Studies on postnatal development were carried out on the same 7- to >60-day-old male mice or >60 days adult of BALB/cJ background that we used and described earlier (Pelletier et al., 2015; Pelletier et al., 2018; Pelletier et al., 2020a; Pelletier et al., 2020b). Three animals were used per age group. Mice were housed at room temperature (RT) with food and water ad libitum and exposed to a 12 h: 12 h light dark cycle.

[Tg (PCSK9)] mice Ten-weeks old Pcsk9 ^−/− male mice that did not or did receive a transgene expressing PCSK9 and wild type (WT) of the same strain as control were used. All procedures were approved by the Clinical Research Institute of Montreal animal care committee. Pcsk9 ^−/− mice lacking the pcsk9 proximal promoter and exon 1 have been described as well as Tg (Pcsk9) mice over expressing Pcsk9 in hepatocytes under the ApoE promotor (Zaid et al., 2008).

To identify the consequences of diabetes and obesity on PCSK9 protein expression we used the same spontaneously diabetic and obese ob/ob and db/db as the ones we used and described in our earlier studies (Pelletier et al., 2018; Pelletier et al., 2020a; Pelletier et al., 2020b). Ten male mice aged of 10 weeks with the leptin receptor (B6.BKS(D)-Leprdb/J homozygotes (db/db) Stock Number (00697) mutation, ten male mice aged of 10 weeks with the leptin (B6.Cg-Lepob/J homozygotes (ob/ob) Stock Number (00632) mutation both experimental group on the C57BL/6J genetic background and three WT mice were used. Mice were purchased from Jackson Lab (Bar Harbor, ME, United States). All procedures were approved by the Animal Care Committee of the Université de Montréal.

The total number of animals per experimental condition was five-to-nine. Western blots were run using three different animals per experimental condition. Different western blots of the same protein, but obtained with different animals, showed similar results. However due to the variability of the intensity of the immunoreactive bands on the films, the results from different blots were neither mixed nor normalized. Therefore, the results showed in the figures are the ones derived from a western blot run with samples from three animals per experimental condition even though the total number of animals per experimental condition is higher than three.

The interstitium and the seminiferous tubules of the testis have different topography and functions. Therefore, all our assays were performed on interstitium- and seminiferous tubule-enriched fractions. In addition, to maximize detection of phosphorylated protein forms within the samples under study, tissue fraction were not pre-exposed to enzymes and processed as described earlier (Akpovi and Pelletier, 2009; Pelletier et al., 2015). Briefly, seminiferous tubules were mechanically teased apart from the interstitium from freshly decapsulated testes in cold phosphate buffered saline (PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4) containing 2 mM PMSF, 1 mM EGTA, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 4 mM Na₃VO₄, 80 mM NaF and 20 mM Na₄P₂O₇ with 10 μM bpV (phen). The resulting seminiferous tubule-interstitium suspension was centrifuged 15 min at 400 rpm (GS-6R Beckman Centrifuge, JH-3.8 Rotor) at 4°C after having been allowed to decant. The interstitial tissue- (ITf) (supernatant) and seminiferous tubule-enriched (STf) (pellet) fractions were centrifuged 10 min at 1,000 rpm (GS-6R Beckman Centrifuge, JH-3.8 Rotor) at 4°C. Each fraction was characterized under the light microscope as described earlier (Pelletier et al., 2018; Akpovi and Pelletier, 2009).

The epididymal spermatozoa were obtained as described earlier (Akpovi et al., 2006). Briefly, epididymides were diced in cold PBS with proteases and phosphatase inhibitors, filtered through a 74 mm mesh, and centrifuged at 2,000 rpm for 15 min in a GS-6R Beckman centrifuge (JH-3.8 Rotor) at 4°C. Then, spermatozoa were recovered and resuspended 5 min in 10 mM Tris-HCl, pH 8, containing 1 mM EDTA to lyse epithelial and blood cells (Herrada and Wolgemuth, 1997), washed twice, and diluted 1:1 in cold PBS with proteases and phosphatase inhibitors. Cells were sonicated in a Fisher Sonic Dismembrator (model 300; Fisher, Farmington, NY) during three 30 s intervals.

