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American Journal of Physiology - Renal Physiology logoLink to American Journal of Physiology - Renal Physiology
. 2019 Aug 28;317(5):F1201–F1210. doi: 10.1152/ajprenal.00629.2018

Inactivation of Tsc2 in Abcg2 lineage-derived cells drives the appearance of polycystic lesions and fibrosis in the adult kidney

Leslie S Gewin 1,2,3, Megan E Summers 4, Julie W Harral 4, Christa F Gaskill 1, Stellor Nlandu Khodo 1, Surekha Neelisetty 1, Timothy M Sullivan 5, Katharina Hopp 6, J Jeffrey Reese 7, Dwight J Klemm 5, Valentina Kon 7, Kevin C Ess 8,9, Wei Shi 10, Susan M Majka 4,11,✉
PMCID: PMC6879939  PMID: 31461347

Abstract

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36–37 with an intact 3′ region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36–37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.

Keywords: ATP-binding cassette G2, polycystic kidney disease, tuberous sclerosis complex 2

INTRODUCTION

Tuberous sclerosis complex 2 (Tsc2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin (mTOR) signaling pathway, the deregulation of which has been associated with cancer, tuberous sclerosis complex (TSC), and lymphangioleiomyomatosis (3–5, 13, 19, 23, 47, 51, 52, 58). Kidney-specific complications associated with loss of Tsc2 function include the progressive formation of cysts and cystic kidney disease, the mechanisms of which remain incompletely understood.

TSC is an autosomal dominant disease known to have a variety of renal manifestations including angiomyolipomas, renal cell carcinomas, and renal cysts, which are the second most common renal TSC manifestation after angiomyolipomas (44). In patients with TSC, renal cystic disease can be subdivided into single/multiple renal cysts presenting with no symptoms, glomerular cysts often diagnosed in the neonatal period, and TSC2/polycystin 1 (PKD1) contiguous gene syndrome (56). The latter is the only scenario in which patients with TSC develop true polycystic kidney disease (PKD), which is generally considered an independent disease entity. TSC2/PKD1 contiguous gene syndrome occurs due to large deletions of TSC2/PKD1 that lie adjacent on chromosome 16p13.3, and patients present with infantile/early childhood PKD (40, 49, 56). This is distinct from the phenotypic presentation of classical PKD, also known as autosomal dominant PKD, which generally is an adulthood disease where renal cysts lead to kidney failure in midlife (54). However, phenotypic variability from infantile onset to adequate renal function until late in life has been previously described (54). The development of both TSC and PKD is partially linked to abnormal activation of mTOR signaling resulting in dysregulation of cell differentiation and growth (6, 15, 19, 23, 27, 44–48, 50, 52, 59, 63). Hence, therapy with mTOR inhibitors slows the progression of cystic disease in TSC and PKD but does not eliminate the underlying pathophysiology (48, 50, 60).

A central question in the pathogenesis of disease is how distinct cell lineages can regulate the onset and severity of cystic kidney disease. This question is typically addressed using developmental models with lineage-specific drivers, e.g., endodermal/epithelial or mesodermal, which function robustly during kidney development and to a lesser extent in adult tissue (12, 22, 25, 27, 42, 50, 62). Developmental phenotypes from targeted drivers are rapidly progressing, and disease may not be modeled into adulthood due to variations in lineage and levels of expression, including those using Tbx4, Twist2/Dermo1, or FoxD1 drivers (9, 46, 64). By comparison, inducing Pkd1 mutation in the developmental or early postnatal periods causes rapid onset PKD, whereas loss of Pkd1 in adulthood results in more slowly progressing PKD (43).

Recently, single cell analysis of kidney identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice (11, 24) as well as a novel previously unidentified cell population (41). The impact in the adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. Therefore, to study the impact of lineage-specific Tsc2 loss on PKD in adulthood, we engineered a novel murine system in which we studied the effect of targeted truncated Tsc2fl36–37 expression in the adult kidney using inducible Cre recombinase driven by expression of the multidrug resistance transporter gene Abcg2. We therefore tested the hypothesis that loss of Tsc2 function in the Abcg2pos renal lineage would result in slowly progressing kidney remodeling and dysfunction. Our results showed that selective expression of Tsc2fl36–37 in the Abcg2pos lineage drives recombination in tetragonolobus lectin-positive (LTLpos) proximal tubule epithelial and rare mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. Taken together, the data illustrate the critical importance of Tsc2 function in adult mesodermal derivatives including the proximal epithelium and mesenchyme and provide a new model to further understand polycystic kidney lesions.

