Loss of Primary Cilia Upregulates Renal Hypertrophic Signaling and Promotes Cystogenesis

Abstract

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease.

Apical cilia on renal epithelial cells serve as mechanosensors that detect and transmit information from the external environment.^1–4 Cilia have been implicated in polycystic kidney disease (PKD) because alterations in the structure/function of cilia leads to renal cyst formation.⁵ Recently, mice with conditional floxed alleles for genes that encode intraflagellar transport proteins, ift88 and kif3a, or cystoproteins, pkd1 and pkd2, have been developed.^6–9 Embryonic knockout of these proteins before approximately postnatal day 12 leads to rapid cyst formation and renal failure. Surprisingly, knockout of pkd1, ift88, or Kif3a in the adult mouse does not lead to a rapid cystic phenotype.^7–10 In the adult ift88 knockout mouse, renal cysts are not observed before 6 months.⁷

This model of delayed cystogenesis offers an opportunity to investigate fundamental mechanisms that control cyst initiation and progression. We reasoned that one means of altering the time course of cystogenesis was to introduce hypertrophic signaling, which results in the activation of growth factors and other compensatory mechanisms. In this regard, there is compensatory hypertrophy that occurs in the remaining kidney when the other kidney is removed.^11,12 This hypertrophic signaling to the remaining kidney results in increased kidney size and mass and elevations in GFR.¹³ Changes in kidney mass occur primarily through hypertrophy of tubular epithelial cells and, to a much lesser extent, via cell proliferation.¹⁴ The advantage of this model is that the remaining kidney is undisrupted in situ, the renal architecture is completely intact, and the only perturbation is the loss of the contralateral kidney. Other studies have shown accelerated cyst formation after ischemic or nephrotoxic kidney injury.^9,15,16 However, recovery from acute kidney injury is a multifactorial process including loss of normal renal architecture, disruption of cell-to-matrix and cell-to-cell interactions, necrosis, apoptosis, and recruitment of a myriad of hormones and autocoids.¹⁷

Recent studies^18,19 have indicated that the mammalian target of rapamycin (mTOR)²⁰ may be one important factor that mediates the hypertrophic response to unilateral nephrectomy. To support this contention, inhibition of the mTOR pathway significantly inhibited renal hypertrophy found in response to unilateral nephrectomy or in diabetes.¹⁸ Although regulated mTOR activity appears to play an important role in hypertrophy, a physiologically adaptive response, dysregulated mTOR is also involved in certain diseases, including tuberous sclerosis complex and cancer^21,22; mTOR has also been implicated in the pathogenesis of PKD.^23–26 This suggests that there may be a link between mTOR signaling, hypertrophy, and cystogenesis. Thus, the purpose of this work was to determine if deletion of cilia would lead to accelerated cyst formation in a model of reduced renal mass and to examine if there is a role for cilia in controlling or altering the degree of renal hypertrophy; a question that, to our knowledge, has not been previously addressed.

RESULTS

After postnatal day 12, deletion of cilia does not lead to significant cyst formation for at least 6 months. It is therefore of interest to determine if the time course of cyst development can be modified. We hypothesized that hypertrophic signaling such as occurs with reductions in renal mass might accelerate cystogenesis. To this end, we performed a unilateral nephrectomy in mice in which cilia had been deleted. In addition, this model also allowed us to determine if cilia play a role in regulating the hypertrophic response to reduced renal mass.

Renal Morphology

All four groups consisting of two-kidney (2-K) and one-kidney (1-K) cre-positive and cre-negative mice received tamoxifen. 1-K groups were nephrectomized 1 week after completion of tamoxifen injections, and mice were studied at 3 weeks postnephrectomy or at 3 months. Mice are designated as cilia (+) for cre-negative mice and cilia (−) for cre-positive mice because tamoxifen, in cre-positive mice, resulted in >80% deletion of cilia.²⁷

Magnetic resonance imaging (MRI) was performed in mice at 3 weeks and at 3 months. No evidence of cyst formation (Figure 1) was detected at 3 weeks or at 3 months in 1-K and 2-K cilia (+) mice. In 2-K cilia (−) mice, there was little evidence for cyst formation at 3 weeks; however, in the 3-month 2-K cilia (−) mouse, tiny focal areas of increased T2 signal were seen that might represent small cortical cysts. With MRI, there were small cysts consistently observed in 1-K cilia (−) mice at 3 weeks; however, there were massive cysts present at 3 months.

