Géraldine Mollet

Proteinuria contributes to the progression of chronic kidney disease and causes protein overload in proximal tubular epithelial cells (PTECs), fibrosis and inflammation. While protein overload is suggested to stimulate PTEC proliferation, its role in inducing cellular senescence remains unclear. This study investigated the effects of protein overload on PTEC proliferation and senescence using both in vivo and in vitro models. Experiments were performed using podocin-knockout (KO) (proteinuric model) and megalin-cubilin-podocin KO mice (proteinuric mice without endocytosis, triple KO). Human immortalized proximal tubular cells (RPTEC/TERT1) were incubated with human serum albumin (HSA) with and without fatty acids. Mouse kidneys and cells were analyzed by western blotting, quantitative RT-PCR, and immunofluorescence. In RPTEC/TERT1 cells, treatment with HSA carrying fatty acids induced proliferation and senescence. In vivo, proteinuric podocin-KO mice exhibited a significant increase in proliferation and senescence markers, particularly within the outer medulla. This response was markedly attenuated in triple-KO mice. Proliferation and senescence occurred in both receptor positive and negative cells. Analyses of cytokines, known to stimulate proliferation (PDGF-B) and senescence (TGFβ and MCP-1), were increased in podocin KO, but diminished in triple KO mice. Mechanistically, TGF-β receptor inhibition prevented HSA-induced senescence, whereas recombinant TGF-β sufficed to induce the phenotype in RPTEC/TERT1. In conclusion, excessive endocytosis of filtered proteins induces a changed cytokine environment with impact for PTEC proliferation and cellular senescence, particularly within the outer medulla.

N ⁶ -threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t ⁶ A) is a universal modification essential for translational accuracy and efficiency. The t ⁶ A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. Structures of the subunits and subcomplexes of human KEOPS are known, but knowledge on the detailed interactions with tRNA is lacking. We present here the first structure of complete hKEOPS and of its complex with a substrate tRNA by cryo-electron microscopy. The CAA tail of tRNA is bound to the TPRKB subunit and the anti-codon loop is positioned at the entrance of the catalytic site of OSGEP subunit. The flexibility of the OSGEP-TP53RK interface allows hKEOPS to fit the surface of the tRNA elbow. We recently identified mutations in all genes encoding for proteins of the t ⁶ A pathway in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. We here expressed and characterized the majority of the Galloway Mowat mutants. All mutants could be purified at high yields and seem to be stable in vitro . The t ⁶ A activity for most of the mutants is above 40% of the WT. Using CRISPR-Cas9 technology we replaced the genes encoding the t ⁶ A pathway proteins in yeast by their human homologues. This yeast construct was perfectly viable and produced WT levels of t ⁶ A modified tRNA. Using this tool, we observed that most of the GAMOS mutants were viable in yeast and yielded comparable t ⁶ A modified tRNA levels. Our data indicate that healthy human cellular development depends on an optimized level of t ⁶ A tRNA modification and is not compatible with a total loss of function of the t ⁶ A machinery.

Kidney organoids are an emerging tool for disease modeling, especially genetic diseases. Among these diseases, X-linked Alport syndrome (XLAS) is a hematuric nephropathy affecting the glomerular basement membrane (GBM) secondary to pathogenic variations in the COL4A5 gene encoding the α5 subunit of type IV collagen [α5(IV)]. In patients carrying pathogenic variations affecting splicing, the use of antisense oligonucleotides (ASOs) offers immense therapeutic hope. In this study, we develop a framework combining the use of patient-derived cells and kidney organoids to provide evidence of the therapeutic efficacy of ASOs in XLAS patients. Using multiomics analysis, we describe the development of GBM in WT and mutated human kidney organoids. We show that GBM maturation is a dynamic process, which requires long organoid culture. Then, using semi-automated quantification of α5(IV) at basement membranes in organoids carrying the splicing variants identified in patients, we demonstrate the efficacy of ASO treatment for α5(IV) restoration. These data contribute to our understanding of the development of GBM in kidney organoids and pave the way for a therapeutic screening platform for patients.

Significance Statement: Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro/ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo. In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. Background: Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. Methods: Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro/ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. Results: The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. Conclusions: In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. Clinical Trial registry name and registration number: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748.

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2 , encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03–mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9–mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches.

The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

Several studies have reported WDR73 mutations to be causative of Galloway–Mowat syndrome, a rare disorder characterised by the association of neurological defects and renal-glomerular disease. In this study, we demonstrate interaction of WDR73 with the INTS9 and INTS11 components of Integrator, a large multiprotein complex with various roles in RNA metabolism and transcriptional control. We implicate WDR73 in two Integrator-regulated cellular pathways; namely, the processing of uridylate-rich small nuclear RNAs (UsnRNA), and mediating the transcriptional response to epidermal growth factor stimulation. We also show that WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins. Altogether, our results suggest that a range of cellular pathways are perturbed by WDR73 loss-of-function, and support the consensus that proper regulation of UsnRNA maturation, transcription initiation and cell cycle control are all critical in maintaining the health of post-mitotic cells such as glomerular podocytes and neurons, and preventing degenerative disease.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1 , NOP10 , or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin–NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin–NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.