METHODS

Mouse models.

The Institutional Animal Care and Use Committee of National Jewish Health and Vanderbilt University Medical Center approved all animal procedures and protocols. Abcg2-CreERT2 mice, a gift of Dr. B. Sorrentino (St. Jude Children's Research Hospital, Memphis, TN) (11), were crossed to an enhanced green fluorescent protein (eGFP) reporter (JAX stock no. 007676, designated mT/mG) to facilitate lineage analysis. These mice were crossed with homozygous Tsc2fl36–37 animals to delete exons 36–37 while the 3′ region and Pkd1 remained intact (13, 14). Age-matched male and female mice were injected intraperitoneally once between 8 and 10 wk of age with a low dose (0.5 mg/18 g) of tamoxifen (T-5648, Sigma, St. Louis, MO) (14). We have previously reported that the recombination efficiency, at two alleles, is ~50% (14). The results shown in Figs. 1–4 were pooled from five independent experiments. Mouse numbers for the indicated end points were as follows: 12 or 20 wk [12 wk: n = 7 female mice (3+/+, 3+/−, and 1−/−) and 7 male mice (3+/+, 3+/−, and 1−/−) and 20 wk: n = 11 female mice (5+/+ and 6−/−) and 12 male mice (6+/+ and 6−/−)], 3 wk [n = 6 female mice (3+/+ and 3−/−) and 6 male mice (3+/+ and 3−/−)], 6 wk [n = 6 female mice (3+/+ and 3−/−) and 7 male mice (3+/+ and 4−/−)], and 9 wk [n = 6 female mice (3+/+ and 3−/−) and 7 male mice (3+/+ and 4−/−)]. While survival was 100% in these experiments, four mice were humanely euthanized between 17 and 20 wk postinduction. In the initial experiments, male and female mice were separated to distinguish sex-specific differences. As the end-point changes from wild-type (WT) mice were similar between male and female mice, the data were pooled for the time course shown in Figs. 4 and 5. With the use of the Dermo1Cre driver crossed to Tsc2fl exon2–4, Tsc2 was depleted in the mesoderm and derivatives during development as previously reported (46). n = 3 for each time point was analyzed.

Fig. 1.

Fig. 1.

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Progressive cyst formation and fibrosis in the adult kidney after expression of truncated tuberous sclerosis complex 2 (Tsc2). A: study timeline. Adult female and male mice were induced between 8 and 10 wk of age with low-dose tamoxifen and euthanized at 12 or 20 wk. Groups were as follows: ATP-binding cassette G2 (Abcg2)CreERT2 Tsc2fl36–37+/+, +/−, or −/−. T, time; HCT, hematocrit. B and C: representative gross anatomy of kidneys correlated to genotype and functional end points (20 wk). WT, wild type. D: hemotyostain of cystic kidney tissue. Scale bar = 100 μm. E: mean body weight. F: mean kidney weight. G: mean kidney length at 20 wk. n = 5+/+ and 6−/− female mice and n = 6+/+ and 6−/− male mice. H, I, K, and M: trichrome stain of Tsc2+/+ and Tsc2−/− kidney tissue (20 wk). Collagen is visualized as blue. Scale bars = 50 μm. J: collagen type I-α1 (Col1A1) expression was quantitated by PCR using total RNA isolated from female and male kidney tissue from 12 and 20 wk. n = 6+/+ and 8−/− female mice and n = 6+/+ and 7−/− male mice. Data are presented as means ± SE. Significance was determined using one-way ANOVA with a Tukey’s post hoc test.

Fig. 4.

Fig. 4.

