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.

Keywords: TFAM; glycolysis; kidney development; mitochondria; polycystic kidney disease.

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.

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.

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.

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.

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.

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.

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.