Disruption of mitochondrial complex III in cap mesenchyme but not in ureteric progenitors results in defective nephrogenesis associated with amino acid deficiency

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)–expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4–mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.


MATERIALS and METHODS
DNA and RNA analysis. Genomic DNA analysis was performed as previously described. 1 RNA was isolated using the RNeasy kit according to the manufacturer's protocol (Qiagen; Hilden, Germany).
Quantitative real-time PCR (qPCR) was performed as described previously. 2 18S rRNA was used for normalization. For the quantification of mRNA expression, the relative standard curve method was used; separate standard curves were generated for each qPCR run. Primer sequences used for the detection of Aqp1, Aqp2, Lhx1, nephrin, podocin, Napi2a, Ncc, Nkcc2, Scnn1a, Six2 and Trpv5 transcripts have been described previously, 3 otherwise primer sequences are listed in Supplementary Table S1.
For RNA FISH, Wnt4 or Qpc were detected in formalin-fixed, paraffin-embedded kidney tissue sections from Six2-Qpc -/and Hoxb7-Qpc -/mice, respectively, using the RNAscope ® Multiplex Fluorescent kit or Basescope™ Colormetric kit according to the manufacturer's instructions (Advanced Cell Diagnostics; Newark, USA). Tissue slides were imaged on an Apiro Versa 200 automated slide scanner (Leica Biosystems; Wetzlar, Germany). RNA sequencing analysis was performed at age E18. 5  Metabolite analysis. For amino acid and nucleotide analysis, snap-frozen kidneys from Crelittermate control and Six2-Qpc -/pups at age P0 were pooled and homogenized in cold water/methanol (9:1) containing 50 mM ammonium acetate (pH 6). A portion of the homogenate (100 μL) was combined with HPLC-grade methanol (300 μL) vortexed and centrifuged at 10,000 x g for 10 min at 4 °C to remove precipitated proteins and other insoluble debris. The supernatant was transferred to a clean tube, dried and reconstituted in 100 μl of acetonitrile/water (2:1) containing 250 μM tyrosine (phenyl-3,5-d2) internal standard. Amino acid species were measured by a targeted HILIC-MS/MS method.
Individual reference standards of all analytes were infused into the mass spectrometer for the optimization of electrospray ionization (ESI) and selected reaction monitoring (SRM) parameters.
LC-MS analysis was performed using an Acquity UPLC system (Waters; Milford, MA) interfaced with a TSQ Quantum Ultra triple-stage quadrupole mass spectrometer (Thermo Scientific, San Jose, CA).
The mass spectrometer was equipped with an IonMax source housing and a heated ESI probe.
Detection was based on SRM using the following optimized source parameters (positive ionization):