Altered renal proximal tubular endocytosis and histology in mice lacking myosin-VI

Abstract

Myosin VI (Myo6) is an actin-based molecular motor involved in clathrin-mediated endocytosis that is highly expressed in the renal proximal tubule brush border. We investigated the renal physiological consequences of loss of Myo6 function by performing renal clearance and physiological measurements on Myo6 functional null Snell’s waltzer (sv/sv) and control heterozygous (+/sv) mice. Sv/sv mice showed reduced body weight and elevated blood pressure compared with controls; no differences were observed for glomerular flow rate, urine volume, blood acid-base parameters, and plasma concentrations and urinary excretions of Na⁺ and K⁺. To assess the integrity of endocytosis-mediated protein absorption by the kidney, urinary albumin excretion was measured, and the proximal tubular uptake of intravenously injected endocytic marker horseradish peroxidase (HRP) was examined. Albumin excretion was increased nearly 4-fold in sv/sv mice relative to controls. Conversely, HRP uptake was reduced and delayed in proximal tubule cells of the sv/sv kidney observed by electron microscopy at 5 and 30 minutes after injection. Consistent with impaired endocytosis, we also observed defects indicating alterations along the endocytic pathway in sv/sv proximal tubule cells: (1) decreased membrane association of the clathrin adaptor subunit, adaptin beta, and Disabled-2 (Dab2) after sedimentation of renal homogenates and (2) reduced apical vacuole number. In addition, proximal tubular dilation and fibrosis, likely secondary effects of the loss of Myo6, were observed in sv/sv kidneys. These results indicate that Myo6 plays a key role in endocytosis-mediated protein absorption in the mouse kidney proximal tubule.

Introduction

Myosin VI (Myo6) is a member of the myosin family of actin filament-based molecular motors that has been implicated in a wide range of functions including clathrin-mediated endocytosis, vesicle trafficking, cell polarity determination, and cell migration (Buss et al. 2004). Myo6 is unique among myosins in that it moves toward the pointed (minus) end of the actin filament, in the direction opposite to that of all other myosins characterized thus far (Wells et al. 1999). As actin filaments in cells are predominantly oriented with their pointed ends toward the cell interior, it has been suggested that Myo6 may use the force generated from its movement on actin filaments to pull or transport bound cargo such as nascent endocytic vesicles at the plasma membrane, uncoated apical endocytic vesicles, and membrane-bound receptors into the cell (Buss et al. 2004; Hasson 2003). In the kidney, Myo6 is highly expressed in the brush border (BB) of proximal tubule (PT) epithelial cells, where it is in part localized to the intermicrovillar (interMV) domain, the site of megalin-dependent, clathrin-mediated endocytosis of plasma proteins filtered through the glomerulus (Biemesderfer et al. 2002). This localization of Myo6 is established during PT differentiation when the cells become endocytosis-competent (Biemesderfer et al. 2002). Some Myo6 is also present within MV (Biemesderfer et al. 2002) and immunoelectron microscopy studies by Yang et al. indicate that as much as 50% of Myo6 is localized to MV in rat PT cells (Yang et al. 2005). Interestingly, these workers observed that there is a redistribution of MV-associated Myo6 to the interMV domain in rats exposed to acute high blood pressure. Thus differences in methods used for tissue preparation (e.g. perfusion-fixation pressure) may account for differences in the relative levels of MV versus interMV localization of Myo6 that has been reported.

The PT-specific, multi-ligand endocytic receptors megalin and its binding partner cubulin, a peripheral membrane protein, mediate the reabsorption of a large number of glomerular-filtered proteins including albumin, the most abundant protein in plasma (Birn and Christensen 2006; Christensen and Gburek 2004). Because Myo6 directly binds to the clathrin-associated adaptor proteins Dab2 and GIPC (GAIP interacting protein, C terminus)/synectin (Buss et al. 2004), which also bind megalin (Lou et al. 2002; Oleinikov et al. 2000), Myo6 may be important for protein endocytosis by PT cells. The importance of functional PT endocytosis is underscored by the correlation between the degree of proteinuria and the rate of progression of various renal diseases and in vitro findings that high protein concentrations induce the production of inflammatory and fibrogenic mediators by tubular cells (Birn and Christensen 2006). In particular, while the glomerular filtration barrier prevents the passage of most of the serum albumin into the tubular lumen, an appreciable amount of albumin is filtered by the glomerulus and subsequently reabsorbed by the PT (Birn and Christensen 2006; Gekle 2005), and the amount of albumin excreted in urine, which reflects the integrity of these two processes, is an important indicator of renal disease.

In vitro studies using dominant negative Myo6 expression in cell lines have shown that Myo6 is essential for clathrin-mediated endocytosis and trafficking of uncoated endocytic vesicles (Aschenbrenner et al. 2003; Buss et al. 2001). Moreover, in Myo6-deficient neurons cultured from the Myo6 functional null Snell’s waltzer (sv/sv) mouse, there is selective inhibition of clathrin-mediated endocytosis, as glutamate but not transferrin receptor internalization is inhibited (Osterweil et al. 2005). These mice also exhibit reduced and delayed apical endocytosis of CFTR in jejunal enterocytes (Ameen and Apodaca 2007). Sv/sv mice are deaf, and their only overt abnormal phenotypes are circling/hyperactive behavior resulting from degeneration of the inner ear neurosensory epithelium (Avraham et al. 1995; Deol and Green 1966) and smaller body size. In this study, we investigated the physiologic and histologic consequences of loss of Myo6 function in the kidney. Physiological measurements and renal clearance studies showed elevated blood pressure in sv/sv mice compared to control animals while maintaining normal glomerular filtration rate (GFR), urine volume, and urine concentrating ability. Urinary albumin levels were elevated in sv/sv mice, and in vivo uptake of HRP was impaired in sv/sv PTs, indicating a role of Myo6 in PT protein endocytosis. In addition, sv/sv kidneys showed decreased association of adaptin β and Dab2 with the BB membrane and reduced apical vacuole number in PT cells. Histologically, sv/sv kidneys exhibited PT dilation and fibrosis with signs of epithelial-mesenchymal transdifferentiation (EMT) of the tubular cells. This study shows the presence of deficits in protein reabsorption and pathology in the sv/sv kidney, with the interesting finding that overall renal function is largely maintained.