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Knockdown of tuberous sclerosis complex 2 (Tsc2) induces epithelial injury that precedes fibrosis. A: adult female and male mice were induced between 8 and 10 wk of age with tamoxifen and euthanized at 3, 6, and 9 wk. Groups were as follows: ATP-binding cassette G2 (Abcg2)CreERT2 Tsc2fl36–37+/+ or −/−. T, time; HCT, hematocrit. B–G: representative hematoxylin and eosin staining of kidney tissue. Scale bars = 100 μm. H: HCT was measured using a standard protocol. n = 8+/+ and 3−/− female mice and n = 4+/+ and 2−/− male mice at 3 wk. I: mean kidney weight. J: mean kidney length. 3 wk: n = 6 female mice (3+/+ and 3−/−) and 6 male mice (3+/+ and 3−/−); 6 wk: n = 6 female mice (3+/+ and 3−/−) and 7 male mice (3+/+ and 4−/−); 9 wk: n = 6 female mice (3+/+ and 3−/−) and 7 male mice (3+/+ and 4−/−). Data are presented as means ± SE. Significance was determined using one-way ANOVA with a Tukey’s post hoc test.

Fig. 5.

Fig. 5.

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Progressive fibrosis was associated with knockdown of tuberous sclerosis complex 2 (Tsc2) in the ATP-binding cassette G2 (ABCG2) lineage. A–D: representative trichrome staining of Tsc2fl36–37+/+ or −/− adult kidney tissue. Scale bars = 100 μm. Arrows indicate interstitial fibrosis.

Evaluation of kidney structure and function.

Mice were euthanized at 3, 6, 9, 12, or 20 wk postinduction, and tissue was processed for paraffin embedding, histology, and standard trichrome staining (Fig. 1A). Blood pressure was measured via tail-cuff plethysmography. Blood urea nitrogen was measured in mouse serum using the Infinity BUN/Urea reagent (ThermoFisher, Waltham, MA). Albuwell M ELISA and Creatinine Companion (Exocell, Philadelphia, PA) were used to calculate the urinary albumin-to-creatinine ratio. Total kidney RNA was prepared with Qiagen RNA Isolation Kit reagents (Qiagen, Valencia, CA) (16, 17, 38). Quantitative RT-PCR assays were performed in triplicate, and levels of analyzed genes were normalized to Gapdh [3-wk kidney injury molecule-1 (Kim1) PCR, n = 14+/+ and 6−/− mice]. Fulton’s index to assess ventricular size was performed as previously described (14).

Imaging.

Images were taken with a Nikon DS-Fi1 camera using Nikon NIS elements AR 4.11.00 acquisition software or the Keyence system using Keyence acquisition software. The fluorochromes used included DAPI and secondary antibodies conjugated to Alexa 488 or Alexa 594 (ThermoFisher, Hampton, NH). Primary antibodies included α-smooth muscle actin (α-SMA; M0851, DAKO), pS6 ribosomal protein (no. 2211, Cell Signaling), and GFP (NB600-308 or Aves, GFP-1010, Novus). LTL was also used for localizing proximal tubules (FL-1321, Vector Laboratories).

Statistical analysis.

Data was analyzed by one-way ANOVA followed by Tukey’s honestly significant difference post hoc analysis using JMP version 9.0.2. Significance was defined as P < 0.05, P < 0.01, or P < 0.001.

RESULTS

Decreased Tsc2 function in the adult kidney Abcg2 lineage drives the formation of renal cystic lesions, parenchymal destruction, and impaired function.

Abcg2, the multidrug resistance transporter largely responsible for the side population phenotype of cells, has been used by our group and others to enrich for putative progenitor cells in many adult tissues in various species (1, 11, 14, 35–37, 53, 55, 65). Abcg2 is expressed in a cell- and tissue-specific manner. In the kidney, Abcg2 is expressed on the apical surface of the proximal tubule epithelium and by a population of “novel” previously undescribed cells (11, 24, 41). Our group previously demonstrated that Abcg2 labels a population of perivascular mesenchymal progenitors in the lung, while localization in pulmonary epithelia is absent (14, 36). Given the important role that Tsc2 expression plays in maintenance of the developing epithelium and mesenchyme (2, 28, 46), we engineered an inducible lineage-labeled murine model to elucidate the role of Tsc2 in regulation of the adult kidney Abcg2pos lineage. Using a low-dose tamoxifen induction strategy, we expressed two copies of the truncated Tsc2fl36–37 allele in the Abcg2 lineage via Abcg2-CreERT2 and colabeled cells with eGFP (13, 14). It is important to note that after recombination, the Tsc2 gene lacks exons 36–37, which encode the GAP domain required for the regulation of mTOR complex 1 signaling, resulting in decreased Tsc2 function and activation of mTOR signaling (13), but the 3′ region and Pkd1 gene remain intact. Hence, observed phenotyps cannot be attributed to Tsc2/Pkd1 contigous gene syndrome.

