin mRNA levels in Angptl3+/+ and Angptl3−/− mice injected with ADR for 12 weeks were also too low to be detected (might be due to detached podocytes undergoing apoptosis). Hence, we detected these mRNAs earlier, 1 week after ADR injection, and positive signals for nephrin and podocin were detected in both Angptl3+/+ and Angptl3−/−mice. Urine podocin mRNA remained elevated throughout the time course before 8 weeks, and returned to normal after 8 weeks of ADR nephropathy progression. In contrast, the nephrin mRNA levels were not correspondingly elevated, as they peaked in week 1 and then decreased, and no further signal was detected in week 12. Both the urine nephrin and podocin mRNA levels in the Angptl3−/− mice were markedly higher than those in the Angptl3+/+ mice in week 1 (P < 0.05). The podocin mRNA level in the Angptl3−/− mice was still higher than that in the Angptl3+/+ mice in week 2 (P < 0.05), but there was no significant difference in the nephrin mRNA level between the Angptl3+/+ mice and the Angptl3−/− mice in week 2 (P > 0.05). After week 2, the nephrin and podocin mRNA levels were comparable between the two groups over the rest of the observed time course (Fig. 5b and c).
Angptl3 knockout attenuates ADR-induced primary podocyte loss, including detachment and apoptosis, in vitro
To further explore the mechanism of podocyte loss, primary podocytes from Angptl3+/+ and Angptl3−/− mice were cultured, and ADR was used to induce podocyte loss, including detachment and apoptosis. The proportions of attached podocytes in the Angptl3+/+ and Angptl3−/− groups without ADR treatment were similar. However, after ADR treatment, a significant difference was noted in the proportion of attached podocytes between the Angptl3+/+ and Angptl3−/− groups; the proportion of attached podocytes in the former group was markedly lower than that in the latter group (Fig. 6a).
Podocyte apoptosis was detected by flow cytometry analysis and TUNEL labeling under the same experimental conditions as those used for the detachment assay. Apoptosis was rarely observed in either the Angptl3+/+ or Angptl3−/− group without ADR treatment. After ADR treatment, the rate of apoptosis detected by flow cytometry in the Angptl3+/+ and Angptl3−/− groups were higher than those in the control groups, and the apoptosis rate in the Angptl3−/− group was significantly lower than that in the Angptl3+/+ group (P < 0.01) (Fig. 6b). A similar and more sensitive finding was obtained using the TUNEL assay (P < 0.001) (Fig. 6c).
Our previous study verified that integrin α3β1, integrin-linked kinase (ILK), and p53 are necessary for Angptl3 to affect PAN-induced podocyte loss [18]. In the current study, the levels of integrin α3β1and phosphorylation of integrin β1, ILK and p53 were detected to re-elucidate the molecular mechanism underlying the amelioration of podocyte loss and glomerulosclerosis in the context of Angptl3 knockout.
The expression of integrinα3 in Angptl3−/− podocytes treated with ADR was weaker than that in control podocytes but stronger than that in Angptl3+/+ podocytes treated with ADR. In comparison to that in the control groups, the expression of integrinβ1, phospho-integrin β1 and ILK in the Angptl3−/− podocyte group treated with ADR were not altered. However, in the Angptl3+/+ podocytes treated with ADR, the expression of integrinβ1 decreased, and phospho-integrin β1 and ILK expression increased (Additional file 2: Figure S2a). No p53 protein was observed in the untreated Angptl3+/+ or Angptl3−/− podocytes, but after ADR treatment, p53 protein expression was observed in the two sets of podocytes, and the level in the Angptl3−/− group was lower than that in the Angptl3+/+ group (Additional file 2: Figure S2b). Taken together, the protein analyses confirmed that following ADR treatment, lower expression of integrinα3 β1 and p53 and higher expression of phospho-integrin β1 and ILK were observed in the Angptl3+/+ podocytes than that in the Angptl3−/− podocytes.