Figure 1: Accelerated cystogenesis at 3 months in a one kidney cilia (−) mouse. MRI was performed in anesthetized, living mice with respiratory gating: (a) 2-K mice and (b) 1-K mice. Within each panel, images to the left are mice at 3 weeks and to the right are from the same mice imaged at 3 months. Arrows denote the presence of a cyst. Images shown are representative mice from the following groups: (a) n = 4, cilia (+); n = 4, cilia (−) and (b) n = 4, cilia (+); n = 5, cilia (−).

Renal histology (Figure 2, a and b) was normal and there was no evidence of renal cystic structures at 3 weeks and 3 months in 1-K and 2-K cilia (+) mice. This was also true for 3-week 2-K cilia (−) mice; at 3 months, we could identify a few small cystic structures. However, the 3-month 1-K cilia (−) group had grossly misshapen kidneys and extensive cystic development. In addition to parenchymal cysts, the kidneys also exhibited juxtaglomerular cysts. As shown in Figure 3, renal epithelial cysts stained positive for the lectin Dolichos biflorus agglutinin (DBA), indicating that these cysts were derived from collecting ducts. There was no evidence for proximal tubular cysts using the lectin Lotus teragonolobus lectin (LTL). Thus, in the absence of cilia, removal of one kidney induced a significant cystic burden.

Figure 2: Glomerular and tubular cysts and immune infiltrates in the one kidney cilia (−) mouse at 3 months. H&E sections (5 μm) from (A) 3-week and (B) 3-month mice. Photomicrographs are representative mouse sections from the following groups: (A) 3 weeks, n = 4, 2-K cilia (+); n = 5, 2-K cilia (−); n = 6, 1-K cilia (+); n = 7, 1-K cilia (−) and (B) 3 months, n = 5, 2-K cilia (+); n = 5, 2-K cilia (−); n = 4, 1-K cilia (+); and n = 4, 1-K cilia (−). Magnification for all panels is 20×. Occasional small cysts are seen in 3-week 1-K cilia (−) and 3-month 2-K cilia (−) mice. Photomicrograph in panel B, lower right, shows massive cysts with the arrow indicating a glomerular cyst. The upper pair of images in panel C represents 5-μm H&E stained sections from 3-month 1-K cilia (−) mice. Arrow in the upper left image denotes the presence of a cortical cyst containing eosinophilic proteinaceous material. This same material is located in cystic structures in collecting ducts from the medulla (upper right image). Both images are at 20×. The lower pair of images in panel C shows examples of intense inflammatory infiltrates associated with cysts (left side) and cuffing vascular structures (right side). Arrow denotes an area of lymphocyte infiltrate. Lower images are at 10×. Panel d is representative 5-μm sections stained with Masson trichrome, 40×, or PAS, 20×, from 3-month 1-K cilia (+) and cilia (−) mice.

Figure 3: Cysts originate from collecting ducts and not in proximal tubules. Images from a 1-K 3-month cilia (−) mouse. (A) Left-side upper and lower panels are FITC-DBA staining for collecting ducts, the middle panels are differential interference contrast microscopy images, and the right-side panels are an overlay of the two images. (B) Similar arrangement of images but using FITC-LTL, which is a marker for proximal tubules. Nuclei are stained with Hoechst.

