Kidney epithelial targeted mitochondrial transcription factor A deficiency results in progressive mitochondrial depletion associated with severe cystic disease.

Abstract

Abnormal mitochondrial function is a well-recognized feature of acute and chronic kidney diseases. To gain insight into the role of mitochondria in kidney homeostasis and pathogenesis, we targeted mitochondrial transcription factor A (TFAM), a protein required for mitochondrial DNA replication and transcription that plays a critical part in the maintenance of mitochondrial mass and function. To examine the consequences of disrupted mitochondrial function in kidney epithelial cells, we inactivated TFAM in sine oculis-related homeobox 2-expressing kidney progenitor cells. TFAM deficiency resulted in significantly decreased mitochondrial gene expression, mitochondrial depletion, inhibition of nephron maturation and the development of severe postnatal cystic disease, which resulted in premature death. This was associated with abnormal mitochondrial morphology, a reduction in oxygen consumption and increased glycolytic flux. Furthermore, we found that TFAM expression was reduced in murine and human polycystic kidneys, which was accompanied by mitochondrial depletion. Thus, our data suggest that dysregulation of TFAM expression and mitochondrial depletion are molecular features of kidney cystic disease that may contribute to its pathogenesis.

Graphical Abstract

INTRODUCTION

Mitochondrial (mt) dysfunction is a well-recognized pathologic feature of common kidney diseases and can trigger cellular injury, inflammation and fibrosis.¹ In the kidney, tubular epithelial cells are highly dependent on ATP generated from oxidative phosphorylation (OXPHOS) as they carry out multiple energy-consuming epithelial transport functions. Sustenance of efficient mt ATP production is therefore essential for normal kidney function and systemic electrolyte homeostasis. In addition, recent evidence indicates that mitochondria have a major role in gene regulation, cellular signaling and cell differentiation via the generation of intermediary metabolites and ROS.² Despite these advances, the role of mt signaling in the pathogenesis of common kidney diseases is not well understood.

To investigate mt function in renal homeostasis and pathogenesis we targeted mitochondrial transcription factor A (TFAM). TFAM is a nuclear-encoded factor that is essential for mt function, maintenance of mt copy number and structural stability of mt DNA as it regulates replication and transcription of the mt genome by bending promoter DNA.^{3, 4} Mammalian mt DNA contains 37 genes, 13 of which encode protein subunits of the respiratory chain complex, 22 encode tRNAs and 2 ribosomal RNAs.³ Thus, TFAM is directly involved in the regulation of mt electron transport and ATP synthesis, via the transcription of genes such as mitochondrially encoded cytochrome b (MT-CYB), mitochondrially encoded cytochrome c oxidase subunit 1 (MT-CO1) and mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6).^{3, 5} Without TFAM, cells lose their ability to produce ATP via OXPHOS, cannot generate significant amounts of mt ROS and become progressively depleted of mitochondria.^6-9 Genetic studies have shown that TFAM is essential for normal embryogenesis as global homozygous Tfam inactivation results in intrauterine lethality by embryonic day (E) 10.5, whereas heterozygous deficiency, although it reduces mt copy number by ~40% and leads to respiratory chain deficiency, does not lead to embryonic lethality.⁶ Thus, genetic targeting of TFAM is a useful experimental strategy for examining the role of progressive mt dysfunction in cellular differentiation and tissue homeostasis. Cell type-specific conditional inactivation of Tfam suggested that OXPHOS and/or mt ROS generation is critical for cellular differentiation, function and normal physiology.^6-10

Primary mt disorders due to nuclear or mt gene mutations may present with kidney disease most commonly manifested as tubulointerstitial injury or isolated tubular dysfunction.^{11, 12} Although implicated in the pathogenesis of certain human diseases such as neurodegenerative disorders,¹³ specific mutations in TFAM causing renal disease have not been reported. More recently reduced TFAM expression has been linked to chronic kidney disease (CKD). Loss of mt integrity due to Tfam inactivation caused tubulointerstitial disease and renal failure in mice, which was partly due to activation of mtDNA stress-induced cGAS-stimulator of interferon genes (STING)-dependent inflammatory responses.¹⁴

Here we report that mice with conditional Tfam inactivation in sine oculis-related homeobox 2 (SIX2)-expressing nephron progenitor cells,¹⁵ develop severe cystic disease and die prematurely from renal failure as young juvenile mice. Tfam^−/− mice were characterized by defects in nephron maturation, which was associated with mt depletion, a reduction in OXPHOS and a metabolic shift towards glycolysis in Tfam^−/− renal epithelium. Given the severity of cystic disease in Tfam^−/− mice, we analyzed 2 mouse models of polycystic kidney disease (PKD), which result from mutations in either polycystin-1 (Pkd1) or cystin-1 (Cys1), as well as human tissues from patients with autosomal dominant polycystic kidney disease (ADPKD). We establish that TFAM is dysregulated in cysts from both murine and human PKD tissues. Taken together, our studies suggest TFAM dysregulation and mt depletion are characteristic features of renal cystic diseases and may have a contributory role in their pathogenesis.

RESULTS

Tfam inactivation in SIX2 lineage cells causes severe cystic disease resulting in renal failure