Materials and Methods

Mice

Myo6^sv/Myo6^sv (sv/sv) mice were generated by crossing +/Myo6^sv (+/sv) and sv/sv mice as previously described (Osterweil et al. 2005). Mice were age- and sex-matched within each experiment, and three to eight mice were observed per genotype, per experimental group. Mice were 15–24 weeks old for HRP uptake studies, 12–28 weeks old for 24-hour metabolic cage studies (urinary volume, osmolality, and albumin; food and water intake), 17–22 months old (Fig. 4A,B) and 5–10 months old (Fig. 4C) for Western blot assays, 17–22 months old for kidney weight measurements, 24–29 months old for retro-orbital blood analysis (Table 1), and 19–21 months old for renal clearance studies (Table 2). All protocols were approved by the Yale University Institutional Animal Care and Use Committee.

Figure 4. Protein expression levels in +/+, +/sv, and sv/sv kidneys.

(A) Myo6 expression levels in +/+ and +/sv kidneys. Western blots of Myo6 in TCA-precipitated total kidney protein of +/+ and +/sv mice. The six lanes in each genotype represent a dilution series of total protein in one kidney, with equal protein loading in each pair of corresponding lanes in the two genotypes. Lane labels indicate relative amounts of protein in the lane. Similar results were obtained from 3 separate pairs of +/+ and +/sv animals. Arrowhead, full-length Myo6. (B) Sedimentation analysis of BB cytoskeletal and endocytic compartment components. Western blots of Myo6, villin, EEA1, and Dab2 in TCA-precipitated fractions from whole kidney homogenates (H) subjected to 1000×g sedimentation (LP, low-speed pellet) and sedimentation of the resulting supernatant at 100,000 × g (UP and US, ultra-speed pellet and supernatant, respectively). Lanes are loaded stoichiometrically with respect to original homogenate volume. Arrowheads indicate the predicted molecular weight of each protein, and bands of smaller size are likely proteolytic products. Proteolytic products of Myo6, EEA1, and Dab2 were observed in the presence of a broad-spectrum cocktail of protease inhibitors during protein preparation as shown here, suggesting that these proteins may be susceptible to cleavage by non-serine or -cysteine proteases, which were not targeted by the inhibitors. Dab2 exists in two alternatively spliced forms, p96 and p67 (see text). (C) Myo6 is required for stabilization of adaptin β at the plasma membrane. Western blots of kidney homogenates from sv/sv and +/sv mice that were sedimented at 4000 × g for 10 min to pellet the BB membrane and probed for adaptin β. Pellets were resuspended in equal volumes as supernatants, and equal volumes were loaded in each lane. Panels shown are representative of three animals per genotype.

Table 1.

Blood parameters of +/+, +/sv, and sv/sv mice

Genotype	N	BW (g)	pH	HCO3− (mM)	Na+ (mM)	K+ (mM)	iCa (mM)	Glucose (mg/dL)	Hct (%)	Hb (g/dL)
+/+	4	33.7 (3.2)	7.25 (0.10)	26.3 (3.0)	148 (2)	5.4 (0.6)	1.20 (0.04)	170 (35)	35 (9)	11.9 (3.2)
+/sv	5	35.5 (3.1)	7.26 (0.06)	26.7 (1.5)	149 (3)	5.4 (0.7)	1.19 (0.05)	151 (19)	37 (2)	12.7 (0.7)
sv/sv	4	27.5 (1.3)*††	7.25 (0.03)	25.1 (1.9)	148 (2)	6.1 (0.8)	1.14 (0.07)	141 (14)	34 (5)	11.7 (1.6)

Mean values and SD (in parentheses) of blood parameters measured from retro-orbital blood samples of mice aged 24 to 29 months.

^*P<0.05 vs. +/+;

^††P<0.01 vs. +/sv.

Table II.

Body Weight, Blood Pressure, Plasma Na⁺ and K⁺, Urine Volume, GFR, and Na⁺ and K⁺ Excretion in +/sv and sv/sv Mice

	Genotype
Physiological Parameters	+/sv	sv/sv
BW (g)	34.8 (5.4)	24.3 (2.8)a
BP (mm Hg)	100.8 (7.7)	116.7 (10.5)b
PNa (mM)	153.34 (5.07)	154.25 (7.27)
PK (mM)	4.29 (0.61)	3.96 (0.40)
UV (µl/min)	0.88 (0.40)	1.19 (0.41)
GFR (ml/min/100g BW)	0.51 (0.25)	0.73 (0.25)
ENa (µEq/min/100g BW)	0.22 (0.17)	0.63 (0.49)
EK (µEq/min/100g BW)	0.46 (0.20)	0.77 (0.43)
FENa (%)	0.31 (0.21)	0.56 (0.32)
FEK (%)	24.44 (13.78)	28.37 (15.13)

Mean values and SD (in parentheses) of physiological parameters measured in renal clearance studies of mice aged 19–21 months (n = 6 per genotype).

^aP<0.01.

^bP<0.05.

Antibodies

The following rabbit polyclonal and mouse monoclonal antibodies were used for Western blotting: rabbit anti-Myo6 tail ((Hasson and Mooseker 1994); 1 µg/ml); mouse anti-adaptin β (BD Transduction Laboratories, San Jose, CA; 1:5000); rabbit anti-early endosome antigen 1 (EEA1) (Upstate, Charlottesville, VA; 1:500); mouse anti-villin (AMAC Inc., Westbrook, ME; 1:2000); and mouse anti-Disabled-2/p96 (BD Transduction Laboratories; 1:1000). For immunofluorescence staining, rabbit anti-vimentin (neural stem cell marker, Abcam Inc., Cambridge, MA; 1:75), rabbit anti-megalin (anti-MC-220 (Zou et al. 2004); 1:1000), mouse anti-villin (Beckman Coulter, Brea, CA; 1:50), rabbit anti-pig villin serum (gift of D. Louvard, Institut Curie; 1:500), rabbit anti-Myo6 tail (10 µg/ml), rabbit anti-EEA1 polyclonal (Cell Signaling Technology, Danvers, MA; 1:100), and mouse anti-Dab2 (1:100) primary antibodies were used, with goat secondary antibodies conjugated to Alexa-488 or -568 (Molecular Probes, Eugene, OR; 1:500).