After recombination, expression of truncated Tsc2 resulted in knockdown of Tsc2 function in the Abcg2 kidney cell subpopulation in vivo. As a result, at 12 or 20 wk postinduction, cysts were identified in the kidney cortex (Fig. 1, A–D). Cystic kidneys were grossly indistinguishable at 12 and 20 wk between male and female mice. We therefore pooled data from the 12- and 20-wk end points after the induction of Abcg2 Tsc2fl36–37 in adult mice (8–10 wk of age). We found that six of eight mice and six of seven mice had kidneys that were grossly cystic at 12 and 20 wk, respectively (Fig. 1, B–D). Heterozygous deletion did not result in cyst formation, and the accompanying renal phenotype and function were indistinguishable from WT (+/+) mice (additional data not shown). Cyst formation was most prominently observed in the renal cortex, and cortical tubules appeared dilated with flattened epithelia (Fig. 1D). Body weight decreased with inducible Tsc2 knockdown in male mice (Fig. 1E); however, both mean kidney weight and length significantly increased in both female and male Abcg2 Tsc2fl36–37 mice (Fig. 1, F and G). The cystic phenotype of the kidney cortex was accompanied by parenchymal tissue destruction, with trichrome staining demonstrating significant fibrosis in Abcg2 Tsc2fl36–37 mice (Fig. 1, H–M). Fibrosis was also present in noncystic regions, suggesting advanced disease (Fig. 1, K–M). Consistent with trichrome staining, the increased collagen type I-α1 expression in the kidney also indicated increased fibrosis in Abcg2 Tsc2fl36–37 mice (Fig. 1J). While mutations in TSC may have multiple organ involvement, gross and histological evaluation did not reveal cysts, tumors, or fibrosis in the lungs, liver, or spleen. These data suggest that Tsc2 regulates epithelial and mesenchymal homeostasis in the adult kidney Abcg2 lineage.

Tsc2 depletion in the Abcg2pos kidney lineage drives proximal tubule injury and myofibroblast accumulation and compromises renal function.

To localize Tsc2 depletion and identify key contributors of this lineage to the various aspects of tissue remodeling in the adult kidney, we performed lineage analysis. eGFP expression was colocalized with the proximal tubule epithelium in cystic structures using LTL staining (Fig. 2, A and B). mTOR activation as a result of Tsc2 depletion was also evident in the proximal tubules by pS6 expression (Fig. 2, C and D). mTOR activation was indicative of decreased Tsc2 activity and suggested a proliferative advantage by these cells. Expression of Kim1, an indicator of proximal tubular injury, was also elevated in both female and male Abcg2 Tsc2fl36–37 mice at 12 and 20 wk (Fig. 2E). Lineage analysis also confirmed that Abcg2-driven eGFP was consistent with a proximal tubule distribution as well as rare perivascular mesenchymal cells (Fig. 2, F–K). eGFP-positive tubules formed cysts as well as some eGFP-negative cells, suggesting that cysts may form in a noncell autonomous manner. Consistent with trichrome staining, the increased α-SMA (Fig. 2I) in the kidney also indicated increased myofibroblast accumulation in Abcg2 Tsc2fl36–37 mice. However, the absence of detectable overlap in GFP and α-SMA suggested that the Abcg2 lineage was not a significant source of myofibroblasts. The significant tubule injury and interstitial fibrosis in Abcg2 Tsc2fl36–37 kidneys indicated that renal function may be compromised (16, 17, 38). Blood urea nitrogen, an indicator of kidney function, and albuminuria were both dramatically elevated in both female and male Abcg2 Tsc2fl36–37 mice at 12 and 20 wk (Fig. 3, A and B). Hematocrit may be reduced in chronic kidney disease due to impaired erythropoietin production by cortical interstitial cells, and levels were significantly reduced in both female and male Abcg2 Tsc2fl36–37 mice (Fig. 3C) (26). Mean systemic blood pressure was increased at 20 wk (Fig. 3D), although there was no significant change in ventricular weights or Fulton’s index (Fig. 3E). These data indicated that proximal epithelial injury in Abcg2 lineage-derived cells, as a result of Tsc2 depletion, drives renal dysfunction and subsequently fibrosis in both male and female mice.

Fig. 2.