In order to investigate mt function in renal epithelium, we inactivated Tfam in SIX2-expressing progenitor cells, which give rise to all nephron segments except collecting duct (CD).¹⁵ For this we crossed the Tfam floxed allele with BAC transgenic mice that express an enhanced GFP/Cre-recombinase fusion protein (eGFP/Cre) under transcriptional control of the Six2 promoter (Figure 1A).¹⁶ Mice homozygous for the Tfam floxed allele and heterozygous for the eGFP/Cre transgene (Six2-eGFP/Cre^tg/+; Tfam^fl/fl) are from here on referred to as Six2-Tfam^−/− mutants. Six2-Tfam^−/− mice were born at expected Mendelian ratios and were not distinguishable from Cre⁻ littermate controls by visual inspection at birth. However, differences in body weight between Six2-Tfam^−/− mutants and Cre⁻ littermate controls became apparent by postnatal day (P) 14 (5.7 ± 0.3 g for mutants vs. 7.5 ± 0.3 g for controls; n=4 each, p=0.004; Supplemental Table S1). Six2-Tfam^−/− mutant mice were characterized by enlarged kidneys compared with control (kidney/body weight ratio of 1.45 ± 0.19 % for mutants vs. 0.60 ± 0.02 % for control; n=4 each, p<0.001; Figure 1B and Supplemental Table S1) and died between age P20 and P30 (Figure 1B). Juvenile lethality in the mutant cohort was associated with renal failure from severe cystic disease with blood urea nitrogen (BUN) levels of 68.40 ± 5.32 mg/dL for mutant mice vs. 16.8 ± 2.0 mg/dL for controls; n=6 and n=7 respectively, p<0.0001 (Figures 1B and 1C). Furthermore, Six2-Tfam^−/− mutants developed significant albuminuria (urine albumin/creatinine ratio of 362.4 ± 75.18 mg/g in Six2-Tfam^−/− mutants vs. 43.58 ± 3.39 mg/g in controls at P14; n=6 and 10 respectively, p<0.0001). This is consistent with the expression pattern of Cre-recombinase in SIX2 nephron progenitor cells, which give rise to cap mesenchyme-derived renal tubules and podocytes.¹⁵ In contrast to Six2-Tfam^−/− mutants, mice with heterozygous Tfam deficiency in SIX2 progenitor cells developed normally, were fertile and did not develop overt kidney disease (Supplemental Figure S1).

Figure 1. Tfam inactivation in SIX2 lineage cells results in severe cystic disease and renal failure.

(A) Schematic illustrating the experimental approach and location of targeted sequences within the Tfam floxed allele. PCR analysis of total genomic kidney DNA isolated from littermate control (Cre⁻) and Six2-Tfam^−/− mice at age P7; the non-recombined Tfam floxed allele is denoted by 2-lox (2); the recombined allele by 1-lox, the wild type allele by wt; + or − indicate the presence or absence of the Six2-eGFP/Cre transgene. (B) Left panels, photographs of kidneys from Cre⁻ control and Six2-Tfam^−/− mice at age P20. Kidney weights are expressed as percentage of body weight (n=4-14). Right panels, blood urea nitrogen (BUN) levels in Cre⁻ littermate control and Six2-Tfam^−/− mice at age P7 (n=7 and n=6 respectively) and Kaplan-Meier survival curves for Cre⁻ control and Six2-Tfam^−/− mice compared with the log-rank test (n=10-13). (C) Representative images of formalin-fixed, paraffin-embedded kidney sections from Cre⁻ control and Six2-Tfam^−/− mice at age P7 and P29 stained with H&E and analyzed by immunohistochemistry (IHC) for α-smooth muscle actin (ACTA2) and cluster differentiation (CD) antigen 31. Number signs depict cystic structures and asterisks depict glomeruli. Scale bars, 1 mm for whole kidney cross-sections, 100 μm for high-power H&E images, and 50 μm for IHC images. Data are expressed as mean ± SEM and were analyzed by 2-tailed Student’s t-test; ***p<0.001.

The analysis of Six2-Tfam^−/− mice, which also expressed the ROSA26-ACTB-tdTomato,-eGFP Cre-reporter allele, hereon referred to a Six2-mT/mG;Tfam^−/− mice, indicated that the vast majority of cystic structures in Tfam^−/− kidneys were derived from cells with a history of Six2-eGFP/Cre expression (Supplemental Figure S2). Cysts in Six2-Tfam^−/− kidneys showed evidence of proliferation as demonstrated by the presence of Ki67-positive cyst lining epithelial cells (on average ~40% of all cyst-lining epithelial cells), whereas cells positive for cleaved caspase 3 were not detected within cysts (Supplemental Figure 3). These findings are consistent with increased levels of phosphorylated mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and β-catenin levels in Six2-Tfam^−/− kidney tissue (Supplemental Figure 3). Taken together, our data indicate that Six2-Tfam^−/− kidneys exhibit molecular features that are frequently associated with renal cystic diseases.

Nephron maturation is defective in Six2-Tfam^−/− mice

Because it can take up to several weeks before mice with tissue-specific Tfam inactivation develop pathology,¹⁷ we next examined the time course of renal disease development in Six2-Tfam^−/− mice. We collected kidneys from control and mutant mice at age P0, P7 and P14 and used histological methods, immunofluorescence staining (IF) and gene expression analysis in whole kidney extracts for assessment. IF for SIX2 and E-cadherin at age P0 demonstrated that kidneys from control and Six2-Tfam^−/− were histologically similar. The formation of cortical nephrogenic zone structures, such as SIX2⁺ cap mesenchyme, E-cadherin-expressing ureteric tips and nascent nephron structures such as renal vesicles, comma-shaped and S-shaped bodies, was not blocked in Six2-Tfam^−/− kidneys (Figure 2A). These histological findings were consistent with the expression levels of genes encoding nephrogenic markers. Six2, paired box 2 (Pax2), LIM homeobox protein 1 (Lhx1), and spalt-like transcription factor 1 (Sall1) mRNA levels were not significantly different between control and Six2-Tfam^−/− mice in total kidney homogenates from P0 kidneys (Figure 2B). This suggested that TFAM inactivation in SIX2 nephron progenitors did not significantly impact the formation of nephrogenic structures.

Figure 2. Nephron maturation is defective in Six2-Tfam^−/− mice.

(A) Shown are representative images of formalin-fixed, paraffin-embedded sections from Cre⁻ control and Six2-Tfam^−/− kidneys at age P0. Kidney sections were stained with toluidine blue and analyzed by immunofluorescence (IF) for sine oculis-related homeobox 2 (SIX2) and E-cadherin (ECAD) expression. Ureteric trees are outlined by dashed white lines. Cap mesenchyme (CM), comma-shaped body (CSB), S-shaped body (SSB) and ureteric tip (UT) are annotated. (B) Left panel, relative mRNA expression levels of developmental markers by qPCR in total kidney homogenates from Six2-Tfam^−/− mutants at age P0 compared with Cre⁻ littermate controls (n=6 each). Right panel, relative glomerular (glom) and nephron segment-specific gene expression in total kidney homogenates from Six2-Tfam^−/− mutants at age P7 compared with Cre⁻ littermate controls (n=4 each). PT, proximal tubule; mTAL, thick ascending limb of Henle; DT, distal tubule; CD, collecting duct. (C) Alcian-blue and periodic acid-Schiff (AB-PAS) stained kidney sections at age P0, P7, and P14. Arrows indicate PAS-positive tubules with brush border; asterisks depict glomeruli, number signs depict cystic tubules. Scale bars, 100 μm for AB-PAS images, 50 μm for toluidine blue-stained images and IF images. Data are expressed as mean ± SEM; 2-tailed Student’s t-test; *P<0.05 **P<0.01 and ***P<0.001. Abb.: Aqp1, aquaporin 1; Aqp2, aquaporin 2; Lhx1, LIM homeobox 1; NaPi2a, sodium-phosphate cotransporter 2A; Ncc, thiazide-sensitive sodium-chloride cotransporter; Nkcc2, sodium-potassium-chloride cotransporter 2; Pax2, paired box 2; Sall1, spalt like transcription factor 1; Scnn1a, epithelial sodium channel 1 alpha subunit; Trpv5, transient receptor potential cation channel subfamily V member 5; Umod, uromodulin.