There were additional pathologic changes observed in 3-month 1-K cilia (−) mice (Figure 2c). As seen in human cystic disease, some cysts in the cortex and in the inner and outer medullary collecting duct system contained eosinophilic proteinaceous material. Similar to chronic kidney disease, lymphocytic infiltrates that also contained eosinophils and macrophages were also identified. These intense inflammatory responses were nested around cystic structures and found surrounding blood vessels. There was also evidence for single-cell apoptosis and necrosis (see supplemental figure). There was no evidence for an increase in collagen content in 3-month 1-K cilia (−) renal tissue (Figure 2d). In the 3-month 1-K cilia (−) mice there appeared to be no renal fibrotic response, no increase in connective tissue, and similarly no gross mesangial expansion.

Enhanced Cell Proliferation

There is enhanced cell proliferation (hyperplasia) in PKD, whereas renal hypertrophy, with reductions in renal mass, largely involves increased cell size. Bromodeoxyuridine (BrdU) was used to assess cell proliferation in response to unilateral nephrectomy in 3-week 1-K mice (Figure 4). There was approximately a 2-fold increase in cell proliferation in the remaining kidney in the cilia (−) mice compared with cilia (+) mice. Interestingly, in the 3-week 1-K cilia (−) kidney sections, cell proliferation was most predominantly found in distal tubular/collecting duct segments, and there was only an occasional BrdU-positive cell found in the proximal tubule.

Figure 4: Cell proliferation is enhanced in the one kidney cilia (−) kidney. BrdU-stained kidney sections from (A) 3-week 1-K cilia (+) and 3-week 1-K cilia (−) mice. Panel B is a summary of the number of BrdU-positive cells per field; there were three mice in each group. Slides were analyzed in a random order and one observer (K.S.) blinded to group counted BrdU-positive cells. *P < 0.05, two-tailed t test, three mice per group, one slide per mouse, ten fields per slide at 40× magnification.

Renal Hypertrophic Responses

Using sequential images from the MRI, overall kidney volume (Figure 5a) in the 2-K groups, with or without cilia, tended to be slightly larger at 3 months compared with 3 weeks. As expected, unilateral nephrectomy led to an increase in kidney volume at 3 months compared with 3 weeks postnephrectomy, with the largest increase in kidney volume occurring in the 1-K cilia (−) mice. To further evaluate hypertrophic responses, we measured the change at 3 months versus 3 weeks in glomerular diameter and proximal tubule and collecting duct epithelial cell heights in 3-month versus 3-week animals (Figure 5, b through d). In 2-K mice, removal of cilia significantly increased glomerular diameter and collecting duct cell height, but it did not increase proximal tubular cell size. Interestingly, the degree of hypertrophy in glomeruli and collecting duct cell height in the 2-K cilia (−) mice was not different from the hypertrophy found with unilateral nephrectomy in the presence of cilia. A major finding is the significantly greater degree of hypertrophy in glomerular diameter and proximal and collecting duct epithelial cell heights in 1-K cilia (−) mice at 3 months versus 3 weeks compared with the other groups.

Figure 5: Structural hypertrophy is enhanced in the absence of cilia. (A) Difference (Δ) in kidney volume as determined by MRI at 3 months versus 3 weeks for each of the four groups. In the 2-K groups, kidney volume is only for the left kidney so that it can be directly compared with the 1-K groups. * P < 0.05, one-way ANOVA, between 3 months and 3 weeks. The number of mice for each group was the same as indicated in Figure 1 for the MRI studies. Hypertrophic responses in (B) glomeruli diameter, (C) height of the proximal tubular epithelial cells, and (D) collecting duct cell height. Each panel is the difference (Δ) between the results obtained at 3 months and those obtained at 3 weeks. There were three mice in each of the eight groups. Two H&E slides were examined per mouse with ten individual measurements of glomerular diameter, proximal tubular epithelial cell height, and collecting duct cell height obtained per slide. Therefore, each average was the mean of 60 measurements from three animals. The same individual (P.D.B.) blinded to group obtained all measurements. *P < 0.05, one-way ANOVA, between 3 months and 3 weeks; **P < 0.05 between groups.