Fig. 2.

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Loss of tuberous sclerosis complex 2 (Tsc2) function in the adult ATP-binding cassette G2-positive (Abcg2pos) kidney lineage drives mechanistic target of rapamycin (mTOR) activation, proximal tubule injury, and myofibroblast accumulation. Adult female and male mice were induced between 8 and 10 wk of age with tamoxifen and euthanized at 12 or 20 wk. Histological evaluation and lineage analysis were performed using Tsc2+/+ and Tsc2−/− kidneys. A: tetragonolobus lectin (LTL) colocalization of the proximal tubule epithelium with enhanced green fluorescent protein (eGFP). B: enlarged area from A. Scale bars = 50 μm. C and D: staining to localize eGFP (green) and pS6 (red) expression as an indication of mTOR signaling. Nuclei were visualized with DAPI. Scale bars = 100 μm. E: kidney injury molecule-1 (Kim1) expression was quantitated by PCR using total RNA isolated from female and male kidney tissue from 12 and 20 wk. n = 6+/+ and 8−/− female mice and n = 6+/+ and 7−/− male mice. Data are presented as means ± SE. Significance was determined using one-way ANOVA with a Tukey’s post hoc. F−K: immunofluorescent staining to localize α-smooth muscle actin (α-SMA; green) and eGFP (red) expression. G and H: eGFP localization in the cortex or medulla. WT, wild type. Magnification: ×4 (scale bar = 100 μm) and ×40 (scale bar = 50 μm).

Fig. 3.

Fig. 3.

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Impaired function is evident after expression of truncated tuberous sclerosis complex 2 (Tsc2 ) in ATP-binding cassette G2 (Abgc2) linage cells. A: blood urea nitrogen (BUN) was measured in mouse serum. n = 7+/+ and 9−/− female mice and n = 15+/+ and 7−/− male mice. B: the albumin-to-creatinine ratio was measured in urine. n = 13+/+ and 12−/− female mice and n = 11+/+ and 7−/− male mice. C: hematocrit was measured using a standard protocol. n = 8+/+ and 10−/− female mice and n = 8+/+ and 6−/− male mice. Groups were pooled from 12- and 20-wk time points. D: mean systemic blood pressure at 20 wk. n = 11+/+ and 8−/− mice. E: Fulton’s index (right ventricle/left ventricle + septum). n = 16+/+ and 26−/− mice. WT, wild type. Data are presented as means ± SE. Significance was determined using one-way ANOVA with a Tukey’s post hoc test.

Epithelial injury progresses gradually and precedes fibrosis in the kidneys after Tsc2 knockdown.

To characterize progressive remodeling and address whether fibrosis occurred as a result of tubule epithelial damage, we next analyzed experimental end points 3, 6, and 9 wk after expression of truncated Tsc2 (Fig. 4A). Gross changes in the kidney architecture were not detected at 3 wk in any mouse; however, they were visible at 6 wk in four of seven Abcg2 Tsc2fl36–37 mice and 9 wk in four of seven Abcg2 Tsc2fl36–37 mice (Fig. 4, B–G). Hematocrit did not differ significantly from control mice at 3 and 6 wk, with the exception of one mouse at 6 wk (Fig. 4H). However, by 9 wk, hematocrit was decreased in five of seven mice, which may indicate progressive parenchymal destruction (Fig. 4H). Kidney weight and kidney length were normal at 3 wk and significantly different by 6 and 9 wk (Fig. 4, I and J). In contrast, expression of Kim1 was significantly elevated 14.5-fold in female and male Abcg2 Tsc2fl36–37 mice at 3 wk relative to control mice (20 ± 8.1 vs. 1.38 ± 0.35, P < 0.002), although the levels were more variable and lower than detected at later time points (Fig. 2E), suggesting that subtle proximal tubule injury may precede cyst formation. Additionally, levels of collagen deposition at 3, 6, and 9 wk progressively increased between Abcg2 Tsc2fl36–37 and control mice (Fig. 5, A–D). These data suggest that subtle epithelial injury results after expression of truncated Tsc and that the progressive nature of epithelial injury and cyst formation negatively impacts the adjacent parenchyma in a progressive manner.