Total kidney protein preparation

Mice were euthanized by CO₂ asphyxiation, and kidneys were removed, placed in ice-cold saline, and then homogenized with a glass Dounce homogenizer on ice in 10 ml brush border homogenization buffer [10 mM imidazole (pH 7.2), 4 mM K-EDTA, 1 mM K-EGTA, 0.02% sodium azide] per kidney. To minimize proteolysis, total kidney protein was prepared by homogenization in trichloroacetic acid (TCA) added to 5%; the precipitated protein was collected by sedimentation at 2800 rpm in a Beckman J-6B centrifuge for 10 min at 4°C. The pellet was briefly washed in cold water to remove acid and resuspended in water for preparation of SDS-PAGE samples and determination of protein concentration by BCA assay (Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer’s protocol.

Kidney homogenate fractionation

Mice were euthanized by CO₂ asphyxiation, and kidneys were removed and placed in ice-cold saline. For determination of adaptin β protein levels associated with isolated BB membranes, kidneys were homogenized in 3 ml cytoskeleton stabilization buffer [10 mM imidazole (pH 7.2), 75 mM KCl, 1 mM K-EGTA, 5 mM MgCl₂, 2 mM DTT, 1 mM Pefabloc SC (Roche Applied Science, Indianapolis, IN)] per kidney, and 1 ml of homogenate was centrifuged at 4000 × g for 10 min at 4°C. The supernatant was collected, and the pellet was resuspended in 1 ml homogenization buffer. 4 µl of each was used for blotting. For determination of levels of other proteins (Myo6, villin, EEA1, Dab2), kidneys were homogenized in 20 volumes (v/w) of Tris/MES buffer (30 mM Tris, 20 mM MES, 100 mM NaCl, pH 7.4) with 1 EDTA-free Complete Protease Inhibitor Tablet (Roche) per 25 ml of buffer using a Potter-Elvehjem homogenizer with a Teflon pestle. Homogenates were spun at 2200 rpm in a Beckman J-6B centrifuge for 10 min, and the resulting supernatant was spun at 36,000 rpm in a Beckman 70.1 Ti rotor for 1 h at 4°C. The low and high speed pellets, which contain BB associated components, were resuspended in BB homogenization buffer (see above) containing Complete Protease Inhibitor. Proteins in the obtained fractions (homogenate, low-speed pellet, ultra-speed pellet and supernatant) were TCA-precipitated as above and loaded stoichiometrically with respect to original homogenate volume on SDS-PAGE gels for immunoblotting.

SDS-PAGE and Western blotting

Protein samples were separated on 5–20% gradient polyacrylamide gels (Laemmli 1970), transferred to nitrocellulose using a Hoefer TE22 transfer unit (Amersham Biosciences, Piscataway, NJ), processed, and detected by using HRP-conjugated goat secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and an enhanced chemiluminescence system (Amersham).

HRP uptake studies

HRP (Sigma-Aldrich; 100µl of 10 mg/ml) was injected via cannulated jugular vein in mice. At 5 or 30 min after injection, kidneys were perfusion-fixed with periodate-lysine-paraformaldehyde (McLean and Nakane 1974) and then harvested and processed for light microscopy (LM; paraffin sections and cryosections) or electron microscopy (EM). Kidneys were embedded in paraffin and cut at 5 µm thickness (Research Histology, Yale School of Medicine) for histology and immunofluorescence staining. For cryosections, kidneys were cryoprotected in 30% sucrose in PBS overnight at 4°C, frozen in liquid isopentane cooled by liquid nitrogen, and cut at 5 µm thickness on a Leica CM3050 S cryostat (Leica Microsystems, Bannockburn, IL) at −20°C. For EM, kidneys were processed conventionally or HRP-reacted. For conventional EM, kidneys were either: (1) post-fixed in 2% glutaraldehyde, 0.2% tannic acid, in 0.1 M sodium phosphate buffer, pH 7.0, for 5 min at room temperature, then 3 h on ice, and further post-fixed in 1% OsO₄ in 0.1 M sodium phosphate buffer, pH 6.0, for 1 h on ice, and stained overnight at 4°C with 1% uranyl acetate; or (2) post-fixed in 4% glutaraldehyde, 2% sucrose, in 100 mM cacodylate buffer, pH 7.4, for 1 h at 4°C, then processed as in (Thomson et al. 2003). For HRP visualization by EM, kidneys were post-fixed as in (2), and cryoprotected and frozen as above. 30 µm-thick frozen sections were cut, rehydrated in TBS, then quenched with 0.5 M ammonium chloride, 0.1% BSA, in TBS for 15 min, blocked with 0.1% BSA, 10% goat serum, in TBS for 15 min, then rinsed with 7.5% sucrose in 50 mM Tris buffer, pH 7.4, and reacted with diaminobenzidine (2 mg/ml) in the Tris-sucrose buffer for 30 min with slow addition of 0.5% H₂O₂. After rinsing with Tris-sucrose buffer, sections were treated with 1% potassium ferrocyanide-reduced OsO₄ in 75 mM cacodylate buffer for 1 h at 4°C in the dark. Tissues were dehydrated in an ethanol series and in propylene oxide and embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA).

Histology

Paraffin sections were stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stain by Research Histology at Yale School of Medicine.

Immunofluorescence staining

Paraffin sections were de-paraffinized and pressure-cooked for 7 min in 10 mM sodium citrate, pH 6.0, for antigen retrieval (Shi et al. 1997). After rinsing with PBS (for vimentin or Dab2/villin) or TBS (for megalin), sections were blocked in 5% normal goat serum, 5% BSA in the respective buffers for 1 h at room temperature, then incubated with primary antibodies in 1% normal goat serum, 1% BSA, 0.1% Triton X-100 in PBS or TBS for 1 h at 37°C or overnight at 4°C. Following washes with 0.1% BSA in PBS or TBS (3 × 5 min), sections were incubated with secondary antibodies for 1 h at room temperature, washed as above, rinsed with PBS or TBS, and mounted in Citifluor (Ted Pella, Inc., Redding, CA) or ProLong Gold (Invitrogen, Carlsbad, CA) for observation by confocal microscopy. Cryosections were rinsed with PBS, incubated with primary antibodies overnight, and processed as above.

Light and electron microscopy

Histologically stained kidney sections were imaged on a Nikon Eclipse E800 microscope with 4x/0.2 Plan Apo, 20x/0.50 Plan Fluor and 40x/1.30 Plan Fluor objectives coupled to SPOT imaging hardware and software (SPOT Image Corporation, Chantilly, VA). EM images were acquired on a JEOL JEM-1230 transmission electron microscope at 80 kV (JEOL, Peabody, MA) equipped with a Hamamatsu ORCA CCD camera (Hamamatsu Corp., Bridgewater, NJ) and AMT imaging software (Advanced Microscopy Techniques, Corp., Danvers, MA). Immunofluorescence staining was imaged by using a Zeiss Axiovert microscope with a 40x/1.0 Plan Apo objective coupled to a laser scanning confocal unit (MRC-1024; Bio-Rad, Hercules, CA) or Zeiss LSM 510 NLO microscope with a 63x/1.40 Plan Apo objective. All images comparing +/sv and sv/sv kidneys were acquired with identical parameters and post-processed in an identical manner by using Adobe Photoshop 5.5.

