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Kidney epithelial targeted mitochondrial transcription factor A deficiency results in progressive mitochondrial depletion associated with the severe cystic disease--Part II

Ken Ishii^1,2,11 et al.

DISCUSSION

Here we establish a critical function for mt transcription factor TFAM in renal tissue homeostasis. We demonstrate that the 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 toward glycolysis. Furthermore, a decrease in cellular TFAM levels and mt dysfunction are characteristic features of murine and human PKD (polycystic kidney disease), 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 the tubulointerstitial disease, mutations in TFAM itself have not been reported in patients with chronic kidney disease. Nevertheless, chronic kidney disease 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 inflammation14 but not a cystic disease. The phenotypic differences between the 2 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 medullary thick ascending limb of Henle segment and ureteric bud–derived CD,25 whereas Six2-eGFP/Cre is expressed in cap mesenchyme and does not target ureteric bud–derived nephron segments.16 Consistent with these findings is the increase in extracellular matrix deposition and absence of cystic disease in 15-month-old Hoxb7-Tfam /mutants; Hoxb7-Cre targets ureteric bud–derived nephron segments (Supplementary Figure S5).26 Furthermore, in keeping with the notion of the 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.

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Defects in nephron differentiation were not completely unexpected in Six2-Tfam^-/- mice because 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^-/-mutants warrants further investigation. Recent studies have shown that mutations in PKD (polycystic kidney disease)1, which are responsible for w85% of ADPKD cases,29 are associated with enhanced glycolytic flux.30 However, the pathophysiologic and therapeutic significance of this finding is not entirely clear because 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 (polycystic kidney disease), 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 Six2-Tfam^-/- tissues share molecular features with PKD (polycystic kidney disease) 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 (polycystic kidney disease) animal models,35,36 cilia are formed in Pkd1^-/-epithelial cells37 and were also detected in renal cysts from Six2-Tfam^-/- mice (Supplementary Figure S3). Several signaling pathways linked to cystogenesis are involved in cilia-associated signaling. These include mitogen-activated protein kinase/extracellular signal-regulated kinase signaling and b-catenin–regulated pathways.33 Both p-ERK and b-catenin levels were elevated in Six2-Tfam^-/- kidneys, suggesting that these pathways were activated. These findings are consistent with observations made in human ADPKD cells and in several murine PKD (polycystic kidney disease)models.38–43.

Peroxisome proliferator-activated receptor-gamma coactivator 1a (PGC-1a), 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 (polycystic kidney disease). A reduction in PGC-1a expression has been proposed to promote cyst proliferation due to increased mt superoxide production in PKD (polycystic kidney disease)1-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 an increase in mt ROS production.9 In addition to the PGC-1a/TFAM axis, recent studies have highlighted a potential role for hypoxia and the hypoxia-inducible factor 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 (polycystic kidney disease). Our findings provide a strong rationale for further investigations into the role of mt health and function in cystogenesis. We propose that therapeutic strategies that aim at improving mt health may be beneficial for the treatment of patients with PKD (polycystic kidney disease).

Figure 7 | Mitochondrial transcription factor A (TFAM) expression in renal cysts from patients with polycystic kidney disease is reduced. (a) Representative images of formalin-fixed paraffin-embedded sections from normal human kidneys and kidneys from polycystic kidney disease (PKD) patients analyzed by immunohistochemistry for TFAM expression, by immunofluorescence (IF) for voltage-dependent anion-selective channel 1 (VDAC) expression, and by RNA fluorescent 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 identify cyst lining epithelial cells, number signs depict cyst lumina, and asterisks depict glomeruli. Bar =100 mm for low-magnification images and 10 mm for high-magnification images. (b) Representative 3-dimensional structured illumination microscopic images of human PKD (polycystic kidney disease) kidney sections analyzed with IF for VDAC expression. 4', 6-diamidino-2-phenylindole (DAPI) 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). Bar = 4 mm. Data are represented as mean +-SEM and were analyzed using the Student's t-test. *P < 0.05. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org

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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 the Supplementary Methods and Materials section. 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) using the Student's t-test. Survival was analyzed using the Kaplan-Meier method, and groups were compared by the 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 National Institutes of Health guidelines for the use and for the care of live animals and were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

DISCLOSURE

All the authors declared no competing interests.

ACKNOWLEDGMENTS

VHH is supported by the Krick-Brooks chair in Nephrology at Vanderbilt University, National Institutes of Health grants R01-DK101791 and R01-DK081646, and a Department of Veterans Affairs Merit Award 1I01BX002348. Further support was provided by National Institutes of Health grants R01-DK103033 (PVT), R01-DK108433 (MS), and R01-DK56942 (ABF); Vanderbilt’s O’Brien Kidney Center (P30-DK114809); Vanderbilt’s Diabetes Research and Training Center (P30-DK20593); the Digital Histology Shared Resource core at Vanderbilt University Medical Center (www.mc.vanderbilt.edu/dhsr); the Translational Pathology Shared Resource core (P30-CA68485); the Vanderbilt Mouse Metabolic Phenotyping Center (U24-DK059637); and the Shared Instrumentation grant S10-OD023475. Information about work performed in the Haase lab can be found at www.haaselab.org.