To complement these experiments, we analyzed a developmental model in which Tsc2 was knocked out in the embryonic mesoderm and its derivative kidney lineages (46). Cyst formation appeared in a rapid progressive manner and was apparent from postnatal days 18−21 (Fig. 6, A–G). The cystic lesions were accompanied with mild interstitial fibrosis, and no fibrosis was evident before cyst formation (Fig. 6). Taken together, these data show that the Abcg2 Tsc2fl36–37 mouse model targets predominantly tubule epithelial and rare mesenchymal compartments to induce epithelial injury driving cyst formation with tubulointerstitial fibrosis and impaired renal function.

Fig. 6.

Fig. 6.

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Progressive cyst formation driven by mesodermal tuberous sclerosis complex 2 (Tsc2) depletion during development is accompanied by interstitial fibrosis. With the use of the Dermo1Cre driver, Tsc2 was depleted in the mesoderm and derivatives during development. Mice were euthanized on postnatal days 7, 18, and 21 (P7, P18, and P21, respectively). A–C: representative trichrome-stained kidneys. D–G: enlarged areas of P18 and P21 indicated by boxes. Arrows indicate interstitial fibrosis. WT, wild type.

DISCUSSION

The present study illustrates the impact of dysregulated Tsc2 function in Abcg2 lineage cells of the adult kidney. We demonstrate that loss of Tsc2 function in proximal tubules can drive PKD as well as fibrosis. These strutural changes were sufficient to compromise kidney function and promote hypertension. Interstingly, the observed PKD was not the typical autosomal dominant PKD as diagnosed in humans, as that is most prominently caused by cysts to the collecting ducts and loop of Henle.

There are many models of cystic kidney disease that modulate Pkd1, Tsc2, or other genes specifically in the tubular epithelia (20, 59). These models suggest that reduced gene expression in tubular epithelia is sufficient to generate cysts (31, 57). Loss of normal Tsc2 function likely leads to both cyst formation and epithelial injury through mTOR signaling activation as well as epithelial injury elevating levels of Kim1 (6, 8, 12, 25, 27, 42, 61). Many studies of these conditional knockout mice using tubule-specific Cre to reduce gene expression have not reported tubulointerstitial fibrosis, a feature of both autosomal dominant PKD and TSC. Yin et al. (61) modeled renal tubule injury in zebrafish by inducible tubule targeted expression of Kim1 in zebrafish. In addition to demonstrating the significance of Kim1 in kidney injury and inflammation, the study linked Kim1 expression to mTOR activation through AKT signaling (61). As with many signaling pathways conserved between development and the adult, mTOR plays a role in tissue repair and regeneration as well as disease. Its activation confers proliferative, survival, and size/mass advantages in kidney cells (10, 34). While controversial, an additional mechanism of cyst formation in the kidney may in part be the the result of mTOR signaling activation and downstream inhibition of Pkd1 or polycystin 1 expression as well as cellular distribution and function (6, 8, 12, 27, 33, 34, 42). Loss of polycystin 1 function can further increase mTOR signaling (33, 34). Truncated Tsc2 in a subset of tubule epithelium increases mTOR activity, drives additional epithelial injury, and causes enhanced expression of Kim1 in adjacent cells, which may also result in enhanced mTOR activity and loss of renal function.

Much attention has focused on the role of Pkd1 or Tsc2 in the tubules, but support for the mesodermal origins of cystic kidney lesions also comes from a recent study by Ren et al. (46) using the developmental mesodermal lineage tag dermo1-cre to inactivate Tsc2 during embryonic development. Developmental inactivation of Tsc2 led to increased lung septal thickening due to arrested development as well as cystic kidneys (46). Dermo1, also known as twist2, is expressed in the developing mesoderm/mesenchyme, which gives rise to vascular smooth muscle, pericytes, and mesenchymal progenitor cells in all adult tissues excluding the head and neck. While the model demonstrates that mesoderm lineage manipulation plays an important role in lesion formation, it did not demonstrate significant fibrosis and was not inducible so it was more relevant to development than the progression of adult PKD (39). Many studies have generated reduced expression and/or function of Pkd1 or Tsc2 that is widely expressed across the kidney (12, 21, 42, 43, 50, 62). While many of these models develop fibrosis, they do not specify which cells contribute to fibrosis formation.