Renal Function and Systemic Hemodynamics

Studies were performed to assess GFR and hemodynamic and excretory function for each group (Table 1). One notable finding was the elevated BP found in the 1-K 3-month cilia (−) mice. In 2-K mice, there were no differences in GFR (Figure 6) in the presence or absence of cilia at 3 weeks or 3 months. As expected, in the presence of cilia, nephrectomy elevated GFR, indicating functional hypertrophy; this elevation in GFR was still present at 3 months. In 1-K cilia (−) mice at 3 weeks there were significantly larger increases in GFR compared with all other groups. Thus, in the absence of cilia, there was a greater degree of functional hypertrophy in response to a reduction in renal mass. At 3 months in 1-K cilia (−) mice, GFR was diminished, most likely because of the degree of renal damage and cyst formation that was present at this later time point.

Table 1: GFR measurements

Figure 6: GFR increases in one kidney cilia (−) mouse at 3 weeks. GFR measurements obtained in 1-K and 2-K cilia (+) and cilia (−) at 3 weeks and 3 months. In the 3-week mouse groups, there were five mice in each of the four groups. In the 3-month mouse groups, there were three 2-K cilia (+), two 2-K cilia (−), five 1-K cilia (+), and five 1-K cilia (−) mice. *P < 0.05, ^§ P < 0.05 one-way ANOVA between 3 weeks and 3 months.

mTOR

The mTOR pathway has been implicated in PKD and unilateral nephrectomy-induced hypertrophy. Using Western blot, phospho-S6 was measured as a surrogate endpoint for mTOR activity in 3-week 1-K and 2-K mice. There was a significant increase in mTOR activity (Figure 7) with removal of cilia in 2-K and 1-K mice, with the highest level of mTOR activity found in the 1-K cilia (−) group.

Figure 7: Renal mTOR signaling is activated with cilia removal in 3-week mice. (A) Representative Western blots from 3-week 2-K cilia (+) and cilia (−) mice and (run on a separate Western blot) 3-week 1-K cilia (+) and cilia (−) mice. (B) Ratio of phospho-S6 over total S6 in all four groups as determined by densitometry analysis. Because 1-K and 2-K groups were run on separate blots, it was not possible to directly compare the 1-K and 2-K groups. *P < 0.05, two-tailed t test.

DISCUSSION

Although in utero deletion of cilia results in massive renal cyst formation and additional developmental abnormalities, the same is not true when cilia are deleted in the adult animal.^7,9,28 Deletion of cilia in the adult mouse, using conditional floxed alleles to disrupt intraflagellar transport proteins, can take up to 6 months for a significant cystic burden to develop. The reason for this delay in cyst formation is not well understood, but the loss of cilia does lead to immediate altered cell function/responsiveness and disruption in planar cell polarity.⁹ In the adult, epithelial cells are quiescent and there is a very low level of proliferation because of cell-to-cell contact inhibition. This may account, at least in part, for the prolonged period of time in which kidney anatomy and function appear to be “normal.”

The primary purpose of this study was to determine if hypertrophic signaling could modify the rate of cystogenesis. We utilized a unilateral nephrectomy model in a mouse with a conditional floxed allele of the ift88 gene. The advantage of this model is that the remaining kidney is untouched and there is no damage to this kidney because of ischemia or nephrotoxins. Previous studies used induction of renal injury with ischemia-reperfusion or a nephrotoxin^9,15,16 to assess accelerated cyst formation. These models involve the repair of renal injury, which is a complex process that involves repopulating damaged renal tubules with regenerated cells. This can be analogous, at least in some respects, to the embryonic condition in which newly formed cells form tubular structures. In contrast, what occurs with the removal of one kidney is purely the activation of hypertrophic signaling in the remaining kidney.¹⁴ In this setting, we determined if deletion of cilia would lead to accelerated cyst formation and examined if there was a role for cilia in controlling or altering the degree of renal hypertrophy; a question that, to our knowledge, has not been previously addressed.