After decapitation, the anterior lobe of the mouse pituitary gland was dissected free from the intermediate and posterior lobes. Anterior pituitaries were placed in PBS-protease and phosphatase inhibitors, sonicated and stored at −80°C until use as previously described (Vitale et al., 2017).

Proteins in samples were assayed using materials from BioRad (BioRad, Mississauga, ON, Canada).

Thirty µg total homogenate of sample were loaded on polyacrylamide gels, separated by 10% or 12% SDS-PAGE, transferred onto nitrocellulose membranes and subjected to western blotting as previously described (Vitale et al., 2009). In all western blot experiments, the membranes were first stained with Ponceau red to ensure equal loading. Briefly, membranes were blocked 1 h at 37°C with 5% skimmed milk in TRIS-buffered saline (TBS: 137 mM NaCl, 27 mM KCl, 25 mM Tris-HCl pH 7.4) then, incubated with the different antibodies. The antibody dilutions were prepared in 5% skimmed milk-TBS as follows: 1:500 for goat polyclonal anti-PCSK9 antibody, 1:500 for anti-ACAT-1, 1:250 for anti-ACAT-2, 1:500 for anti-HSL, 1:2,000 for anti-SR-BI, 1:1,000 for anti-SR-BII, 1:500 for anti-ABCA1, 1:250 for anti-IR-α, 1:800 for anti-IR-β, 1:2000 for IL-17, 1: 100 for anti-IL-17RA and 1:1500 for anti-LDL-R. Next, membranes were washed in TBS containing 0.05% Tween 20 and incubated 1 h with the corresponding secondary antibody conjugated to HRP at room temperature. The antigen-antibody complexes were detected by chemiluminescence. The intensity of the immunoreactive bands was quantified by laser scanning with the public Scion Image Software (Scioncorp, MD, United States).

Free cholesterol (FC) and total cholesterol (TC) in interstitial tissue and seminiferous tubule-fractions (volume equivalent to 1 mg of total protein) and in serum (80–100 µl) were measured using the method we described in detail earlier (Akpovi et al., 2006). Briefly, tissue fraction homogenates and serum were extracted with hexane-isopropanol (3:2) and evaporated at 37°C with N-EVAP nitrogen evaporators (Organomation Associates, Inc. Berlin, MA, United States). An enzymatic kit (Wako Chemical USA, Richmond, VA, United States) was used to measure FC and TC. Esterified cholesterol (EC) was determined by subtracting FC from TC. FC and EC contents are expressed in mg of cholesterol/dl of serum or in mg of cholesterol/mg of total protein of enriched tissue fraction.

Serum testosterone concentrations were measured by EIA with a Cayman Chemical (Cayman Chemical Company, Ann Arbor, MI, United States) enzyme immunoassay kit according to the manufacturer’s instructions. The method sensitivity was 6 pg/ml. All samples were assessed in duplicate in a single assay with coefficient of variation ranging from 0.85 to 7.27% between duplicates.

Serum glucose concentrations were measured within 5 h following an overnight (18 h) fasting in mice fed a standard chow. Serum, ITf and STf glucose concentrations were measured with a colorimetric enzymatic (glucose-oxidize) assay (Mutarotase-GOD) (Autokit Glucose Wako, Wako, TX, United States) according to the manufacturer’s instructions. Tissue factions were prepared as described (Moraes et al., 2004) with modifications. Briefly, STf and ITf were sonicated in 6N perchloric acid while in an ice bath. The acid homogenates were centrifuged at 14,000 g and the supernatant used for glucose determination. 10 μl of serum or ITf or STf homogenates were mixed with 1.5 ml color reagent and incubated for 10 min. The absorbance of samples and standards was measured at 505 nm against the blank.

Serum and tissue fraction insulin concentrations were measured with commercially available ELISAs as we described earlier (Pelletier et al., 2020a). Briefly, STf and ITf were homogenized with a tissue grinder in an acid ethanol solution (180 mM HCl in 70% ethanol; 0.01 ml/mg tissue) on ice (Vijay et al., 2008). Tissue lysates were sonicated (Fisher Sonic Dismembrator) 3 × 15 s and centrifuged 5 min at 10,000 g at 4°C. The supernatant was recovered for insulin determination. We measured total insulin (insulin T) which is insulin 1+ insulin2 levels using an ALPCO Diagnostics (Salem, NH, United States) ELISA kit. In addition, we used the Aviva Systems Biology Ins2 ELISA kit (San Diego, CA, United States) which measures only insulin2.