Tubule diameter measurements

Kidney paraffin sections from HRP studies were stained for megalin, and confocal images of 15 random fields in the renal cortex per animal were acquired using a 40x objective. The diameters of PTs (defined as the shorter inner diameter of circular/elliptical megalin staining with a major to minor axis ratio < 2.2) in each image were measured using ImageJ software (U.S. National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/).

Fibrosis Quantification

The number of dilated PTs and the number of those exhibiting adjacent fibrosis was determined in a total of 20 bright-field images of renal cortex, acquired using a 20x objective, in PAS-stained sv/sv kidney sections from four mice. Four to six images per mouse were analyzed. The percentage of dilated PTs with adjacent fibrosis was calculated for each mouse and then averaged.

Blood parameter measurements

Blood was drawn from the retro-orbital sinus of anesthetized animals into heparinized capillary tubes. Samples were immediately analyzed using an i-STAT blood gas/chemistry analyzer (Heska Corporation, Loveland, CO) for pH, concentrations of HCO₃⁻, Na⁺, K⁺, ionized calcium, glucose, hematocrit, and hemoglobin.

Renal clearance studies

Renal clearance studies were carried out on mice as described previously (Wang et al. 1998). Mice were anesthetized by intraperitoneal injection of 100 to 150 mg/kg body weight of Inactin (Sigma-Aldrich) and placed on a thermostatically controlled surgical table to maintain body temperature at 37°C. After tracheotomy, the left jugular vein was exposed and cannulated with a PE-10 catheter for intravenous infusion. A carotid artery was catheterized with PE-10 tubing for blood pressure recording and blood collections. The bladder was exposed and catheterized for urine collection via a suprapubic incision with a 10 cm piece of PE-50 tubing. Upon completion of surgery, 0.3% body weight of isotonic saline was given intravenously to replace surgical fluid losses. Subsequently, a priming dose of 5 µCi of ³H-methoxy-inulin (New England Nuclear, Boston, MA) was given in 0.05 ml isotonic saline followed by a maintenance infusion of 0.9% NaCl and 4 mM KCl containing 10 µCi/ml of ³H-inulin (infusion rate = 0.41 ml/hr). An equilibration period of 60 min was allowed before beginning the experiment. Two 30-minute urine collections were performed, and blood samples were taken at the beginning and end of each urine collection period. Mean volumes of measurements obtained from two collections were used for each mouse. Urine volume, GFR, absolute excretion rates (ENa, EK), fractional excretion rates (FENa, FEK) and plasma Na⁺ and K⁺ concentrations were determined. Na⁺ and K⁺ concentrations in plasma and urine were measured by standard flame photometry (type 480 Flame Photometer, Corning Medical and Scientific, Corning, NY). The absolute and fractional renal excretion rates were calculated by standard methods (Wang et al. 2000).

Urinary albumin and osmolality

Mice were placed individually in silicone-coated metabolic cages for 24-hour urine collection. They were acclimatized to cages for 19–26 hours before the collection period. Albumin concentrations were measured using the Mouse Albumin ELISA Quantitation Kit (Bethyl Laboratories, Inc., Montgomery, TX) according to the manufacturer’s protocol. BSA (A7030; Sigma-Aldrich) was used for the blocking solution. All samples were analyzed in duplicate, and total albumin in each sample was calculated from the urine volume and mean albumin concentration. Urine osmolality was determined by using a Wescor 5100c vapor pressure osmometer (Wescor Inc., Logan, UT).

Statistics

Data are expressed as the mean ± SE unless otherwise noted. Statistical significance was determined by Student’s two-tailed heteroscedastic t-test or one-way ANOVA followed by Tukey’s test, and a P-value of < 0.05 was considered statistically significant.

Results

Renal physiology of the sv/sv mouse

Measurement of blood parameters, including acid-base parameters (pH, [HCO₃⁻]) and the concentrations of Na⁺ and K⁺, in retro-orbital blood samples from +/+, +/sv, and sv/sv mice showed no significant differences (Table 1). As +/sv mice express +/+ levels of Myo6 protein in the kidney (Fig. 4A) and +/sv and sv/sv mice are more genetically similar to each other than to +/+ (C57BL/6J) mice due to their mixed background, we compared renal function by renal clearance studies in +/sv and sv/sv mice. No significant differences were found in urine volume, GFR, and absolute and fractional excretions of Na⁺ and K⁺ (Table 2). 24-hour urine volume, food and water intake, urine osmolality, and total kidney weight were also not different between +/sv and sv/sv mice (Table 3). The sole differences found in basic physiological parameters were reduced body weight and elevated mean arterial blood pressure in sv/sv mice; sv/sv body weights were ~70–80% of +/+ and +/sv weights (Tables 1 and 2), and blood pressure was elevated by ~20% compared to +/sv mice (Table 2). No obvious deleterious effects of this mild level of blood pressure elevation were noted, and the mutant mice have life spans comparable to their heterozygous littermates. The reduced body weight, also characteristic of mice with other deafness mutations, is likely due to the dramatically greater activity exhibited by these mice. These results indicated overall maintenance of renal function in sv/sv mice.

Table 3.

Total kidney weight, 24-hour urine volume, urine osmolality, and food and water intake in +/sv and sv/sv mice.

