Background
Chronic kidney disease (CKD) is a substantial worldwide burden on patients and society. Pathologically, glomerulosclerosis accounts for the vast majority of CKD cases leading to end-stage renal disease (ESRD), and podocyte loss is closely related to the occurrence and progression of glomerulosclerosis [
1‐
3]. The mechanism of glomerulosclerosis and therapeutic interventions aimed at the prevention or reversion of glomerulosclerosis have been intensively investigated. Despite decades of extensive research, no specific treatments are available to prevent or reverse glomerulosclerosis.
Angiopoietin-like protein 3 (Angptl3) is a secreted protein that is mainly produced by the liver and minimally expressed in the normal kidneys [
4]. Angptl3 plays important roles in the regulation of lipid metabolism [
4], angiogenesis [
5], the stem cell proliferation process [
6], insulin resistance [
7], hepatocellular carcinoma [
8] and some other biological functions [
9‐
11]. Our previous work revealed increased Angptl3 expression in the glomeruli of children with nephrotic syndrome (including minimal change disease and glomerulosclerosis) and animal models of Adriamycin (ADR) nephropathy, and in ADR- or puromycin aminonucleoside (PAN)- treated cultured podocytes [
12‐
16]. Moreover, we found that Angptl3 overexpression stimulates podocyte F-actin rearrangement in vitro [
17], increases podocyte motility [
16] and accelerates podocyte loss (including podocyte detachment and apoptosis) [
18], which may be related to promoting proteinuria. To further clarify the role of altered Angptl3 expression as a regulatory or modulatory factor in renal proteinuria, we used Angptl3 gene knockout mice. Our previous results showed that Angptl3 knockout was associated with renoprotection in the early stage of ADR nephropathy [
19]. However, ADR nephropathy usually progresses to end stage kidney disease, which is phenotypically characterized by glomerulosclerosis [
20]. Here, we found that Angptl3 knockout not only ameliorates ADR nephropathy in the early stage but also protects against its progression. Angptl3 knockout ameliorated glomerulosclerosis by attenuating podocyte loss via rescuing podocytes detachment and apoptosis. The current study proposes that Angptl3 antagonists or inhibitors are potential and attractive therapeutic candidates for podocyte injury and proteinuria in the occurrence and progression of nephropathy, which has not been proposed in other studies.
Methods
Mouse models
All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of the Institute of Developmental Biology and Molecular Medicine (IDMIACUC) at Fudan University. As our previous protocol described [
19], Angptl3−/− mice were bred with two Angptl3+/− mice (B6; 129S5-Angptl3tm1Lex, No. 032146-UCD, Mutant Mouse Regional Resource Centers, Davis, CA), the mouse genotypes were identified by polymerase chain reaction (PCR), and female 8-week-old Angptl3+/+ and Angptl3−/− mice received an intravenous ADR (Sigma-Aldrich, St. Louis, USA; 25 mg/kg dissolved in isotonic saline to a final concentration of 5 μg/μL) on day 0. The control mice received an identical volume of saline intravenously. The ADR-injected mice were given two daily intraperitoneal injections of 2 ml of glucose-electrolyte solution at 36 h after the ADR injections from day 2 to day 6, to prevent weight loss due to low appetite [
19]. At weeks 1, 2, 4, 6, 8 and 12, spot urine and blood samples were collected for biochemical studies. The mice were sacrificed at the indicated time points, and the kidneys were removed after saline perfusion for histological studies. There were 6 mice in each group at each time point (Additional file
2: Figure S1a). All mice were euthanized via cervical dislocation following the IDMIACUC Animal Protocol.
Morphometric analysis
Kidneys were re-moved from the euthanized mice. Tissue samples (0.4 cm × 0.3 cm × 0.3 cm) were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 μm, stained with periodic acid-schiff (PAS), and examined under a light microscope. For the transmission electron microscope (TEM), the renal cortices were cut (1 mm3) and fixed in 2.5% glutaraldehyde overnight, post fixed in 1% osmium tetroxide, dehydrated and embedded according to routine procedures.
Images from at least 10 (until glomerulosclerosis was observed, otherwise the whole kidney was sectioned) sequential glomerular cross-sections (4 μm thickness) approximately at the glomerular equator (0.4 cm × 0.3 cm) were collected for each histological section and assessed by a blinded observer. The number of cells in each glomerular Bowman’s capsule cross section was counted as described by Kim YH et al. [
21], with a minor modification. Thirty glomeruli from each mouse of the 6 mice per group were assessed via light microscopy, and 1 glomerulus from each mouse of the 6 mice per group was evaluated under a TEM.