Furthermore, it is important that genetic deletion is inducible to avoid developmental phenotypes or rapidly progressive cystic disease that does not accurately mimic human PKD and precludes the use of therapeutic agents. Our inducible model avoids these problems and generates cystic disease with significant fibrosis 3–5 mo after inducing recombination. Another benefit of the low-dose tamoxifen approach is that recombination is not induced in the majority of cortical tubule cells (Fig. 2). In PKD, it is thought that the heterozygous loss of function of PKD1/PKD2 leads to the formation of cysts and disease progression due to somatic mutations in a minority of tubules (32). Thus, our model phenocopies this process better than widespread deletion of a gene in the majority of tubule cells. Our model also has the unique feature of affecting subpopulations of epithelial and mesenchymal components. As epithelial-stromal interactions are important for noncystic chronic kidney disease progression, it is likely relevant to the progression of the disease for PKD (18). A caveat to these findings includes the variability in recombination after a single low-dose induction, which may alter our interpretation of the progression of cyst formation. However, in combining multiple time points and making a comparison with a mesodermal driven developmental model, one may conclude that epithelial injury and cyst formation precedes parenchymal destruction and fibrosis. Additionally, it is difficult to evaluate the contribution of stromal Tsc2 depletion to cyst formation in these experiments. Given the increase in α-SMA expression and timeline of fibrosis in Tsc2 knockdown mice, it is likely that these injured epithelia affect surrounding mesenchymal cells through paracrine signaling. Our previous study of perivascular Abcg2pos mesenchymal progenitor cells in the lung (14) as well as others in the kidney (14, 29, 30) have demonstrated that dysfunction or loss of microvascular supporting cells leads to capillary rarefaction, exacerbated fibrosis with injury, and progressive tubule injury with fibrosis. Thus, our model is a useful tool for future studies of epithelial/stromal interactions in PKD.

The benefit of using Abcg2-specific Tsc2 deletion is that we can label and isolate this small cell population from various mouse and human tissues by flow cytometry using lineage tags in mice and cell surface antibodies for human cells. Expression of Abcg2 is not limited to one tissue. However, in the lung and skin, we have previously demonstrated that Abcg2 is specific to perivascular mesenchymal progenitors (14) and absent in differentiated pericytes, vascular smooth muscle cells, and the epithelium. In the kidney, the proximal tubule is rich in transporters given its high resorptive capacity, and this may explain why it is targeted by the Abcg2 transporter. The narrower focus on the Abcg2-labeled lineage will facilitate identification of specific targets in adult kidney tissue and define interactions between the epithelium and mesenchyme during the early stages of lesion formation. Our model, therefore, allows the study of inductive interactions between epithelial and mesenchymal compartments during cystic lesion formation.

GRANTS

This work was funded by Department of Defense Tuberous Sclerosis Complex Research Program Grant W81XWH-18-1-0719 and National Institutes of Health (NIH) Grants R01-HL-091105 and R01-HL-11659701 (to S. M. Majka). Additional funding was provided by NIH Grant R01-DK-108968-01 and Veterans Affairs Merit Grant 1I01BX003425-01A1 (to L. S. Gewin). Funding was provided from NIH Grant R01-HL-146541-01. Funding was also provided by the Zell Family Foundation (to K. Hopp).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.J.K., W.S., and S.M.M. conceived and designed research; L.G., M.E.S., J.W.H., C.F.G., S.N.K., S.N., T.M.S., K.H., W.S., and S.M.M. performed experiments; L.G., M.E.S., J.W.H., C.F.G., S.N.K., S.N., T.M.S., K.H., J.J.R., W.S., and S.M.M. analyzed data; L.G., M.E.S., J.W.H., C.F.G., S.N.K., S.N., T.M.S., K.H., D.J.K., V.K., K.C.E., and S.M.M. interpreted results of experiments; M.E.S., J.W.H., C.F.G., S.N.K., and S.M.M. prepared figures; L.G., M.E.S., C.F.G., S.N.K., K.H., K.C.E., and S.M.M. drafted manuscript; L.G., M.E.S., J.W.H., C.F.G., S.N.K., K.H., J.J.R., D.J.K., V.K., K.C.E., W.S., and S.M.M. edited and revised manuscript; L.G., M.E.S., J.W.H., C.F.G., S.N.K., S.N., T.M.S., K.H., J.J.R., D.J.K., V.K., K.C.E., W.S., and S.M.M. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Jeanine D’Armiento for critical review of the article as well as Cathy Xu and David Heinz for expert technical assistance.

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