MRI²⁹ was used to assess the time course for the development of cysts. Three-week postnephrectomy was chosen because most renal hypertrophic signaling has been initiated.¹⁴ Three months was chosen because major cyst formation occurred only after 6 months in the ift88 mouse.⁷ Although a few small cysts were found in cilia (−) mice at 3 weeks and were also detected in 2-K cilia (−) mice at 3 months, the major finding is the marked difference in the degree of cyst formation between 3-month 1-K and 2-K cilia (−) mice, in which there was massive cyst formation in the 1-K cilia (−) mice. These results strongly suggest that the hypertrophic signaling that occurs with nephrectomy can elicit an accelerated cystogenic response in the absence of cilia.

Histologic analysis confirmed the MRI results. In the 3-month 1-K cilia (−) group, there were massive cysts that included glomerulocysts^30,31 and tubular cysts, most notably in DBA-positive collecting ducts. Such cystic development can be associated with human disease found in autosomal dominant PKD and disrupted mTOR signaling in tuberous sclerosis complex.³¹ There was little evidence for fibrogenesis or an increase in collagen or other connective tissue elements such as myofibroblasts. This may be due to the rate of disease onset or other variables not yet clearly understood. There were intense inflammatory infiltrates that were adjacent to cysts and surrounded the blood vessels. As pointed out by Mrug et al.³² there is an upregulation of the innate immune response genes in certain forms of PKD. It is possible that the normal infiltrative response in chronic kidney disease that occurred in these mice also may be modulated by changes in cilia expression.

Cell proliferation plays an important role in cystogenesis^3,8,16, whereas a reduction in renal mass predominantly leads to increased cell size. We found that the hypertrophic signaling from reducing renal mass led to an increase in cell proliferation when cilia are absent compared with when cilia are intact. Cell proliferation was highest in distal tubule/collecting ducts compared with proximal tubules. The reason for the preferential increase in proliferative cells in distal nephron segments is not clear, although it may be related to segment specific responsiveness or sensitivity to hypertrophic signaling.

Cilia (+) mice responded to nephrectomy with an appropriate increase in renal mass. Interestingly, nephrectomy in the presence or absence of cilia in the 2-K group led to similar increases in glomerular diameter or collecting duct cell size. The fact that cystic development, cell proliferation, and hypertrophic responses were all enhanced in the distal tubule/collecting duct strongly suggests that these segments are exquisitely sensitive to hypertrophic signaling. The other finding is that increased kidney volume, glomerular diameter, proximal tubule cell size, and collecting duct cell height were all increased in the 3-month 1-K cilia (−) mice relative to the other groups, suggesting that cilia participate in regulating cell hypertrophy in response to hypertrophic signaling.

As expected, we found that unilateral nephrectomy led to a significant increase in GFR at 3 weeks that was maintained at 3 months (compensatory renal hypertrophy). A significant finding was the remarkable elevation in GFR at 3 weeks in the 1-K cilia (−) group. We are not aware of other experimental situations or conditions that have invoked such a large increase in GFR in the mouse. This increase in GFR in the 1-K cilia (−) mice was not sustained at 3 months, most likely because of the destruction of renal tissue and a decline in the number of functioning nephrons. The mechanism for this large increase in GFR in the 1-K cilia (−) group is presently unknown. Reductions of renal mass lead to the activation of several growth factors,^14,33–35 changes in renal nerve activity,³⁶ and alterations in mechanisms that control renal hemodynamics such as the tubuloglomerular feedback mechanism.^37,38 Augmented functional hypertrophy in the 1-K cilia (−) group at 3 weeks may represent an abnormal/exaggerated response to growth factor/vasodilator signaling acting at the level of the afferent arteriole or the macula densa in the absence of cilia.

Recent studies by Chen et al.^18,19 have provided persuasive evidence that elevated mTOR activity is involved in renal hypertrophic responses to unilateral nephrectomy or to the induction of diabetes. Importantly, knockout of S6 kinase, a key enzyme downstream from mTOR, effectively eliminates most hypertrophy in response to nephrectomy. The mTOR pathway plays an important role in cell growth, cell cycle regulation, and protein translation and is responsive to cellular nutrient and energy levels and several growth factors.^20,21,39 Derangements in the mTOR pathway have been implicated in cancer, PKD, and tuberous sclerosis.