Urine ribonucleic acid preparation and quantitation
Urine was collected overnight (average 12 h), and the total urine pellet ribonucleic acids (RNA) was isolated using the protocol of the RNeasy Mini Kit (Qiagen, Hilden, Germany) [
22]. The steady-state amount of nephrin, and podocin messenger ribonucleic acids (mRNAs) was analyzed by real-time PCR using the Stratagene Mx3000p real-Time PCR System (Santa Clara, CA, United States).The analysis was conducted as described in the instructions of the SYBR FAST qPCR Kit (KAPA). RT-qPCR was conducted 3 times in duplicate using each of the cDNA samples. The amplified transcripts were quantitated with the comparative method using glyceraldehyde-phosphate dehydrogenase (GAPDH) as an internal control. The primers were designed using Primer express software (Primer premier 5.0) based on GenBank accession numbers. The sequences used were: Nephrin, 5′- CCC AGG TAC ACA GAG CAC AA-3′ and 5′- CTC ACG CTC ACA ACC TTC AG-3′; Podocin, 5′- TCT CCT GGA AAG GAA GAG CA-3′ and 5′- GTC TTT GTG CCT CAG CTT CC-3′; GAPDH, 5′- TGC GAC TTC AAC AGC AA CTC-3′ and 5′-ATG TAG GCC ATG AGG TCC AC-3′.
Podocyte culture and treatment
The kidney cortex of mice was removed, minced and subjected to standard differential three-step sieving under sterile conditions to isolate the glomeruli as previously reported [
23]. The isolated glomeruli were plated in 10 cm plates coated with type I collagen. The culture medium consisted of K1 medium and NIH 3 T3 medium mixed in a 1:1 ratio as described [
24]. The outgrowth of podocytes started between days 3 and 4. After 6 days of primary culture, the glomeruli were removed and the podocytes were trypsinized and replated onto plates coated with type I collagen for further growth, expansion and analysis (Additional file
2: Figure S1b). We used immunoflurescence staining and flow cytometry analysis to identify the purity of the primary podocytes. We confirmed that most (more than 90%) of the cells were podocytes (Additional file
2: Figure S1d and e). We performed a 3-[
4,
5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay to quantify cell viability, and the assay demonstrated that the podocyte cellular activity of passage 1 day (P1) podocytes increased significantly on day 3, peaked on day 7, and then slowly decreased (Additional file
2: Figure S1c). Therefore, we used P1 podocytes at days 3 to 9 in our in vitro study. Podocytes were treated with ADR (1 μmol/ml, 48 h) to induce podocyte loss, and control podocytes were treated with phosphate buffer solution.
The urine protein excretion assay, serum biochemistry analysis, PAS histostain, transmission electron microscopy, immunofluorescence labeling, MTT assay, detachment assay, apoptosis detection assay and Western blot analysis were described in Additional file
1: Supplementary Methods.
Statistical analysis
Most analyses and calculations were performed using Prism® version 5 (GraphPad Software, La Jolla, CA, USA) and IBM SPSS Statistics version 19.0 (IBM Corporation, Armonk, NY, USA). Values, which are expressed as the mean ± SEM, were compared by Student’s t-test, one-way analysis of variance or the nonparametric Kruskal-Wallis test, followed by the Student Newman-Keuls posthoc test. Statistical significance was set at P < 0.05.
Discussion
The ADR-induced nephropathy mouse model is ideal for clarifying the underlying mechanisms of the response of podocytes to injury in kidney disease [
20]. The successful establishment of the ADR nephropathy mouse model is closely related to many factors, such as the mouse susceptibility to ADR, the dosing of ADR, the method of administration, and the frequency and interval time of the method. In the current study, we successfully established an ADR nephropathy mice model on a B6; 129S5 genetic background with ADR (25 mg/kg) administered intravenously once via tail vein injection and monitored for long-term follow-up. Our ADR nephropathy mice model on a B6; 129S5 genetic background was manifested with minimal change disease in the early stage (1 week after ADR injection) and glomerulosclerosis formation in the end stage (12 week post ADR injection), which further facilitated the in vivo study of the pathophysiology and treatment of chronic proteinuric renal disease.
Angptl3 knockout was observed to play a crucial protective role (attenuated proteinuria and hypoproteinemia, and improved renal structure and function as well as the general condition and survival of mice) in the whole process of ADR nephropathy. In the early stage, the Angptl3 knockout greatly attenuated proteinuria and maintained the integrity of the podocyte foot process, which was consistent with the observations of our previous study [
19]. In the end stage, Angptl3 knockout effectively delayed glomerulosclerosis formation. The present results suggested that antagonists or inhibitors such as small molecule drugs or antibodies targeting Angptl3 might be specific and effective candidate therapeutic approaches for podocyte injury and proteinuria in nephropathy, and if those therapeutic approaches were used in treatments, continuously maintaining a low level of Angptl3 (long-term medication) would have better effectiveness.