	+/sv	sv/sv	P
Total kidney weight (g)	0.46 ± 0.02 (n=3)	0.56 ± 0.04 (n=6)	NS
Urine volume (ml·24 h−1)	0.86 ± 0.14 (n=11)	1.02 ± 0.07 (n=12)	NS
Urine osmolality (mOsm·kg−1 H2O)	1536 ± 102 (n=12)	1735 ± 65 (n=12)	NS
Food intake (g·24 h−1)	3.9 ± 0.3 (n=8)	4.8 ± 0.4 (n=8)	NS
Water intake (ml·24 h−1)	4.7 ± 0.3 (n=11)	4.8 ± 0.2 (n=12)	NS

Sv/sv mice have albuminuria

To determine the overall integrity of protein filtration at the glomerulus and endocytosis in the PT, we measured urinary albumin levels of +/sv and sv/sv mice by mouse albumin ELISA on 24-hour urine samples. Under normal conditions, albumin is reabsorbed efficiently by PT cells via receptor-mediated endocytosis by megalin and cubulin, and its level in urine reflects glomerular and tubular function and correlates with the progression of renal disease (Birn and Christensen 2006; Gekle 2005). Sv/sv mice had nearly four-fold greater albumin excretion compared to +/sv mice at 12–28 weeks of age (Fig. 1; 192.9 ± 27.0 vs. 49.5 ± 7.1 µg • 24 h⁻¹, n_sv/sv = 12, n_+/sv = 11, P < 0.0005). For both sv/sv and +/sv mice, urinary albumin levels did not differ statistically between age groups 12–15 weeks and 21–28 weeks. Together with the observation of morphologically normal glomeruli in sv/sv kidneys (Fig. 8A), these results suggest a defect in PT protein endocytosis in sv/sv mice and involvement of Myo6 in the uptake of albumin.

Figure 1. Albuminuria in sv/sv mice.

Mean albumin excretion in 24-hour urine of +/sv and sv/sv mice. Urinary albumin concentration was measured by ELISA and multiplied by 24-hour urine volume to determine total excretion. Sv/sv mice exhibit ~4-fold increase in albumin excretion compared to +/sv mice at age 12–28 weeks. ***P < 0.0005, n_+/sv = 11, n_sv/sv = 12.

Figure 8. PAS staining showing fibrosis and signs of cellular proliferation and de-differentiation in the sv/sv kidney.

(A) +/+ and +/sv kidneys show normal tubular structure and with occasional interstitial cells. Sv/sv kidneys exhibit PT dilation and basement membrane thickening (arrow) associated with larger clusters of interstitial cells. Glomeruli were observed to be normal in all three genotypes. (B) Diffuse to dense appearance of fibrosis in sv/sv kidneys. Fibrosis is often located adjacent to PT cells with flattened and/or crowded nuclei and attenuated brush border (see also A and C). The middle panel shows a focus of epithelial-mesenchymal transdifferentiation (EMT) with disrupted tubular basement membrane and tubular cells that appear to be migrating into the interstitium (arrow). (C) In addition to those forming a cap-like area (A and B), some fibroses encircled PTs, and enlarged nuclei were often observed in the adjacent tubular cells. Mice observed were 12–23 weeks old (n_+/+ = 4, n_+/sv = 5, n_sv/sv = 6). Bar = 50 µm.

Endocytosis of intravenously injected endocytic marker HRP by PT cells is reduced and delayed in the sv/sv kidney

To detect qualitative differences in endocytosis of the fluid-phase marker HRP (Bacic et al. 2006; Sundin et al. 1997) by the sv/sv kidney, we compared its uptake and trafficking in PT cells in 15- to 24-week-old +/sv and sv/sv mice at 5 or 30 min after intravenous injection by electron microscopy. HRP labels accessible endocytic compartments, from small apical vesicles to lysosomes, which can be visualized cytochemically (Graham and Karnovsky 1966; Straus 1962).

At 5 min in +/sv kidneys, most PT cells contained HRP in apical vesicles, tubules, and early endosomes (Fig. 2A). The most prevalent vacuolar structures stained in the +/sv kidney at 5 min were electron-lucent apical vacuoles with electron-dense rims, presumably early endosomes. At 30 min, staining was found additionally in later endosomal and lysosomal structures deeper in the cell (Fig. 2A), which were identified by their multivesicular, mottled, or lamellate appearance and were less electron-lucent compared to vacuoles observed at 5 min, indicating progression in HRP trafficking.

Figure 2. HRP uptake in +/+, +/sv, and sv/sv kidneys.

(A) Electron micrographs showing the subcellular localization of HRP in +/sv and sv/sv PT cells at 5 and 30 min after injection. (B) Representative electron micrographs of sv/sv PT cells in dilated tubules exhibiting the highest levels of HRP uptake in the sv/sv kidney at 5 and 30 min. 15- to 24-week-old mice (n_+/sv = 4, n_sv/sv = 8) were injected with 1 mg HRP. Refer to text for details. Bar = 1 µm.

In sv/sv kidneys, most PT cells contained no or very few HRP-labeled apical vesicles at 5 min, and at 30 min, staining was largely restricted to apical tubules and vesicles (Fig. 2A). However, a subset (<25%) of sv/sv PT profiles, which were often dilated (see below), were stained comparably at 5 min to those in the +/sv kidney. These tubules commonly showed staining in two kinds of apical endocytic compartments (Fig. 2B): endosome-like structures similar to those observed in +/sv kidneys at 5 min, and membranous vacuoles resembling lysosomes. Stained apical vesicles and early endosomes were less frequently observed, suggesting that HRP in these cells may be reaching later compartments in the endocytic pathway by 5 min, by perhaps rapidly traversing or bypassing earlier compartments. At 30 min, staining in these high-uptake sv/sv cells mostly remained apical in compartments similar to those at 5 min (Fig. 2B), indicating a trafficking defect. Slightly more apical tubular staining was present at 30 min than at 5 min (Fig. 2B), which may be due to recycling, and some cells had vacuolated or blebbing apical domains and did not appear healthy, possibly in response to increased protein load or apical accumulation of HRP resulting from impaired trafficking toward the cell interior.

One function proposed for Myo6 is the downward retrieval/retention of megalin into the interMV coated pit (Hasson 2003). Consequently, the observed impairment of HRP uptake could result from reduced megalin within the coated pit. This appears not to be the case since megalin is properly localized to the interMV domain in the sv/sv PT; there is also faint megalin staining on lumenal membrane protrusions that are likely the result of disrupted BB membrane organization in the sv/sv tubules (Fig. 3). Thus, the endocytic defect in the PT cells of the sv/sv kidney is not due to mislocalization of megalin.

Figure 3. Megalin localization in sv/sv PTs.

Confocal immunofluorescence images of megalin in +/+ (top) and sv/sv (bottom) PTs. Note that in the sv/sv, some megalin is present on the protrusive membranes facing the lumen (arrowheads), consistent with the apical membrane disruption observed at the ultrastructural level. Mice observed were 15–24 weeks old (n_+/+ = 3, n_sv/sv = 4). Bar = 10 µm.