Consistent with the studies of Chen et al.,^18,19 we found that unilateral nephrectomy increased mTOR activity. Additionally, we found that mTOR activity also increased upon deletion of cilia. If glomeruli and collecting ducts are highly sensitive to mTOR activation, this may explain why there were equivalent increases in cell height and glomerular diameter in 1-K cilia (+) and 2-K cilia (−) mice. A new finding from our work is that in the 3-week 1-K cilia (−) mice there was a large increase in mTOR activity. Thus, it is possible that the marked increase in mTOR activity in the 3-week 1-K cilia (−) mice contributed to the pathologic and functional changes in this same group of mice at 3 months. It should be noted that other pathways also contribute to hypertrophy and cystogenesis found in the study presented here. Future studies are needed to determine the role of chronic mTOR pathway inhibition of hypertrophic signaling in the absence of cilia. However, Shillingford et al.²⁴ reported that in an orpk-rescued mouse (derived from the TG737/ift88 hypomorph), which develops cysts in the adult mouse, rapamycin profoundly attenuated cyst formation.

The study presented here supports a model in which cilia help to regulate and control mTOR and temper the response of this pathway to growth factors. Although the link between cilia per se and mTOR regulation is not completely understood, Shillingford et al.²⁵ have reported that polycystin 1 helps to regulate the mTOR pathway, and clearly polycystin 1 is an important component of cilia and is necessary for normal ciliary function. Thus, reduced renal mass with cilia intact or in the presence of both kidneys but with the loss of cilia leads to increases in mTOR activation and compensatory hypertrophy. However, we speculate that with a reduction in renal mass in the absence of cilia, activation of growth factors may lead to a marked increase in mTOR activation that results in exaggerated structural and functional hypertrophy and the stimulation of cell proliferation and cystogenesis. In addition, because the mTOR pathway is involved in immune activation, it could help to explain the intense immune responses found in the 3-month 1-K cilia (−) mice.⁴⁰ Thus, in the absence of ciliary regulation of mTOR activity, hypertrophic signaling induces inappropriate or exaggerated activation of mTOR, which may contribute to increased cell proliferation and cyst formation, which ultimately leads to accelerated renal failure.

CONCISE METHODS

ift88 Mice

Development of ift88 floxed allele mice has been previously reported (see references ^7,27 for details). Mice were maintained in accordance with the Institutional Animal Care and Use Committee regulations at the Medical University of South Carolina. Genotyping was performed as described previously.²⁷ To induce cre activity, tamoxifen was administered once a day for 5 consecutive days when male and female mice were approximately 8 weeks of age. Male and female mice were randomly assigned to the various groups. Tamoxifen (Sigma, St. Louis, MO) dissolved in corn oil (Sigma) was administered intraperitoneally (0.5 ml of 10 mg/ml tamoxifen) to cre (+) and cre (−) mice.

Nephrectomy

Mice were anesthetized (5% induction and 1% to 2% maintenance) with isoflurane using a nose cone and monitored for depth of anesthesia and respiratory rate. Using sterile techniques, a 2-cm incision was made in the left lumbar region, the kidney was exteriorized, the renal hilum was tied off using 4.0 Vicryl Plus (Ethicon), and the kidney was removed. Deep tissues were approximated and skin was closed with 4.0 Vicryl Plus (Ethicon). Mice were given 1 ml of warm saline and 0.5 ml of Carprofen at 5 mg/kg subcutaneously after the procedure with another dose given at 24 hours.

GFR

Mice were anesthetized with isoflurane and placed on a heated table to maintain body temperature at 37°C. The right jugular vein was cannulated for infusion of replacement fluid (1% bovine serum albumin in 0.9% NaCl) at 0.2 ml/h. The left femoral artery was cannulated and BP was measured with a Digi-Med BP analyzer system (Micro Med, Louisville, KY). The urinary bladder was cannulated to allow collection of urine. Animals were given a 0.2-ml bolus of ¹⁴C-inulin (9 μCi in 1% bovine serum albumin in 0.9% NaCl) followed by an infusion at 0.2 ml/h. After a 60-minute equilibration period, two 30- to 40-minute clearance periods were obtained.