Although nascent nephron structure formation was not inhibited, staining with alcian blue-periodic acid-Schiff (AB-PAS) and with lotus tetragonolobus lectin (LTL) indicated defective terminal nephron maturation in Six2-Tfam^−/− mice at age P0. AB-PAS, which stains tubular basement membranes and brush border and LTL, which identifies specific oligosaccharides in the brush border of proximal tubule (PT) cells, were both decreased in mutant kidneys. Figure 2C shows AB-APS staining for the P0, P7 and P14 time points. LTL histochemistry and IF for Wilms’ tumor 1 protein (WT1) for P0, P7 and P14 time points are shown in Supplemental Figure S4. At age P0 the relative area which stained positively with LTL was 2.10 ± 0.51% and 0.39 ± 0.1 % at age P7 vs. 6.25 ± 0.28 and 6.2 ± 1.1 % for controls respectively; n=3-4, p=0.0004 and 0.0007 respectively (Supplemental Figure S4). Consistent with theses histological findings is the significant decrease in the expression of genes encoding glomerular and nephron segment-specific markers podocin, nephrin, aquaporin 1 (Aqp1), sodium-phosphate cotransporter-2a (NaPi2a), uromodulin, sodium-potassium-chloride cotransporter 2 (Nkcc2), and thiazide-sensitive sodium chloride cotransporter (Ncc) in Six2-Tfam^−/− mice (Figure 2B). Taken together these data suggest that Six2-Tfam^−/− kidneys exhibit a progressive reduction in the number of mature proximal nephron segments and glomeruli.

As Six2-eGFP/Cre activity results in TFAM-deficient CM-derived nephron segments, we predicted that maturation of CD epithelial cells, which are ureteric bud (UB)-derived, would not be affected in Six2-Tfam^−/− kidneys. Consistent with this notion is that staining with dolichos biflorus agglutinin (DBA), which reacts with N-acetyl-D-galactose in distal tubule and CD, indicated a relative overrepresentation of DBA-positive structures in Six2-Tfam^−/− kidneys. At age P7, positively stained areas for DBA comprised 13.91 ± 0.9 % of total area for mutants vs. 1.93 ± 0.1 % for control; n=3, p=0.0002 (Supplemental Figure S4). mRNA expression of sodium channel epithelial 1 alpha subunit (Scnn1a) or aquaporin 2 (Aqp2), which are both expressed in CD epithelial cells was not significantly decreased compared with control (Figure 2B). In contrast to Six2-Tfam^−/− kidneys, Tfam inactivation in HOXB7 progenitor cells, which give rise to CD epithelial cells, resulted in loss of CD nephron marker expression, mild tubular dilatation, but not cystogenesis (Supplemental Figure S5). Taken together our data indicate that loss of TFAM function in SIX2- progenitor cells does not block the development of nascent nephron structures but inhibits terminal nephron maturation.

Tfam^−/− cysts are deficient in common nephron segment markers

To characterize the histogenetic origin of Tfam^−/− renal cysts, we performed IF analyses of Tfam^−/− kidneys at age P14 and examined the expression of nephron segment markers megalin (specific for PT), uromodulin (medullary thick ascending limb of Henle, mTAL), NCC (distal tubule) and aquaporin 2 (CD). The majority of cysts with a maximal diameter of >50 μm did not express these segment-specific markers indicating lack of cellular differentiation (Figure 3A and Supplemental Figure S6). Furthermore, approximately 50% of cysts with a maximal diameter of >50 μm were characterized by intraluminal deposits of uromodulin, which suggested origination from nephron segments distal to the PT and descending loop of Henle (Figure 3B).

Figure 3. Tfam^−/− cysts do not express common nephron segment-specific markers.

(A) Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Six2-mT/mG;Tfam^−/− mice at age P14 stained for for enhanced green fluorescent protein (eGFP), megalin, uromodulin, thiazide-sensitive sodium chloride cotransporter (NCC) and aquaporin 2 (AQP2) by immunofluorescence (IF). DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear staining (blue fluorescence). Arrows depict tubular structures expressing respective nephron segment-specific markers. Nephron segment marker expression was assessed in cysts with a maximal diameter of >50 μm. (B) Representative images of formalin-fixed, paraffin-embedded kidney sections from Six2-mT/mG;Tfam^−/− mice at age P14 stained for eGFP and uromodulin by IF. Asterisks depict cysts with intraluminal uromodulin. The presence of intraluminal uromodulin was examined in cysts with a maximal diameter of either 50–100 μm or in cysts larger than 100 μm in maximal diameter. Scale bars in panels A and B, 100 μm.

Progressive abnormalities in mt function and morphology in Tfam^−/− epithelial cells

To characterize the time course of the metabolic consequences of Tfam deletion in renal epithelial cells we first examined mRNA levels of Tfam and of TFAM-regulated mt-Co1, mt-Cyb, and mt-Atp6 in Tfam^−/− kidneys at age P0, P7 and P14. As expected, Tfam, mt-Co1, mt-Cyb and mt-Atp6 mRNA levels were significantly reduced (Figure 4A). Tfam^−/− epithelial cells tagged with eGFP (Six2-mT/mG;Tfam^−/− mice) exhibited a significant reduction in MT-CO1 protein expression (Supplemental Figure S7). Mt DNA copy number was reduced by 63%, which is consistent with mt depletion, a hallmark of TFAM deficiency (Figure 4A). In contrast, the expression of nuclear genes encoding NADH:ubiquinone oxidoreductase core subunit 3 (Ndufs3) and succinate dehydrogenase complex flavoprotein subunit A (Sdha) was not affected at age P0, but was reduced at age P7 and P14 (Figure 4A). These findings from mt gene and protein analysis are consistent with progressive loss of mt copy number in Tfam^−/− epithelium.