Testes were perfusion-fixed with Bouin’s fixative (Kabbaj et al., 2001; Kabbaj et al., 2003). For morphological studies tissue sections were stained with PAS. Immunolabeling Endogenous peroxidase activity was inhibited with 0.6% hydrogen peroxide (H₂O₂) in 70% ethanol (Pelletier et al., 1999). Unspecific binding was blocked with 0.5% skim milk in TBS containing 0.1% Tween-20 (TBST) for 1 h at 37°C. Next, tissue sections were incubated overnight at room temperature with either (1:50) rabbit polyclonal anti-PCSK9 or (1:1,000) the rabbit polyclonal anti LDL-R, then, 1 h with biotinylated anti-rabbit IgG (1:2,000 in TBST) next, for 1 h with HRP-conjugated streptavidin 1:200 as we described earlier (Pelletier et al., 1997; Pelletier et al., 2011) Tissue sections were washed in TBST after each incubation and exposed to TBS containing 0.01% H₂O₂, 0.05% diaminobenzidine tetrachloride (DAB) (pH 7.7) for 10 min at room temperature (Kabbaj et al., 2001). Controls included the use of the primary antibody alone and, c) the use of the second antibody alone. The sections were mounted in Permount and viewed with a Carl Zeiss Axiophot 2 microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada) and image capture was carried out with Northern Eclipse software (Empix Imaging Inc. Mississauga, ON, Canada).

Analyses were done with Stata software (Stata Corporation, College Station, TX, United States). Data were evaluated with the Student’s t-test or the one-way ANOVA followed by the Tukey honestly significant difference test (THSDT).

The PCSK9 antibody recognized a 75 kDa band corresponding to the uncleaved precursor pro-PCSK9, a 62 kDa band corresponding to the cleaved mature catalytic PCSK9 fragment and a weak 55 kDa immunoreactive band in the wild type (WT) mouse liver used as a positive control (Figure 1A). Neither proPCSK9 nor PCSK9 were detected in the Pcsk9 ^−/− mouse tubule- (Figure 1A) and interstitial tissue-fractions and spermatozoa (not shown). The 75- and 62 kDa PCSK9 were detected in WT mouse seminiferous tubule-fractions where the 62 kDa PCSK9 was the intense band (Figure 1A STf). Unlike tubule-fractions, the 75 kDa PCSK9 band was more intense than 62 kDa band in WT mouse spermatozoa (Figure 1A SPZ). The intensity of the 62 kDa band was increased and the PCSK9-55 kDa form was apparent in the Tg (PCSK9) tubule-fractions (Figure 1A).

21-day-old mice contained traces of 75 kDa pro-PCSK9 and 62 kDa PCSK9, the levels of both significantly increased in the 28–42- and >60-day-old mice (Figure 1B). The pro-PCSK9 and PCSK9 level profiles were similar though 75 kDa pro-PCSK9 levels were ∼50% higher than 62 kDa PCSK9 levels from 28 to >60 days (Figure 1B).

Pro-PCSK9 and PCSK9 profiles were similar from 21 to 42 days; the level of both significantly increased from 28 days. Thereafter, pro-PCSK9 significantly increased and the PCSK9 levels became threefold higher compared to pro-PCSK9 in >60-day-old-mice (Figure 1B).

Free cholesterol (FC) and esterified cholesterol (EC) levels dropped ∼50% in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 3A). FC and EC levels doubled in Tg (PCSK9) mice compared to the WT mice (Figure 3A) suggesting over expressing the gene had an opposite effect compared to its deletion.

Unlike serum cholesterol, FC and EC concentrations significantly escalated in the interstitial tissue- and tubule-fractions (Figure 3A) in Pcsk9 ^−/− compared to WT mice. FC and EC levels were comparable in the Tg (PCSK9) and WT mice (Figure 3A).