The interMV domain of the BB cytoskeleton is destabilized in the absence of Myo6

We compared Myo6 protein levels in renal homogenates from +/+, +/sv and sv/sv mice and confirmed that +/sv kidneys express Myo6 at +/+ levels (94 ± 12% of +/+ levels, n=3 pairs of +/+ and +/sv; Fig. 4A) and Myo6 is absent from sv/sv kidneys. Comparison of total protein levels of BB and subapical components (villin, megalin, Dab2, early endosome antigen 1 (EEA1)) in renal homogenates from +/+, +/sv and sv/sv mice also showed no significant differences. Consistent with this, the subcellular localization of these proteins did not differ in PT cells of +/sv and sv/sv kidneys (Fig. 5). While EEA1 staining intensity was slightly reduced qualitatively in sv/sv compared to +/sv PTs, staining was localized comparably in the subapical region (Fig. 5B). However, sedimentation analysis of these homogenates revealed elevated levels of soluble p67 isoform of clathrin adaptor protein Dab2 in sv/sv kidneys, suggesting that the interMV coated pits may be destabilized in the absence of Myo6 (Fig. 4B). The lower molecular weight immunogens detected are likely proteolytic fragments of Dab2 since the number and intensity of these bands is significantly reduced in immunoblots of total kidney protein prepared by homogenization in 5% TCA. Myo6 is associated with the clathrin-coated pit via its tail domain binding to Dab2, which binds the clathrin adaptor AP-2 (Inoue et al. 2002; Morris et al. 2002a). Dab2 exists in two alternatively spliced protein forms, p96 and p67 (Xu et al. 1995), and both isoforms can bind Myo6 (Inoue et al. 2002; Morris et al. 2002a). Thus, one possible effect of the loss of Myo6 in PT cells may be a weakened linkage between plasma membrane-associated endocytic complexes and the actin cytoskeleton and hence a structurally destabilized apical membrane domain. To test this idea, +/sv and sv/sv kidney tissues were homogenized in a cytoskeleton-stabilizing buffer, and crude BB membranes and soluble fractions were collected by sedimentation. The content of the interMV membrane component, adaptin β, an AP-2 subunit, in the crude BB membrane was significantly greater in the +/sv preparation compared to sv/sv (~90% vs. 50%) (Fig. 4C), suggesting that the interMV coated pit domain is destabilized in the absence of Myo6.

Figure 5. Myo6, villin, EEA1, and Dab2 localization in +/sv and sv/sv PTs.

Confocal immunofluorescence of (A) villin (red) and Myo6 (green); (B) villin (red) and EEA1 (green); and (C) villin (green) and Dab2 (red). (A and B), cryosections; (C), paraffin sections. Bar = 10 µm.

Myo6 is required to maintain proper apical vacuole number

We examined the ultrastructure of PT cells in sv/sv kidneys for any defects in cellular architecture that may result from the loss of Myo6. Sv/sv PT cells showed regions of apical membrane lacking BB microvilli more often than +/+ or +/sv cells, although sv/sv BBs indistinguishable from those of +/+ and +/sv were also observed, indicating that while Myo6 is not essential for BB maintenance, its loss may render the BB more susceptible to de-stabilization (Fig. 6). The striking difference consistently observed between +/+ or +/sv and sv/sv PT cells was a reduction in the number of large apical vacuoles resembling endosomes in the sv/sv PT cells in a given micrograph (Fig. 6). Many sv/sv PT cells were observed to contain no apical vacuoles per cell, while nearly all +/+ and +/sv PT cells contained at least one and ranged up to four or more per cell. While the number of profiles per unit area does not necessarily reflect the number of objects per unit volume in a non-homogeneous structure (Delesse 1847) such as the cell, we estimate that Myo6 is required for proper maintenance of the apical endosome compartment in PT cells, although as noted above the distribution of the early endosome marker, EEA1, is similar in sv/sv and +/sv tubules (Fig. 5B).

Figure 6. Reduced apical vacuole number in PT cells of the sv/sv kidney.

PT cells in sv/sv kidneys (right) have fewer large apical vacuoles (arrows) compared with those in wild-type kidneys (left). Note the partial loss of microvilli in the sv/sv. Mice observed were 15–24 weeks old (n_+/+ = 4, n_sv/sv = 4). Bar = 500 nm.

Sv/sv kidneys exhibit PT dilation, fibrosis, and signs of EMT

Sv/sv kidneys showed dilated PTs located primarily within the superficial and mid-cortex (Fig. 7A). The dilations appeared to affect mainly the proximal convoluted segment. To assess this phenotype, kidney paraffin sections were stained for the PT marker megalin, and the lumenal diameters of PTs in the cortex were measured. As paraffin embedment results in tissue shrinkage (Iwadare et al. 1984), the measurements represent relative rather than absolute values. As shown by the histogram in Fig. 7B, the frequency distribution of PT diameters was altered in sv/sv mice. In +/+ and +/sv mice, no diameters measured were greater than 20 µm, in contrast to 27 ± 5% of tubules in the sv/sv mouse, where diameters ranged up to 73 µm. Concomitantly, the fraction of tubules with diameters of 5–10 µm, which constitute the majority in +/+ and +/sv kidneys, was reduced to about half in the sv/sv kidneys (Fig. 7B). The median diameter tended to be greater in sv/sv compared to +/+ kidneys (sv/sv: 12.8 ± 1.8 µm vs. +/+: 8.2 ± 0.2 µm; P > 0.05), while mean diameter was nearly twice that of +/+ (sv/sv: 15.5 ± 1.1 µm vs. +/+: 8.4 ± 0.3 µm; n_sv/sv = 4, n_+/+ = 3; P < 0.01). Mean and median diameters were not statistically different between +/+ and +/sv kidneys.

Figure 7. PT dilation in sv/sv mice.

(A) H&E staining of +/+, +/sv, and sv/sv kidneys showing tubule dilations restricted to the cortex in sv/sv kidneys. Bar = 500 µm. (B) Histogram of PT diameters in +/+, +/sv, and sv/sv mice. The lumenal diameters of PTs in +/+, +/sv, and sv/sv mice (15–24 weeks old; n_+/+ = 3, n_+/sv = 3, n_sv/sv = 4) were measured in 15 random fields within the renal cortex per animal at 400X magnification on paraffin sections stained for megalin, a PT-specific marker (see Methods). >133 tubules were measured in each +/+ and +/sv mouse, and >87 tubules in each sv/sv mouse. The percentage of tubules with a given diameter (bin size = 5 µm) was calculated for each mouse. Error bars, SE.