Figure 4. Tfam^−/− renal epithelium is characterized by progressive mitochondrial depletion.

(A) Mitochondrial (mt) gene expression by qPCR at age P0, P7 and P14 (fold change over control, n=6 each) and mt DNA content (P7) in total kidney homogenates from Cre⁻ littermate control (co) and Six2-Tfam^−/− mice. (B) Oxygen consumption rate (OCR) in primary proximal tubular epithelial cells (PTEC) isolated from Cre⁻ littermate control and Six2-Tfam^−/− kidneys at age P7 on a Seahorse XFe24 platform. Representative OCR measurements in control and Tfam^−/− PTEC treated with oligomycin A (olig A, ATP synthase inhibition), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; uncoupler) and inhibitors of oxidative phosphorylation rotenone and antimycin A. Also shown are calculated basal and maximal respiration, ATP-linked respiration and spare respiratory capacity (n=3 each). (C) Ultrastructural analysis of mitochondria by transmission electron microscopy (TEM). Shown are representative TEM images of kidney sections from Cre⁻ control and Six2-Tfam^−/− mice at age P0 and P7. Red arrows depict mitochondria. Scale bar, 500 nm. (D) 3D structured illumination microscopy images of kidney sections from Six2-mT/mG (co) and Six2-mT/mG; Tfam^−/− mice at age P7. Enhanced green fluorescent protein (eGFP), cytochrome c oxidase subunit 4 (COXIV) and voltage-dependent anion-selective channel 1 (VDAC) were detected by immunofluoresecence. DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear staining (blue fluorescence). White dashed lines outline renal tubule in control and cyst lumen in mutant kidney, number signs depict tubular lumen in control or cyst lumen in mutant kidney. Mt area was quantified with Imaris software (n=3 each); Shown are total (tot) mt volume (vol) per eGFP-positive cell (25-40 cells/sample analyzed), the ratio of total mt volume per total cell volume, and the maximal (max) size of the mt network per cell. Mt network size was determined in a tubular cross section with 5 cells per cross section. Scale bars, 4 μm. Data are represented as mean ± SEM and were analyzed by Student’s t-test; *P<0.05 **P<0.01 and ***P<0.001. Abb.: mt-Atp6, mitochondrially encoded ATP synthase membrane subunit 6; mt-Co1, mitochondrially encoded cytochrome c oxidase 1; mt-Cytb, mitochondrially encoded cytochrome B; Ndufs3, NADH:ubiquinone oxidoreductase core subunit S3; Sdha, succinate dehydrogenase complex flavoprotein subunit A; Tfam, mitochondrial transcription factor A.

We next investigated the metabolic effects of Tfam inactivation on primary proximal tubule epithelial cells (PTEC) isolated at age P7. Tfam^−/− PTEC exhibited significant reductions in basal oxygen consumption rates (40.77 ± 4.10 for mutants vs. 61.75 ± 5.18 pmol/min/10⁴ cells for controls; n=3 each, p=0.034), ATP linked respiration (33.11 ± 3.91 for mutants vs. 46.92 ± 4.91 pmol/min/10⁴ cells for controls; n=3 each, p=0.093), maximal respiration (116.8 ± 14.19 for mutants vs. 225.5 ± 13.55 pmol/min/10⁴ cells for controls; n=3 each, p=0.005) and spare respiratory capacity (76.05 ± 10.18 for mutants vs. 163.7 ± 8.61 pmol/min/10⁴ cells for controls; n=3 each, p=0.003) (Figure 4B).

To further characterize the degree of mt depletion and damage, we examined Tfam^−/− kidneys by transmission electron microscopy (TEM) and 3D structured illumination microscopy (3D SIM). At age P7 mitochondria exhibited irregular shapes and ballooning, which is consistent with previous findings in Tfam knock-out mice. TEM analysis indicated that structural abnormalities of mitochondria, such as increased size and abnormal cristae, progressed postnatally, as the morphological differences between mutants and control were less apparent at age P0 and became more severe with age (Figure 4C). 3D SIM was used to examine mt volume and network size in Six2-mT/mG;Tfam^−/− mutants compared with Six2-mT/mG control mice at age P7. Mt volume was measured in cross-sections of 5-8 tubules per section, examining 25-40 eGFP-positive cortical epithelial cells stained for VDAC by IF. We found that total mt volume per eGFP-positive cell was significantly decreased (62.66 ± 16.46 μm³/cell for mutants vs. 177.4 ± 30.17 μm³/cell for controls; n=3 each, p=0.0289) and was associated with a change in the ratio of total mt volume per total cell volume from 0.230 ± 0.01 in controls to 0.089 ± 0.016 in Six2-Tfam^−/− mutants (n=3 each, p=0.0017) (Figure 4C). Maximal mt network size, which measures the largest mt network encountered in all eGFP-positive cells examined, was decreased in Six2-Tfam^−/− kidneys (143.2 ± 23.2 μm³ for mutants vs. 318.3 ± 49.45 μm³ for controls; n=3 each, p=0.0327). Taken together ultrastructural and SIM findings and the findings from mt gene and protein analysis are consistent with progressive loss of mt copy number in Tfam^−/− epithelium.

TFAM deficiency shifts renal epithelial metabolism towards glycolysis

PTEC in the kidney use fatty acid β-oxidation and OXPHOS for ATP generation, and are gluconeogenetic.¹⁸ To gain additional insights into metabolic alterations associated with Tfam inactivation, we performed RNA sequencing analysis of kidneys from Six2-Tfam^−/− and Cre⁻ littermate control mice at age P7. We found that key regulatory genes involved in glycolysis, such as hexokinase 2 (Hk2) and enolase 2 (Eno2), were upregulated. In contrast, the expression of most genes involved in TCA cycle was decreased, e.g. isocitrate dehydrogenase 1 (Idh1), as was the expression of genes involved in fatty acid β-oxidation, such as acetyl-Coenzyme A acyltransferase 1B (Acaa1b), acyl-Coenzyme A dehydrogenase, and medium chain (Acadm) (Figure 5 A and B; Supplemental Figure S8). Consistent with decreased expression of genes involved in TCA cycle and β-oxidation genes was the accumulation of neutral lipids as detected by oil red O staining in frozen kidney sections at age P14 (Figure 5C).