Arterial blood (approximately 80 μl) was collected at the midpoint of each urine collection. Urine volume was measured gravimetrically. Aliquots of urine and blood were counted in a Beckman LS6500 liquid scintillation counter, and the data obtained were used to calculate inulin clearance.

Longitudinal MRI Scans

Mice were scanned using a 7-T Bruker Biospec USR system (Bruker, Inc., Billerica, MA) with a 35-mm quadrature volume coil. Vital signs of the anesthetized mice (respiration, heart rate, and body temperature) were monitored using a MRI-compatible monitoring and gating system (model 1025, SA Instruments, Inc., Stony Brook, NY). Respiratory gating was used to increase image quality.

After acquiring scout images, respiratory-gated T2-weighted fast spin echo images were acquired with a RARE factor of 8 from the abdominal area in coronal and axial planes using TR/TE = 4000 ms/41 ms and four averages. The field of view was 40 × 20 mm and was digitized to a matrix of 256 × 128 for the coronal images. For the axial images, the field of view was 20 × 20 mm and was digitized to a matrix of 256 × 256. In both planes, the slice thicknesses were 0.5 mm with no gap in between the slices. MRI data were acquired using the scanner's control software, Paravision (Bruker, Inc., Billerica, MA). Contrast and brightness were adjusted to enhance the visualization of the kidney. The region of interest was carefully outlined on each image manually and the number of pixels was calculated. The number of pixels within the selected region of interest on each slice was converted to volume information by multiplying the in-plane area with the slice thickness. The total volume of each selected region was estimated by adding the volumes associated with this region in the neighboring slices. This quantification method yielded volume measurements in absolute values.

Histologic Analysis

For light microscopy, 5-μm sections were cut from paraffin-embedded kidneys and stained with hematoxylin-eosin (H&E), Masson trichrome, or periodic acid–Schiff (PAS). For BrdU staining, mice were injected intraperitoneally with BrdU 2 hours before sacrifice. BrdU staining was performed using a BrdU staining kit (Invitrogen, Carlsbad, CA) according to protocol. A single observer blinded to groups counted BrdU-positive cells. BrdU-positive cells in ten fields of view (40× magnification) were counted per slide (n = 3 per group).

Western Blot Analysis

Mouse kidney tissue was homogenized and proteins were extracted. Protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL) were added to each sample. Samples (60 μg) were boiled with Tris(2-carboxyethyl)phosphine and separated on SDS-PAGE (10% to 20%), transferred to nitrocellulose, and immunoblotted with anti-phospho-S6 ribosomal protein (Ser235/236) or anti-S6 ribosomal protein antibodies (Cell Signaling Technology, Danvers, MA). Immunoblots were visualized with ECL Plus (GE Healthcare, Piscataway, NJ) on a Typhoon Trio instrument (GE Healthcare).

Immunofluorescence

Sections of mouse kidney were stained with FITC-conjugated DBA to visualize collecting ducts or with FITC-conjugated LTL to visualize proximal tubules. Images were collected using a Leica confocal microscope.

Statistical Analysis

Data were expressed as mean ± SEM; statistical significance of differences in mean values was assessed by t test or ANOVA with Tukey's post hoc test, as appropriate. Differences between means were considered significant at values of P < 0.05.

DISCLOSURES

None.

This project was supported by VA Merit Grants (P.D.B.), P30DK074038 (L.G.W.), DK32032 (P.D.B.), P30 AR 46031 (Siegal Core PI), and P30 DK 079337 (Siegal Core PI), DK35534 (L.G.W.), DK065655 (B.Y.) and DK061458 (J.B.) in addition to funds from Dialysis Clinic, Inc.