AUTHOR CONTRIBUTIONS

VHH conceived the project. KI, HK, and VHH designed the research studies, analyzed and interpreted data, wrote the manuscript, and made figures. KI, HK, NG, KT, AL, CT, OD, and CRB performed experiments and acquired and analyzed data. MS, NSC, and PVT provided mouse reagents and mouse tissues and conceptual input and assisted in the interpretation of data. ABF and MEK provided human tissues.

Cistanche is good for polycystic kidney disease

SUPPLEMENTARY MATERIAL

Supplementary File (PDF)

Figure S1. Associated with Figure 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 β/ mice at (A) 3 months of age and (B) >10 months of age. Sections were stained with alcian blue/periodic acid–Schiff (AB-PAS) and analyzed by immunohistochemistry (IHC) for a smooth muscle actin (ACTA2) expression. Asterisks depict glomeruli. Bars ¼ 100 mm. Right panels show blood urea nitrogen (BUN) levels and renal mt DNA content in Cre littermate control and Six2-Tfamþ/mutant mice at 3 months of age (n ¼ 5 and 6, respectively) and age>10 months (n =4 and 3, respectively). Data are represented asmean0.01. SEM and were analyzed by the 2-tailed Student’s t-test; **P <0.01.

Figure S2. Associated with Figure 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 the 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 eGFP-expressing cells (green fluorescence); number signs depict 2 small cysts derived from nontargeted, tdTomatoexpressing cells (red fluorescence). Red arrows depict eGFP-negative cells (no recombination). White arrows depict eGFP-positive cyst lining cells (indicates recombination). Bar=100 mm. (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. Bar=25 μm.

Figure S3. Associated with Figure 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 were analyzed for Ki67 expression by immunohistochemistry (IHC). Red arrows depict Ki67-positive cells in control and Tfam^-/- kidneys. Bar =100 mm. (B) Immunoblot analysis of ERK, phospho-ERK (p-ERK), and b-catenin expression in whole kidney homogenates from Cre littermate control and Six2- Tfam / mutant mice at age P14. (C) Cleaved caspase 3 expressions 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–positive cells to illustrate tissue distribution at low-power magnification. Red arrows depict cleaved caspase 3–positive cells in high-power magnification images. Bars =1 mm (top) and 100 mm (bottom). (D) Cilial axoneme labeling by immunofluorescence with anti-acetylated a-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. Bars =100 mm (top) and 10 mm (bottom).

Figure S4. Associated with Figure 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). The section was 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 LTLβ and DBAβ tubules were quantified with ImageJ (National Institutes of Health, Bethesda, MD); the number of glomeruli was counted manually. White arrows depict nephrons reacting with LTL or DBA, and asterisks depict glomeruli. Bars ¼ 100 mm. Data are represented as mean SEM and were analyzed by a 2-tailed Student’s t-test. **P < 0.01. ***P < 0.001.

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Figure S5. Associated with Figure 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-Tfamþ/ and Hoxb7- Tfam^-/- mutant mice. Sections were stained with Masson trichrome (MTrichrome) and analyzed by immunofluorescence (IF) for tdTomato (TDT) and cytochrome oxidase IV (COX IV) expression. Number signs depict dilated tubules in MTrichrome- stained sections, and asterisks depict tdT-positive HOXB7 progenitor cell-derived collecting ducts. (B) IF and RNA fluorescent in situ hybridization (RNA-FISH) images of formalin-fixed, paraffin-embedded kidney sections from 3-month-old heterozygous Hoxb7-Tfamβ^-/- 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 TD expressing tubules (collecting ducts). In Hoxb7-Tfam^-/- mutant mice tdT-expressing tubules do not express AQP2 and mt-Co1. Bar =100 mm. (C) Representative images of kidney sections from 15-month-old control and Hoxb7-Tfam^-/-mice stained with MTrichrome. Bar =100 mm. 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 a 2-tailed Student's t-test.

Figure S6. Associated with Figure 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. Bar=100 μm.

Figure S7. Associated with Figure 4. Team^-/- 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. Bar =100 μm.

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

Figure S9. Associated with Figure 6. TFAM expression is reduced in Cyscpk/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, and number signs depict cyst lumina. Bars ¼ 100 mm (low-power magnification) and 10 mm (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 (COX IV) and VDAC expression. Bar ¼ 10 mm (low-power magnification images) and 2 mm (high-power magnification images). Asterisk depicts an interstitial cell nucleus.

Figure S10. Associated with Figure 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.

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Excerpted from: ' Kidney epithelial targeted mitochondrial transcription factor A deficiency results in progressive mitochondrial depletion associated with severe cystic disease ' by Ken Ishii1,2,11 et al.

---Kidney International (2021) 99, 657–670

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