Figure 5. Renal epithelial TFAM deficiency is associated with increased glycolysis and abnormal fatty acid metabolism.

(A) Analysis of metabolic gene expression by RNA sequencing of whole kidney cortex isolated from Six2-Tfam^−/− and Cre⁻ littermate control (co) mice at age P7 (n=4 each). Shown are differentially regulated metabolic genes involved in glycolysis and TCA cycle. Genes with statistically significant increase in mRNA expression are shown in red, genes with decreased expression are shown in green. (B) mRNA expression levels of selected differentially regulated genes by qPCR (n=4-6). (C) Altered fatty acid metabolism in Six2-Tfam^−/− mutant mice. Shown are representative images of oil red O stained kidney sections from Six2-Tfam^−/− and Cre⁻ littermate control mice at age P14. Arrows point to oil red O-positive cells and number sign depicts cyst. Scale bar, 10 μm. (D) Glycolytic flux analysis of primary proximal tubular epithelial cells (PTEC) isolated from Cre⁻ littermate control and Six2-Tfam^−/− kidneys at age P7 (n=4 each) on a Seahorse XFe24 platform. Shown are representative measurements of glycolytic proton efflux rate (glyco PER) in control and Tfam^−/− PTEC treated with 2-deoxyglucose (2-DG) and oxidative phosphorylation inhibitors rotenone and antimycin A (AA). Also shown are average glyco PER at baseline and the ratio of mitochondrial (mt) oxygen consumption rate (OCR) over glyco PER. Data are represented as mean ± SEM and were analyzed using Student’s t-test; *P<0.05, **P<0.01 and ***P<0.001. Abb.: Acaa1b, acetyl-CoA acyltransferase 1B; Acadm, medium-chain acyl-CoA dehydrogenase; Eno2, enolase 2; Hk2, hexokinase 2; Idh1, isocitrate dehydrogenase 1; Pcx, pyruvate carboxylase.

To assess to what degree changes in metabolic gene expression affected glucose metabolism in epithelial cells, we performed metabolic flux analyses of primary PTEC isolated from control and mutant kidneys at age P7 using a Seahorse XFe24 platform. Mutant PTEC were characterized by a significant increase in basal glycolysis [proton efflux rate (PER) of 117.4 ± 12.07 pmol/min for mutants and 73.34 ± 6.33 pmol/min for control; n=3, p=0.0032] and a decrease in the ratio of mt OCR over glycolytic PER from 0.73 ± 0.08 in control to 0.34 ± 0.02 in mutant PTEC; n=3, p=0.0074. Taken together these data indicate that Tfam^−/− epithelial cells had undergone a metabolic shift from OXPHOS and fatty acid β-oxidation towards glycolysis (Figure 5D).

Reduced TFAM expression in murine and human PKD tissues is associated with mt depletion

Because Six2-Tfam^−/− kidneys bore strong resemblance to PKD kidneys, we next assessed whether TFAM was dysregulated in PKD tissues. We first examined TFAM and TFAM-regulated gene expression in two well-established genetic PKD mouse models. Tfam mRNA levels were significantly reduced in whole kidney homogenates from Pkd1^−/− and Cys^cpk/cpk mice, which carry mutations in either Pkd1 or Cys1. This was associated with a decrease in the expression of mitochondrially and nuclear-encoded mt genes as well as dysregulated glycolytic gene expression. Similar to Six2-Tfam^−/− kidneys, Pkd1^−/− and Cys^cpk/cpk kidneys were characterized by elevated Eno2 and Hk2 and significantly decreased phosphoglycerate kinase (Pgk) 1, pyruvate dehydrogenase kinase (Pdk) 1 and Pdk4 transcript levels (Figure 6A). TFAM protein expression, as assessed by immunofluorescence (IF), was reduced in cyst-lining epithelial cells compared with epithelial cells from adjacent non-cystic tubules (Figure 6B). This was associated with reduced mt-Co1 and mt-Atp6 expression by RNA FISH (Figure 6B and Supplemental Figure S9). Mt volume, as determined by 3D SIM, was reduced by 55% compared with either epithelial cells from adjacent, non-cystic tubules or with PTEC from normal control kidneys. Differences in mt volume between normal control PTEC and PTEC from non-cystic tubules were not found (Figure 6C).

Figure 6. TFAM expression is reduced in Pkd1^−/− and Cys^cpk/cpk renal cysts.

(A) Shown are relative mRNA expression levels (fold-change over Cre⁻ littermate control) of mitochondrially and nuclear-encoded mitochondrial and glycolytic genes in Pkd1^−/− and Cys^cpk/cpk kidneys (n=3-5). (B) Representative images of kidney sections from Pkd1^−/− and littermate control mice analyzed at age P22. Shown are immunofluorescence (IF) staining for mitochondrial transcription factor A (TFAM) and RNA fluorescent in situ hybridization (RNA-FISH) for mitochondrially encoded cytochrome c oxidase 1 (mt-Co1) and mitochondrially encoded ATP synthase membrane subunit 6 (mt-Atp6) transcripts. White arrows identify cyst-lining epithelial cells with strongly reduced TFAM, mt-Co1 or mt-Atp6 expression. Glomeruli are marked by gl. TFAM-expressing tubular structures are identified by red fluorescence. Asterisks depict tubular epithelial cells with detectable mt-Co1 and mt-Atp6 transcripts. DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear staining (blue fluorescence). Scale bars, 25 μm for IF images and 10 μm for RNA-FISH images. (C) 3D structured illumination microscopy (SIM). Shown are images of kidney sections from of Pkd1^−/− kidneys analyzed by histochemistry with lotus tetragonolobus lectin (LTL) and by IF for cytochrome c oxidase subunit 4 (COXIV) and voltage-dependent anion-selective channel 1 (VDAC) expression. Mitochondrial (mt) volume was quantified with Imaris software based on COXIV staining. Arrows depict cyst-lining epithelial cells, number signs depict cyst lunima and asterisks depict non-cystic tubules. Cyst-lining epithelial cells are outlined by dashes lines. Scale bars, 10 μm for left panel and 3 μm for right panel. Data are represented as mean ± SEM and were analyzed using Student’s t-test; *P<0.05, **P<0.01 and ***P<0.001. Abb.: Eno2, enolase 2; Hk2, hexokinase 2; mt-Cytb, mitochondrially encoded cytochrome B; Ndufs3, NADH:ubiquinone oxidoreductase core subunit S3; Pdk1, pyruvate dehydrogenase kinase 1; Pdk4, pyruvate dehydrogenase kinase 4; Ppargc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Pgk1, phosphoglycerate kinase 1; PTEC, proximal tubule epithelial cells; Sdha, succinate dehydrogenase complex flavoprotein subunit A.