Histology of PAS-stained sv/sv kidneys at 12 to 23 weeks of age revealed the presence of fibrosis adjacent to PT cells (Fig. 8A; n_+/+ = 4, n_+/sv = 5, n_sv/sv = 6 mice). Renal fibrosis can be thought of as the result of a failed wound-healing process mediated primarily by renal fibroblasts and tubular epithelial cells in response to chronic injury sustained by kidney tissue and is characterized by an excessive accumulation of extracellular matrix (ECM) mainly produced by activated fibroblasts in the interstitium (Liu 2006). The fibrotic foci were located next to tubular cells with flattened and/or crowded nuclei suggestive of cellular de-differentiation and proliferation and were frequently associated with dilated PTs (Fig. 8A). 85 ± 4 % of observed fibroses were associated with dilated PTs, and 29 ± 8% of dilated PTs exhibited adjacent fibrosis (n = 4 mice). Usually only one and sometimes two such foci were seen in a given dilated tubular cross-section. Most fibrosis formed a cap-like area adjacent to tubular cells (Fig. 8B); less frequently, they encircled the tubule with a more uniform thickness, and enlarged nuclei were often observed in the associated tubular cells (Fig. 8C). Staining of the accumulated ECM ranged from diffuse (Fig. 8B, left and center) to dense (Fig. 8B, right), indicating maturation of fibrotic scars. Fibrotic foci were also present in the kidneys of older, 13- to 14-month-old female sv/sv mice (n = 4), where essentially all were cap-like and densely stained, and were not observed in age- and sex-matched +/sv mice (n = 4). Similarly to kidneys of younger male sv/sv mice, those of older female sv/sv mice showed normal glomerular morphology and no gross histological abnormalities other than PT dilation and basement membrane thickening.

Morphologic features consistent with EMT were observed at the light and electron microscopic levels. Focal areas of tubular basement membrane disruption were observed in which tubular cells appeared to be entering the interstitium (Fig. 8B (center, arrow)), and tubular cells adjacent to fibrosis often showed cell flattening and extensive loss of the apical brush border (Fig. 8A–C). At the ultrastructural level, reduced mitochondria and basolateral infoldings and impaired apical endocytosis were also noted in the affected cells, and ECM deposits were sometimes observed to contain or be located adjacent to fibroblast-like cells with abundant rough endoplasmic reticulum and pericellular fibers (results not shown).

To assess for expression of EMT markers, immunofluorescence staining was performed for the mesenchymal marker vimentin and myofibroblastic marker alpha smooth muscle actin (αSMA). In addition to the expected staining of glomeruli and blood vessels (Bachmann et al. 1983), sv/sv kidneys showed focal vimentin staining of enlarged clusters of interstitial cells, and some of these were adjacent to vimentin-positive cells within dilated tubules or regions of flattened tubular cells, suggesting EMT of the tubular cells (Fig. 9). Staining for αSMA in vascular smooth muscle cells was normal in sv/sv kidneys, but there was some increased staining in perivascular and peritubular interstitial areas of sv/sv kidneys although the differences were much less pronounced than that observed for vimentin.

Figure 9. Vimentin in +/+, +/sv, and sv/sv kidneys.

In +/+ and +/sv kidneys, vimentin staining was present in glomeruli and blood vessels. Sv/sv kidneys showed additional staining in clusters of interstitial cells, some of which were adjacent to stained tubular cells (arrow). Such vimentin-positive tubular cells were observed focally in dilated tubules and regions of flattened tubular cells. BV, blood vessel; G, glomerulus. Bar = 50 µm.

Discussion

Assessment of the primary defects resulting from loss of Myo6 function

Our data show that Myo6 is involved in both fluid-phase and receptor-mediated endocytosis in PT cells, and consistent with a defect in the latter, sv/sv mice are impaired in renal albumin reabsorption. Because Myo6 interacts with components of the interMV / clathrin-associated endocytic machinery and Golgi complex (Buss et al. 2004), the primary defects in the sv/sv kidney are likely those in receptor-mediated endocytosis and Golgi traffic.

The reduced and delayed endocytic uptake and trafficking of HRP (Fig. 2) in sv/sv mice provides direct evidence that, in polarized cells in vivo, Myo6 is involved in both endocytic vesicle formation and movement of endocytic vesicles toward early endosomes. That Myo6 contributes to stabilizing the plasma membrane association of adaptin β (Fig. 4C), which directly binds clathrin (Gallusser and Kirchhausen 1993), suggests that Myo6 may facilitate endocytic vesicle formation by stabilizing the AP-2 complex at the plasma membrane. Interestingly, Myo6 is also required to stabilize the membrane association of p67, but not p96, Dab2 (Fig. 4B). As p67 contains endocytic motifs but lacks the clathrin- and AP-2-binding (DPF motif) regions of p96 and has relatively less efficient endocytic function in vivo (Maurer and Cooper 2005), Myo6 may also play a role in stabilizing non-clathrin associated endocytic complexes at the membrane and/or modulate as-yet-unknown, non-endocytic functions of p67. Dab2 is a putative tumor suppressor whose expression is down-regulated in a majority of human breast and ovarian carcinomas (Fazili et al. 1999; He et al. 2001; Mok et al. 1998; Sheng et al. 2000; Smith et al. 2001). While total renal Dab2 expression levels in sv/sv mice were not different from those of controls, the increase in soluble p67 Dab2 may affect mitogenic signaling in sv/sv PT cells.

The reduction in number of large apical vacuoles in sv/sv PT cells (Fig. 6) suggests that Myo6 is involved in maintenance of an apical endosomal compartment (Zhai et al. 2003). As EEA1 is properly localized and expressed in sv/sv PT cells, the affected compartment may perhaps be recycling endosomes. In vitro studies have demonstrated that Myo6 is required for efficient transport of nascent endocytic vesicles from cell peripheries to the early endosome prior to their fusion with EEA1-positive early endosomes (Aschenbrenner et al. 2003). These studies showed that Myo6 is absent from endosomes, as has been noted in rat PTs (Biemesderfer et al. 2002). Since Myo6 does not stably associate with the endosome (Aschenbrenner et al. 2003; Biemesderfer et al. 2002), the reduction in the large apical vacuoles we presume to be components of the endosomal compartment may be a downstream consequence of impaired delivery of uncoated vesicles to the early endosome by Myo6. Reduction of apical endosomes has also been observed in PT cells of megalin knockout mice ((Willnow et al. 1996)), indicating the importance of endocytic machinery components in the proper maintenance of the endosomal compartment.