The loss of cells in glomeruli beyond a critical level results in widespread glomerulosclerosis leading to the progressive loss of renal function culminating in end-stage of kidney disease [
1,
2]. The most important of the lost cells are podocytes, and if progressive podocyte loss is allowed to occur over time, then this loss is associated with progressive glomerulosclerosis [
2,
3]. In this partial study, we found that Angptl3 knockout reduced the number of detached cells in glomerular Bowman’s capsule and attenuated the number of apoptotic cells in the renal tissue of mice with end-stage ADR nephropathy. We demonstrated that Angptl3 knockout attenuated podocyte loss (alleviated the decrease in podocyte density in the renal tissue and reduced the urine podocyte mRNA levels) in mice with end-stage ADR nephropathy, and we suggested that most of the lost cells were podocytes. These findings revealed that Angptl3 knockout could ameliorate glomerulosclerosis formation in mice with ADR nephropathy by attenuating podocyte loss.
Podocyte detachment and apoptosis are two risk factors that cause podocyte loss [
21,
22]. Podocytes adhere to the GBM principally via integrinα3β1 [
28], and ILK plays a key role in integrinα3β1-mediated podocyte adhesion [
29]. The tumor suppressor protein p53 is involved in the crucial processes of podocyte apoptosis [
30]. Our previous study confirmed that Angptl3 is a novel factor that is involved in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro. Knockdown of Angptl3 by small interfering RNA (siRNA) markedly ameliorated podocyte loss, and the observed effects were partially correlated with the altered integrinα3β1, ILK and p53 [
18]. Culturing primary podocytes seemed to be a bridge between cell lines and in vivo growth, and produces cells with characteristics closer to the biological characteristics of podocytes in vivo [
31]. In the current study, we cultured primary podocytes from Angptl3+/+ and Angptl3−/− mice in vitro to further explore whether Angptl3 knockout ameliorates podocyte loss. We demonstrated that Angptl3 knockout effectively rescued primary podocytes from detachment and apoptosis induced by ADR, and Integrin α3β1, ILK, and p53 were altered markedly in the observed process. These results powerfully support our previous viewpoint that lowering Angptl3 expression or knockout Angptl3 may ameliorate the loss of podocytes via reductions in podocyte detachment and apoptosis in vitro, and the observed effects partially correlated with the alterations in integrinα3β1, ILK and p53. Notably, comparing with our previous study [
18], this study showed that Angptl3 knockout tended to protect podocyte loss more effectively than the Angptl3 lower by siRNA, which might invite speculation regarding a “dose-dependent effect” of Angptl3 on podocyte loss. Generally, the lowering the expression of Angptl3 resulted in a better effect on renal protection. However, the exact phenomenon and mechanism need to be further studied.
Recent years have seen dramatic advances in the understanding of the roles of Angptl3 in podocyte injury. Our findings provide more information on the identification of potential and specific therapeutics targeting Angptl3 in podocyte injury and proteinuria. However, our studies are limited. Firstly, the PAN model may also be used as a model of glomerulosclerosis or minimal change disease based on the amount of PAN injected [
32]. Our results need to be confirmed in the PAN model. Second, we previously demonstrated that Angptl3 is involved in the development of proteinuria by triggering integrin β3 and the downstream FAK/PI3K signaling pathway in podocytes. Either decreasing ANGPTL3 expression or interfering with the ANGPTL3-integrin β3 interaction might be beneficial for protecting and decreasing proteinuria [
17,
19]. However, in the current study, our finding indicated that Angptl3 knockout could attenuate proteinuria, but could not alleviate proteinuria completely. The possible cause may be that even though we inhibited or deleted Angptl3, integrin β3 might have been activated by other factors, such as urokinase receptor (uPAR), to promote the effacement of podocyte foot processes [
33] and produced proteinuria [
34]. The results suggested that candidate therapeutic approaches targeting on Angptl3 should be combined with other therapeutic options to produce potent therapeutic effects on proteinuria. Finally, ADR nephropathy is a highly reproducible model of renal injury. It is also a “robust” model in that the degree of tissue injury is severe while being associated with acceptable mortality and morbidity [
20]. In the current study, we observed that in week 1 post ADR injection, a small number of mice including both Angptl3+/+ and Angptl3−/− mice started to die, although we did not observe the heart function and pathology of these dead mice, the putative cardiotoxicity of doxorubicin might be then main causes of death [
35]. In addition, we observed the liver function and structure of weak mice (close to death) and found that their blood alanine aminotransferase (ALT) levels were abnormally high and that their liver structure showed pathologic vacuoles (data not shown but available from authors). Therefore, we speculated that the cause of death in mice receiving ADR injection was not only cardiac dysfunction, but also abnormal liver function. Previous studies suggest Angptl3 is mainly produced by the liver [
4] and associated with liver health [
36]. A higher proportion of Angptl3+/+ mice than Angptl3−/− mice died throughout our observation period, which might indicate that the Angptl3 knockout also played an important role in hepatic protection. In-depth studies regarding the hepatic protection mediated by Angptl3 knockout are also necessary.
Acknowledgments
The authors acknowledge Prof. Guo Muyi and Prof. Zhigang Zhang from Department of Pathology and Key Laboratory of Molecular Medicine, Shanghai Medical College of Fudan University for histological assistance. And thanks Dr. Martin Bitzan from McGill University Health Center for his guidance during manuscript writing.