To examine whether loss of TFAM expression is a common molecular feature of human PKD, we analyzed nephrectomy specimens from five ADPKD patients by IHC, IF, RNA-FISH and 3D SIM. Reduced TFAM expression was observed in 75.2 ± 7.5 % of renal cysts analyzed by immunohistochemistry (IHC). This was associated with decreased expression of MT-CO1 and MT-ATP6 by RNA-FISH (Figure 7A and Supplemental Figure S10). Mt volume in cyst-lining epithelial cells, was diminished by approximately 70% as assessed by 3D SIM (Figure 7B). Taken together, the analysis of two PKD mouse models and human ADPKD tissues suggested that TFAM deficiency and mt depletion are common findings in PKD tissues and are likely to impact the pathogenesis of renal cystic disease.

Figure 7. TFAM expression in renal cysts from patients with polycystic kidney disease is reduced.

(A) Shown are representative images of formalin-fixed paraffin-embedded sections from normal human kidneys and kidneys from polycystic kidney disease (PKD) patients analyzed by immunohistochemistry (IHC) for mitochondrial transcription factor A (TFAM) expression, by IF for voltage-dependent anion-selective channel 1 (VDAC) expression and by RNA in situ hybridization for mitochondrially encoded cytochrome c oxidase 1 (MT-CO1) and mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) mRNA expression. Arrows point identify cyst-lining epithelial cells, number signs depict cyst lumina, and asterisks depict glomeruli. Scale bar, 100 μm for low-magnification images and 10 μm for high-magnification images. (B) Shown are representative 3D structured illumination microscopy images of human PKD kidney sections analyzed with immunofluorescence for VDAC expression. DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear staining (blue fluorescence). Dashed lines mark tubules, and number signs depict tubular or cyst lumina. Mitochondrial (mt) volume was quantified using Imaris software (n=5). Scale bar, 4 μm. Data are represented as mean ± SEM and were analyzed using Student’s t-test; *P<0.05.

DISCUSSION

Here we establish a critical function for mt transcription factor TFAM in renal tissue homoeostasis. We demonstrate that inactivation of TFAM in SIX2, but not in HOXB7 progenitor cells resulted in the development of severe postnatal cystic disease, which was associated with mt depletion and a metabolic shift from OXPHOS towards glycolysis. Furthermore, a decrease in cellular TFAM levels and mt dysfunction are characteristic features of murine and human PKD, suggesting that a reduction in TFAM activity may contribute to and/or modulate the development of renal cystic disease.

Patients with mt disease syndromes are prone to develop kidney pathology. Kidney disease in this setting frequently manifests as tubular dysfunction and/or tubulointerstitial disease, whereas renal cyst formation is rare.^{12, 19-22} Although mutations in TFAM-regulated genes, such as MT-CO1,²³ have been identified in patients with tubulointerstitial disease, mutations in TFAM itself have not been reported in patients with CKD. Nevertheless, CKD progression has been recently associated with decreased TFAM activity, which resulted in the activation of fibrotic and inflammatory pathways due to mt stress.^{14, 24} In contrast to Six2-Tfam^−/− mutants, mice with Ksp-Cre-mediated Tfam inactivation developed renal fibrosis and inflammation,¹⁴ but not cystic disease. The phenotypic differences between the two models are likely a reflection of which renal cell types were targeted as well as the differentiation state of Cre-expressing cells. Ksp-Cre mediates recombination in the distal nephron with prominent Cre activity in the mTAL segment and UB-derived CD,²⁵ whereas Six2-eGFP/Cre is expressed in cap mesenchyme and does not target UB-derived nephron segments.¹⁶ Consistent with these findings is the increase in extracellular matrix deposition, but absence of cystic disease in 15-month-old Hoxb7-Tfam^−/− mutants; Hoxb7-Cre targets UB-derived nephron segments (Supplemental Figure S5).²⁶ Furthermore, in keeping with the notion of developmental stage- and cell type-dependence is the observation that inactivation of Tfam using Nphs2-Cre (Podocin-Cre) did not result in developmental or adult renal phenotypes,²⁷ whereas Six2-Tfam^−/− mice developed significant albuminuria.

Defects in nephron differentiation were not completely unexpected in Six2-Tfam^−/− mice, as cellular differentiation has been associated with increased reliance on OXPHOS for ATP generation, whereas undifferentiated pluripotent cells prefer glycolysis over OXPHOS to meet energy demands.²⁸ To what degree the progressive loss of OXPHOS activity per se contributed to cystogenesis in Six2-Tfam^−/− warrants further investigation. Recent studies have shown that mutations in PKD1, which are responsible for ~85% of ADPKD cases,²⁹ are associated with enhanced glycolytic flux.³⁰ However, the pathophysiologic and therapeutic significance of this finding is not entirely clear as the effects of glucose deprivation on cyst proliferation and PKD progression are controversial.^{31, 32}

Although we do not propose that TFAM dysfunction represents a primary event in the development of PKD, our studies raise the possibility that TFAM dysfunction may have a contributory role in its pathogenesis and/or progression. We demonstrate that TFAM protein levels are reduced in cyst-lining epithelial cells from murine and human PKD tissues and found that Six2Tfam^−/− tissues share molecular features with PKD tissues that are linked to cystogenesis. Abnormal cilia function has been implicated in the pathogenesis of renal cystic diseases.^{29, 33, 34} Although, the absence of cilia has been reported for some PKD animal models,^{35, 36} cilia are formed in Pkd1^−/− epithelial cells,³⁷ and were also detected in renal cysts from Six-Tfam^−/− mice (Supplemental Figure S3). Several signaling pathways linked to cystogenesis are involved in cilia-associated signaling. These include MAPK/ERK signaling and β-catenin-regulated pathways.³³ Both phospho-MAPK/ERK and β-catenin protein levels were elevated in Six2-Tfam^−/− kidneys, suggesting that these pathways are activated. These findings are consistent with observations made in human ADPKD cells and in several murine PKD models.^38-43

Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, an upstream transcriptional regulator of TFAM and driver of mt biogenesis, was decreased in cell lines isolated from patients with ADPKD and would, in addition to TFAM itself, represent a potential therapeutic target for PKD. A reduction in PGC-1α expression has been proposed to promote cyst proliferation due to increased mt superoxide production in PKD1-defective cells.⁴⁴ Although we did not measure mt ROS production in our model, tissue-specific TFAM inactivation in other cell types was associated with a decrease, and not increase in mt ROS production.⁹ In addition to the PGC-1α/TFAM axis, recent studies have highlighted a potential therapeutic role of hypoxia and the hypoxia-inducible factor (HIF) pathway in the therapy of mt diseases.^{45, 46} To what degree hypoxia-associated pathways can be exploited therapeutically for the treatment of diseases that are associated with mt dysfunction, such as PKD, requires further investigation.

In summary, our data demonstrate that mt transcription factor TFAM is required for normal nephron differentiation and that loss of TFAM activity in renal epithelial cells reproduces molecular and metabolic features associated with PKD. Our findings provide strong rational for further investigations into the role of mt health and function in cystogenesis. We propose that therapeutic strategies which aim at improving mt health may be beneficial for the treatment of patients with PKD.

METHODS

The generation of the conditional Tfam allele has been described elsewhere.⁹ A detailed description of mouse lines and experimental methods can be found in Supplemental Methods and Materials. RNAseq data sets are shared at geo@ncbi.nlm.hih.gov; accession number GSE147189.

Statistical analysis.

Data are reported as mean ± SEM. Statistical analyses were performed with Prism 6 software (GraphPad Software Inc., San Diego, CA, USA) using Student’s t test or 1-way ANOVA with Tukey’s post hoc analysis. Survival was analyzed using the Kaplan-Meier method and groups were compared by log-rank test. P-values of less than 0.05 were considered statistically significant.

Study approval.

All procedures involving mice were performed in accordance with NIH guidelines for the use and for care of live animals and were approved by the Vanderbilt University Institutional Animal Care and Use Committee (IACUC).

Supplementary Material

1

Supplemental Figure S1 (associated with Fig. 1). Heterozygous Tfam inactivation in SIX2 progenitor cells does not associate with kidney disease. Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Cre⁻ littermate control and heterozygous Six2-Tfam^+/− mice at 3 months of age (A) and >10 months of age (B). Sections were stained with alcian blue-periodic acid-Schiff (AB-PAS) and analyzed by immunohistochemistry (IHC) for α-smooth muscle actin (ACTA2) expression. Asterisks depict glomeruli. Scale bars, 100 μm. Right panels show blood urea nitrogen (BUN) levels and renal mt DNA content in Cre⁻ littermate control and Six2-Tfam^+/− mutant mice at 3 months of age (n=5 and n=6 respectively) and age >10 months (n=4 and n=3 respectively). Data are represented as mean ± SEM and were analyzed by the 2-tailed Student’s t-test; **p<0.01.

Supplemental Figure S2 (associated with Fig. 1). Tfam^−/− renal cysts are derived from cells with Six2-eGFP/Cre expression. Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Six2-mT/mG;Tfam^−/− mice analyzed by immunofluorescence (IF) with antibodies against enhanced green fluorescent protein (eGFP) and tdTomato red fluorescent protein. eGFP expression indicates Six2-eGFP/Cre-mediated recombination of the mT/mG Cre-reporter allele. (A) IF analysis of tdTomato and/or eGFP expression in kidneys at age P7, P14 and P29. Asterisks depict large cysts derived from Six2-eGFP/Cre-targeted GFP-expressing cells (green fluorescence), number signs depict two small cysts, derived from non-targeted, tdTomato-expressing cells (red fluorescence). Red arrows depict eGFP-negative cells (no recombination). White arrows depict eGFP-positive cyst-lining cells (indicates recombination). Scale bar, 100 μm. (B) Analysis of TFAM expression by IF in Cre⁻ control and Six2-Tfam^−/− mutants at age P7. White arrows depict TFAM-positive tubular structures (red fluorescence); gl, glomerulus. Scale bar, 25 μm.

Supplemental Figure S3 (associated with Fig. 1). Tfam^−/− kidneys are characterized by increased proliferative activity. (A) Representative images of kidney sections from Cre⁻ littermate control and Six2-Tfam^−/− mice at age P14 analyzed for Ki67 expression by immunohistochemistry (IHC). Red arrows depict Ki67-positive cells in control and Tfam^−/− kidneys. Scale bar, 100 μm. (B) Immunoblot analysis of ERK, phospho-ERK (p-ERK) and β-catenin expression in whole kidney homogenates from Cre⁻ littermate control and Six2-Tfam^−/− mutant mice at age P14. (C) Cleaved caspase 3 expression in formalin-fixed, paraffin-embedded kidney sections from Cre⁻ littermate control and Six2-mT/mG; Tfam^−/− mice at age P14 analyzed by IHC. Red dots were placed over cleaved caspase 3-postive cells to illustrate tissue distribution at low-power magnification. Red arrows depict cleaved caspase 3-positive cells in high-power magnification images. Scale bars, 1 mm (top) and 100 μm (bottom). (D) Cilial axoneme labeling by immunofluorescence with anti-acetylated α-tubulin staining. Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Cre⁻ littermate control and Six2-Tfam^−/− mutant mice at age P14. #, ##, ### depict small, intermediate and large-sized cysts respectively. White arrows depict cilia. Scale bars, 100 μm (top) and 10 μm (bottom).