HMGCoA-reductase levels significantly augmented in Pcsk9 ^−/− mice not in Tg (PCSK9) mice compared to the WT counterparts (Figure 3C, ITf). ACAT2 protein levels significantly increased in Pcsk9 ^−/− mice compared to WT, whereas Tg (PCSK9) and WT mice had comparable ACAT2 levels (Figure 3C, ITf). In contrast, ACAT1 and HSL levels changed little in Pcsk9 ^−/− and Tg (PCSK9) compared to WT mice (Figure 3C, ITf).

HMGCoA-reductase and HSL protein contents were not significantly different in Tg (PCSK9), Pcsk9 ^−/− and WT mice (Figure 3C, STf). However, ACAT1 and ACAT2 protein contents significantly increased in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 3C, STf). Tg (PCSK9) and WT mice had comparable ACAT1 and ACAT2 levels (Figure 3C, STf).

SR-BI levels were halved in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 3D, ITf). SR-BI levels were not significantly different in Tg (PCSK9) and WT mice (Figure 3D, ITf). SR-BII and ABCA1 levels in Pcsk9 ^−/− mice were not significantly different in Tg (PCSK9), Pcsk9 ^−/− and WT mice (Figure 3D, ITf).

SR-BI and SR-BII levels changed little in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 3D, STf). SR-BI and SR-BII levels were not different in Tg (PCSK9) and WT mice (Figure 3D, STf). By contrast, ABCA1 levels dropped 50% in Pcsk9 ^−/− compared to the WT whereas ABCA1 levels were similar in Tg (PCSK9) and WT mice (Figure 3D, STf).

Neither IL-17A levels nor IL-17A receptor (RA) levels changed significantly in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 3E, ITf). But in Tg (PCSK9) mice, IL-17RA levels significantly upsurged compared to WT mice despite no changes in IL-17A levels (Figure 3E, ITf).

Unlike the interstitial tissue-fractions, the 33- and 18–15 IL-17A and IL-17RA levels significantly escalated in Pcsk9 ^−/− compared to the WT mice (Figure 3E, STf). In Tg (PCSK9) mice, 18–15 IL-17A significantly fell whereas IL-17RA levels increased six fold compared to WT mice (Figure 3E, STf).

The LDL-R antibody detected ∼160-, 95-, 75- and 65 kDa immunoreactive bands in the WT mouse liver used as a positive control (Figure 4A, Liver). The 160- and 95 kDa LDL-R were detected in WT mouse interstitial tissue- and tubule-fractions (Figure 4A, ITf and STf).

The 160- and 95 kDa LDL-R levels significantly increased in Pcsk9 ^−/− and Tg (PCSK9) mice compared to WT mice (Figure 4A, ITf).

Only the 160 kDa LDL-R levels significantly increased in Pcsk9 ^−/− mice compared to the WT mice (Figure 4A, STf). Instead in Tg (PCSK9) mice, 160 kDa levels significantly dropped whereas 95 kDa increased compared to the WT mice (Figure 4A, STf).

120 kDa LDL-R levels significantly increased whereas the level of lower molecular weight bands decreased in Pcsk9 ^−/− mice compared to the WT counterparts. In Tg (PCSK9) mice, 120 kDa significantly dropped compared to the WT mice but the levels of the lower molecular weight bands virtually recovered WT values (Figure 4A, SPZ).

Blood glucose concentrations in Pcsk9 ^−/−, Tg (PCSK9) and WT counterparts mice did not significantly differ (Figure 5A, Serum).

Glucose concentrations were not significantly different in the Pcsk9 ^−/−, Tg (PCSK9), and WT counterparts mice (Figure 5A, ITf).

By contrast, glucose concentrations significantly fell in the Pcsk9 ^−/− mice compared to the WT counterparts (Figure 5A). Tg (PCSK9) and WT mice had similar glucose concentrations (Figure 5A, STf).

Glucose concentrations were significantly lowered in Pcsk9 ^−/− and Tg (PCSK9) compared to the WT mice (Figure 5A, SPZ). Although still lower than WT, glucose levels in Tg (PCSK9) were higher than in Pcsk9 ^−/− (Figure 5A, SPZ).

Total insulin (insulinT) and insulin2 levels were not significantly different in Pcsk9 ^−/− mice and WT counterparts (Figure 5B Serum). Tg (PCSK9) mice had significantly elevated insulinT and insulin2 concentrations compared to the WT mice (Figure 5B, Serum).