Albuminuria in the absence of glomerular defects in sv/sv kidneys indicates decreased tubular albumin reabsorption and suggests that Myo6 is involved in the clathrin-dependent, megalin/cubulin-mediated endocytosis of albumin. This role contrasts with that of podocyte-expressed Myosin 1e (Myo1e), whose loss of function in mice causes glomerular lesions accompanied by a much higher level of proteinuria consistent with a glomerular filtration barrier defect (Krendel et al. 2009). The magnitude of albuminuria observed in sv/sv mice is similar to those reported in megalin- and cubulin-deficient animal models. Megalin knockout mice exhibit a ~1.5-fold increase, while cubulin-defective dogs show a 7-fold increase, in albumin excretion (Birn and Christensen 2006; Birn et al. 2000). In addition, Dab2 and GIPC/synectin knockout mice exhibit low molecular weight proteinuria similar to that observed in megalin knockout mice (Leheste et al. 1999; Morris et al. 2002b; Naccache et al. 2006). However, in contrast to a number of proteinuric diseases and mouse models, in which renal megalin expression is decreased (Nagai et al. 2005; Obermuller et al. 2001; Piwon et al. 2000; Tojo et al. 2001), sv/sv renal megalin expression levels were comparable to those of controls. Furthermore, megalin was properly localized subapically in sv/sv PTs (Fig. 3), indicating that Myo6 is not required for targeting megalin to the apical membrane and that localization of megalin at the apical endocytic apparatus is not sufficient for proper rates of internalization of albumin in the absence of Myo6.

Secondary, downstream consequences of loss of Myo6 function

Primary endocytic and Golgi-associated defects may cause membrane compositional defects at the plasma membrane, endosome, and Golgi complex that in turn could lead to altered renal physiology and consequently elevated renal stress responses. The observed renal fibrosis in sv/sv mice may be a secondary effect of the loss of Myo6.

The PT dilation in sv/sv kidneys (Figs. 7, 8) was associated with the presence of flattened and/or crowded tubular nuclei indicating tubular cell proliferation. The dilation is likely not due to a general increase in intraluminal hydrostatic pressure such as that caused by intratubular obstruction because, in both chronic ureteral obstruction in neonatal mice and clinical obstructive nephropathy, tubular dilation is greater in the distal nephron than in the proximal nephron, possibly due to the greater mechanical compliance of the former (Cachat et al. 2003). Rather, the restriction of dilation to the PT segment in sv/sv kidneys may be a direct effect of the loss of Myo6 function in the nephron segment. Clues to the causes of tubular dilation in sv/sv kidneys may come from studies of renal cystic disease, which have revealed four major contributing factors: (1) hyperplasia; (2) fluid secretion into the tubular lumen; (3) abnormalities of the ECM that alter the epithelial microenvironment; and (4) primary cilia defects (Murcia et al. 1999; Singla and Reiter 2006). In addition, tubular dilation in rodent models of polycystic kidney disease (PKD)(Murcia et al. 1999) has been linked to defects in mitotic orientation and division of tubular cells during postnatal nephron maturation (Fischer et al. 2006). As Myo6 is required for proper spindle orientation in mitotic Drosophila neuroblasts (Petritsch et al. 2003), it would be interesting to examine spindle positioning during sv/sv tubular development.

Surprisingly, while most sv/sv PT cells exhibited reduced and delayed uptake of HRP, a subset of cells in dilated tubules showed enhanced HRP uptake in apical vacuolar compartments. The lack of appreciable progression in trafficking from the compartments toward the cell interior (Fig. 2B) suggests that Myo6 may be involved in trafficking from those vacuoles to later endocytic compartments or that those vacuoles lack features required for efficient movement along the endocytic pathway. Increased uptake by cells in dilated PTs may be a Myo6-independent, secondary mechanism that compensates for endocytic deficits in the sv/sv kidney. Similar to macropinocytosis in growth factor-stimulated cells (Norbury 2006), the enhanced uptake may be due to autocrine factors secreted by the tubular cells. Alternatively, clathrin-mediated endocytosis may have become rate-limiting in the absence of Myo6, and the rate of non-specific membrane internalization by bulk endocytosis (e.g., macropinocytosis) may have been increased in sv/sv dilated tubules. In this regard, it is interesting to note that macropinocytosis is greatly enhanced in bone marrow-derived dendritic cells of the sv/sv mouse (Holt et al. 2007).

Blood pressure of sv/sv mice was elevated by ~20% compared to +/sv mice (Table 2). While hypertension arises from a variety of etiologies, the kidney is the organ central to long-term regulation of arterial pressure and development of hypertension (Zandi-Nejad et al. 2006). In the rat, adult blood pressure is sensitive to prenatal protein restriction during nephrogenesis (Woods et al., 2004), and thus hypertension in sv/sv mice may be at least in part due to reduced protein uptake during renal development. Another possible cause of hypertension is increased plasma catecholamines resulting from sympathetic nervous system overactivity, which may be associated with hyperactivity of sv/sv mice (Guyenet 2006; Lohmeier 2001). The catecholamines dopamine and norepinephrine inhibit tubular sodium reabsorption (reviewed in (Aperia et al. 1996)), consistent with the trend for slightly elevated sodium excretion observed in sv/sv mice (Table 2; ENa and FENa). The novel link between blood pressure increase and loss of Myo6 function may have important implications in human cardiac disease. An autosomal dominant missense mutation affecting the motor domain of Myo6 in humans has been associated with familial hypertrophic cardiomyopathy, which is diagnosed in the absence of hypertension

It is remarkable that sv/sv mice live just as long as +/+ and +/sv mice despite the myriad of renal defects observed. Albuminuria is an independent risk factor for the progression of renal disease, and in vitro evidence suggests that albumin-induced renal inflammation and fibrosis are dependent on the initial endocytosis of albumin by PT cells (Birn and Christensen 2006; Wohlfarth et al. 2003). Thus, an interesting question is whether inhibition of endocytosis protects against renal damage caused by excess albumin in the tubular lumen. A comparison of the responses of sv/sv and wild-type PT cells to albumin overload may provide insight into the mechanism of proteinuria-induced renal injury as well as clarify whether the fibrosis observed in sv/sv kidneys is attributable to potentially increased albumin uptake by cells in dilated PTs.