Supplemental Figure S4 (associated with Fig. 2). Inactivation of Tfam in SIX2 lineage inhibits nephron maturation. Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Cre⁻ littermate control and Six2-Tfam^−/− mutant mice at age P0, P7, and P14 (n=4-6). Section were analyzed by lectin histochemistry using lotus tetragonolobus (LTL) lectin and dolichos biflorus agglutinin (DBA) lectin. Wilms’ tumor 1 (WT1) protein expression was analyzed by immunofluorescence. Areas with LTL⁺ and DBA⁺ tubules were quantified with ImageJ, the number of glomeruli was counted manually. White arrows depict nephrons reacting with LTL or DBA, asterisks depict glomeruli. Scale bars, 100 μm. Data are represented as mean ± SEM and were analyzed by 2-tailed Student’s t-test; **P<0.01 and ***P<0.001.

Supplemental Figure S5 (associated with Fig. 2). Inactivation of Tfam in HOXB7 progenitor cells does not result in cyst development. (A) Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from 3-month-old heterozygous Hoxb7-Tfam^+/− and Hoxb7-Tfam^−/− mutant mice. Sections were stained with Masson’s trichrome (MTrichrome) and analyzed by immunofluorescence (IF) for tdTomato (tdT) and cytochrome oxidase IV (COXIV) expression. Number signs depict dilated tubules in MTrichrome- stained sections, asterisks depict tdT-positive HOXB7 progenitor cell-derived colleting ducts. (B) IF and RNA-FISH images of formalin-fixed, paraffin-embedded kidney sections from 3-month-old heterozygous Hoxb7-Tfam^+/− and Hoxb7-Tfam^−/− mutant mice. Sections were analyzed for tdT and AQP2 protein expression by IF and tdT RNA and mitochondrially encoded cytochrome c oxidase subunit 1 (mt-Co1) RNA expression by RNA-FISH. Asterisks depict tdT-expressing tubules (collecting ducts). In Hoxb7-Tfam^−/− mutant mice tdT-expressing tubules do not express AQP2 and mt-Co1. Scale bar, 100 μm. (C) Representative images of kidney sections from 15-month-old control and Hoxb7-Tfam^−/− mice stained with MTrichrome. Scale bar, 100 μm. Right panel, Blood urea nitrogen (BUN) from Cre⁻ littermate control mice and Hoxb7-Tfam^−/− mutants (n=6 each). Data are represented as mean ± SEM and were analyzed using 2-tailed Student’s t-test.

Supplemental Figure S6 (associated with Fig. 3). Lack of nephron segment marker expression in cysts from Six2-Tfam^−/− kidneys. Representative images of formalin-fixed, paraffin-embedded kidney sections from Six2-mT/mG;Tfam^−/− mice at age P14. Sections were analyzed by immunofluorescence with antibodies specific for enhanced green fluorescent protein (eGFP), megalin, uromodulin, thiazide-sensitive sodium chloride cotransporter (NCC) and aquaporin 2 (AQP2). Merged images are shown on the right. Arrows indicate tubular structures expressing the respective nephron segment markers. Scale bar, 100 μm.

Supplemental Figure S7 (associated with Fig. 4). Tfam^−/− epithelial cells are deficient in MT-CO1. (A) Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Six2-mT/mG;Tfam^−/− mice at age P7. Kidney sections were analyzed by immunofluorescence for the expression of enhanced green fluorescent protein (eGFP) and mitochondrially encoded cytochrome c oxidase subunit 1 (MT-CO1). eGFP expression indicates Six2-eGFP/Cre-mediated recombination of the mT/mG Cre-reporter allele. Asterisks depict eGFP-negative tubules (no recombination), which express MT-CO1; number signs depict eGFP-positive tubules (recombined), which do not express MT-CO1, indicating loss of TFAM function. Scale bar, 100 μm.

Supplemental Figure S8 (associated with Fig. 5). Inactivation of Tfam in SIX2 lineage cells alters the expression of metabolic genes. Genome-wide RNA expression analysis by RNAseq was performed with whole renal cortex isolated from Cre⁻ control littermate and Six2-Tfam^−/− mutant mice at age P7. Shown are heatmaps illustrating changes in the expression patterns of genes involved in oxidative phosphorylation, glycolysis, glucose transport, fatty acid metabolism and TCA cycle (n=4 each)..

Supplemental Figure S9 (associated with Fig. 6). TFAM expression is reduced in Cys^cpk/cpk renal cysts. (A) Shown are representative images of formalin-fixed, paraffin-embedded kidney sections from Cys^cpk/cpk mice at age P18. Sections were analyzed by RNA fluorescent in situ hybridization for mitochondrially encoded cytochrome c oxidase subunit 1 (mt-Co1) and mitochondrially encoded ATP synthase membrane subunit 6 (mt-Atp6) expression, by immunofluorescence (IF) for voltage-dependent anion-selective channel 1 (VDAC) expression and by lectin histochemistry with lotus tetragonolobus lectin (LTL). White arrows depict cyst-lining epithelial cells, dashed lines outline cyst-lining epithelial cells, number signs depict cyst lumina. Scale bars, 100 μm (low-power magnification) and 10 μm (high-power magnification). (B) 3D structured illumination microscopy (3D SIM) of littermate wild type kidney at age P18. Shown are representative images of kidney sections stained with LTL and analyzed by IF for cytochrome c oxidase subunit IV (COXIV) and VDAC expression. Scale bar, 10 μm (low-power magnification images) and 2 μm (high-power magnification images). Asterisk depicts an interstitial cell nucleus.

Supplemental Figure S10 (associated with Fig. 7). TFAM expression is decreased in renal cysts from patients with polycystic kidney disease. Relative TFAM expression levels in renal cysts from 5 patients with polycystic kidney disease were assessed by immunohistochemistry (n=5). Shown is the proportion of cysts with low or high TFAM expression in cyst-lining epithelium. The number of cysts counted per section is shown in white.

TRANSLATIONAL STATEMENT.

We have used mouse genetics to understand the role of mitochondrial dysfunction in kidney homeostasis. Inactivation of mitochondrial transcription factor TFAM in SIX2 epithelial progenitor cells resulted in renal failure due to severe polycystic kidney disease. Our findings demonstrate that progressive mitochondrial dysfunction is associated with defective epithelial differentiation and renal cystogenesis. Furthermore, we established that TFAM expression and mitochondrial number was reduced in human polycystic kidney tissue. Our studies suggest that therapeutic strategies, which aim at improving mitochondrial health may be beneficial in the treatment of patients with ADPKD.