InsulinT concentrations showed a tendency to increase though differences were not significant whereas insulin2 significantly augmented in Pcsk9 ^−/− mice in comparison to the WT (Figure 5B, ITf). In Tg (PCSK9) mice, insulinT and insulin2 significantly increased compared to the WT mice (Figure 5B, ITf).

IinsulinT concentrations were significantly higher in Pcsk9 ^−/− than the WT counterparts (Figure 5B, STf). WT insulinT and insulin2 concentrations were recovered in Tg (PCSK9) mice (Figure 5B, STf).

The 75 kDa pro-PCSK9 levels dropped six fold whereas the 62 kDa PCSK9 concentration changed little in db/db compared to the WT counterparts (Figure 5C, ITf). As well, in ob/ob mice, 75 kDa pro-PCSK9 significantly dropped but 62 kDa PCSK9 upsurged eightfold in comparison to the WT (Figure 5C, ITf).

Neither 75 kDa pro-PCSK9 nor 62 kDa PCSK9 levels in db/db mice significantly differ from the WT counterparts (Figure 5C, STf). Conversely, in ob/ob mice, pro-PCSK9 levels significantly increased but PCSK9 significantly dropped compared to the WT (Figure 5C, STf).

Only the 95 kDa LDL-R levels significantly dropped in db/db mice compared to the WT counterparts. In ob/ob mice, 160 kDa LDL-R levels decreased while the levels of the remaining LDL-R bands did not change (Figure 5D, ITf).

The 160 kDa LDL-R levels and the levels of other breakdown bands significantly decreased in db/db and ob/ob mice compared to the WT counterparts (Figure 5D, STf).

120- and 65 Da LDL-R levels significantly increased whereas 95 kDa levels significantly fell in db/db mice compared to the WT counterparts (Figure 5D). As well, 120 kDa significantly augmented and 95 kDa levels significantly decreased in ob/ob mice compared to the WT but the level of the 75- and 65 kDa remained unchanged (Figure 5D, STF).

The antibody detected a ∼135 kDa full length IR-α band and several degradation products of 90-, 75- and 50 kDa (Figure 6A, ITf). The level of 135 kDa and of some breakdown products significantly increased in Pcsk9 ^−/− compared to the WT counterparts (Figure 6A, ITf). In Tg (PCSK9) mice, 135 kDa and most breakdown products significantly augmented compared to the WTs (Figure 6A, ITf).

The 135 kDa IR-α and most breakdown bands significantly increased in Pcsk9 ^−/− and Tg (PCSK9) mice compared to the WT counterparts (Figure 6A, STf).

The 135 kDa IR-α was not detected in spermatozoa. (Figure 6A, SPZ). The other bands were not significantly different in Pcsk9 ^−/−, Tg (PCSK9) and WT.

The antibody detected the 110-98 kDa full length IR-β and 75-, 66- and 50 kDa IR-β degradation products (Figure 6B, ITf). The 110-98 kDa band levels significantly decreased in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 6B, ITf). Full length IR-β levels did not recovered in Tg (PCSK9) mice (Figure 6B, ITf).

Both, 110–98- and 75 kDa IR-β levels significantly upsurged in Pcsk9 ^−/− and Tg (PCSK9) mice compared to the WT counterparts (Figure 6B, STf).

Mouse epididymal spermatozoa exhibited 50 kDa IR-β and traces of 66- and 75 kDa (Figure 6B, SPZ). The 66- and 50 kDa levels significantly fell in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 6B, SPZ). Conversely, all IR-β band levels significantly upsurged in Tg (PCSK9) mice compared to Pcsk9 ^−/− and WT mice (Figure 6B, SPZ).

The 135 kDa full-length IR-α levels were not affected but other low molecular weight band levels significantly decreased in the Pcsk9 ^−/− mice compared to the WT counterparts (Figure 6C, AP). Conversely, in Tg (PCSK9) mice, 135 kDa levels significantly increased in comparison to the WT counterparts (Figure 6C, AP).

The 110–98- and 50 kDa IR-β levels were halved in Pcsk9 ^−/− mice compared to the WT counterparts (Figure 6D, AP). By contrast, in Tg (PCSK9) mice, these levels significantly increased compared to Pcsk9 ^−/− and WT mice (Figure 6D, AP).

Table 1 summarizes the changes in cholesterol, enzymes, transporters, IL-17A, IL-17RA, LDL-R, glucose, Insulin Total, Insulin 2, IR-α, IR-β, testosterone in serum, interstitial tissue- and seminiferous tubule-fractions, epididymal spermatozoa and anterior pituitary in Pcsk9 -/-, db/db, and ob/ob mice.

The accumulation of cholesterol concurred with an increase in HMGCoA-red and LDL-R levels and a drop in SR-BI in Pcsk9 ^−/− mice indicating imbalances in cholesterol synthesis and transport. Our immunohistochemical studies localised LDL-R and PCSK9 to the wall of blood vessels. Adding LDL-R molecules at the cell membrane could enhance endocytosis of LDL-cholesterol and increase intracellular cholesterol. Normal cholesterol levels recovered in the interstitium in Tg (PCSK9) though the 160 kDa LDL-R levels remained elevated suggesting that local factors other than PCSK9 are involved in the expression of LDL-R in testicular vessels. Like LDL-R and PCSK9, HSL, ACAT1, and SR-BII also localise to wall of the vessels (Kabbaj et al., 2001; Akpovi et al., 2006, and unpublished results). However, the HSL, ACAT1, and SR-BII protein expression was not affected by the lack of Pcsk9.

Our finding that Leydig cells were neither PCSK9- nor LDL-R-immunoreactive agrees with the report of low levels of LDL-R expression in rat Leydig cells (Reaven et al., 2000). Cellular cholesterol is synthesized from acetyl-CoA or up taken from the surroundings. The endogenous cholesterol is used for testosterone synthesis whereas HDL and/or LDL are not the source of cholesterol in Leydig cells (Reaven et al., 2000). In addition, 25-OH-cholesterol contributed by macrophages serves as a substrate for testosterone production in Leydig cells (Nes et al., 2001). The unchanged testosterone levels suggest that the lowering of cholesterol in serum activates several mechanisms for the mobilisation of the cholesterol used in testosterone synthesis in Pcsk9 ^−/− mice. Our finding of increased HMGCoA-red levels in Pcsk9 ^−/− mouse interstitium agrees with the report that endogenous cholesterol is the source of cholesterol for testosterone production (Reaven et al., 2000). The levels of ABCA1, the transporter for the active free cholesterol efflux in Leydig cells, were not affected. Our results indicate a coordination of the synthesis, the uptake and the esterification to regulate cholesterol levels in Leydig cells.

A part of cholesterol is imported from blood through HDL (Fofana et al., 2000) by multiligand transporters (Akpovi et al., 2006; Pelletier et al., 2009), another part arises from by-products resulting from the phagocytosis of lipid-rich apoptotic germ cells’ membranes remnants and lipid-containing residual bodies (Pelletier and Friend, 1983; Pelletier and Vitale, 1994; Pelletier, 2011). The overload of apoptotic figures in these mice could contribute to the accumulation of cholesterol in tubules in Pcsk9 ^−/− mice. In addition, the higher LDL-R levels in the wall of the vessels could add more cholesterol by its transfer from vessels to cells in tubules. The innumerable vacuoles in Sertoli cells reflect the increased cholesterol levels and lipid droplets dissolved during the dehydration process in alcohols and xylene series.

Sertoli cells and germ cells express SR-BI and SR-BII (Akpovi et al., 2006). The unchanged SR-BI and SR-BII levels suggest that the cholesterol accumulation was not due to passive cholesterol influx in the Pcsk9 ^−/− mouse tubules. Sertoli cells participate in the efflux of cholesterol. Sertoli cells express ABCA1 and Abca1-deficient mice exhibit lipid accumulation in Sertoli cells (Selva et al., 2004). The 50% reduction in ABCA-1 in the Pcsk9 ^−/− mouse tubules may contribute to the cholesterol accumulation and suggests that the efflux and reverse cholesterol transport are mediated by ABCA1 in tubules. By contrast, SR-BI is protagonist in the interstitium (Akpovi et al., 2006; Pelletier et al., 2009).