Andrew Beenken

The proximal tubule maintains a robust endocytic pathway that is necessary for the recovery of proteins and vitamins from the ultrafiltrate and for the rapid regulation of ion transport. The large multiligand receptor megalin is essential to this process. In the absence of functional megalin, membrane and fluid flux through endocytic compartments is profoundly compromised, whereas the binding of filtered drugs to megalin is a major cause of nephrotoxicity. Insights into the structure of megalin offers clues as to how megalin might drive endocytic dynamics through pH-dependent conformational rearrangements, and findings from focused ion beam–scanning electron microscopy and machine learning-assisted segmentation challenge the traditional model of proximal tubule endocytosis and instead support a model in which a continuous network of dense apical tubules, together with the endoplasmic reticulum, regulates the control of ligand passage, fission and dissociation. In addition to its function as an endocytic receptor, megalin maintains proximal tubule function through other mechanisms. Genomic analyses and animal studies show that predicted alterations in megalin expression have complex effects on disease progression and protection. Consistent with these observations, megalin expression affects proximal tubule transcription and cell metabolism, with important consequences for disease pathology.

Donnai-Barrow syndrome (DBS) arises from loss-of-function (LoF) variants in the endocytic receptor low-density lipoprotein receptor–related protein 2 (LRP2; or megalin) and is characterized by low–molecular weight proteinuria and developmental abnormalities. Urinary proteomics of 9 patients with DBS revealed that the urinary proteome of a DBS patient with the missense variant LRP2 p.C1400R was indistinguishable from that of patients with splice site, nonsense, or frameshift mutations. A CRISPR mouse model of the variant was generated to determine the mechanism of LoF and proteinuria. The mutant LRP2 was expressed and observed to dimerize and localize to the proximal tubule apical membrane. However, both fluid-phase and receptor-mediated endocytosis was impaired in the context of a general perturbation of endocytic flux. Immunofluorescence revealed aberrant endocytic recycling with mislocalized RAB11 ⁺ and TFR1 ⁺ compartments and enlarged lysosomes. Structural modeling showed that the LRP2 assembly likely tolerates the cysteine-to-arginine substitution at the cell surface, but at endosomal pH the variant introduced steric clashes that may disrupt intramolecular interfaces and disturb receptor recycling. These findings point to the importance of LRP2 recycling for global endocytic flux and offer a blueprint for leveraging patient-specific alleles to dissect proximal tubule function.

Key Points: Transferrin receptor 1 is critical for perinatal nephron growth and maturation; its deletion results in widespread cystic hypodysplasia. Nontransferrin bound iron traffic is a physiologic source of iron in the midgestation embryo and complements transferrin receptor 1. Cystic hypodysplasia can be rescued by exogenous iron or by the activation of systemic iron traffic with hypoxia inducible factor activators. Background: Periconceptual maternal iron deficiency (FeD) is a worldwide cause of premature births and low birth weights. Yet, it is unknown whether FeD affects all developing tissues equally or rather target- s pecific lineages. In addition, since FeD restricts both transferrin bound and nontransferrin bound iron species, their unique contributions to organogenesis are indeterminant. Methods: To address questions of iron traffic and kidney development, we examined the deletion of the singular transferrin receptor ( TfR1−/− ), created green flourescent protein-labeled TfR1−/− embryonic stem cells for inoculation into wild blastocysts, and created TfR1 -floxed mice to generate cell autonomous deletions of TfR1 in mesenchymal, ureteric, and stromal derivatives. Finally, we created a model of global FeD with iron poor diets, for comparison with cell autonomous TfR1 deletions. Results: Transferrin receptor deletions only modestly suppressed tubulogenesis, had little, if any effect on the growth of the ureteric bud and no gross effects on kidney stroma at mid gestation. By contrast, nutritional FeD nearly abolished kidney development, highlighting the limited phenotypes induced by transferrin receptor deletion. Yet, in the second postnatal week, the critical function of TfR1 became evident by the growth of residual TfR1+ cells that had escaped Cre-mediated deletion and by tubular segment-specific polycystic transformation. Timed treatment with iron or systemic activators of iron trafficking prevented both cystic dysplasia and the terminal loss of kidney function, reversing extensive malformations of the kidney. Conclusions: TfR1 is the critical iron species targeting postnatal tubulogenesis, but in the embryo, TfR1 must be complemented by alternative iron species called nontransferrin bound iron. Iron-deficient kidney disease is reversible postnatally.

Spns1 mediates a novel iron conductance essential during embryogenesis. Spns1 knockout leads to endocytic and lysosomal defects, accompanied by iron overload in the kidney. Reversal of iron overload by restricting dietary iron or by concurrent knockout of the iron transporter, DMT1 rescued the endocytic and organellar defects and reverted markers of iron overload. These data suggest feedback between iron and proximal tubule endocytosis.

Key Points: We conducted a clinical, genetic, and pathological analysis on 64 cases from 39 families with TRPC6-associated podocytopathy (TRPC6-AP). Analysis of 37,542 individuals excluded a major contribution of loss-of-function variants to TRPC6-AP, legitimating current drug discovery approaches. This study identifies key features of disease that can help intervention studies design and suggests similarities between TRPC6-AP and primary FSGS. Background: Understanding the genetic basis of human diseases has become integral to drug development and precision medicine. Recent advancements have enabled the identification of molecular pathways driving diseases, leading to targeted treatment strategies. The increasing investment in rare diseases by the biotech industry underscores the importance of genetic evidence in drug discovery and approval processes. Here we studied a monogenic Mendelian kidney disease, TRPC6-associated podocytopathy (TRPC6-AP), to present its natural history, genetic spectrum, and clinicopathological associations in a large cohort of patients with causal variants in TRPC6 to help define the specific features of disease and further facilitate drug development and clinical trials design. Methods: The study involved 64 individuals from 39 families with TRPC6 causal missense variants. Clinical data, including age of onset, laboratory results, response to treatment, kidney biopsy findings, and genetic information, were collected from multiple centers nationally and internationally. Exome or targeted sequencing was performed, and variant classification was based on strict criteria. Structural and functional analyses of TRPC6 variants were conducted to understand their effect on protein function. In-depth reanalysis of light and electron microscopy specimens for nine available kidney biopsies was conducted to identify pathological features and correlates of TRPC6-AP. Results: Large-scale sequencing data did not support causality for TRPC6 protein-truncating variants. We identified 21 unique TRPC6 missense variants, clustering in three distinct regions of the protein, and with different effects on TRPC6 3D protein structure. Kidney biopsy analysis revealed FSGS patterns of injury in most cases, along with distinctive podocyte features including diffuse foot process effacement and swollen cell bodies. Most patients presented in adolescence or early adulthood but with ample variation (average 22, SD ±14 years), with frequent progression to kidney failure but with variability in time between presentation and kidney failure. Conclusions: This study provides insights into the genetic spectrum, clinicopathological associations, and natural history of TRPC6-AP. Clinical Trial registry name and registration number: A Study to Test BI 764198 in People With a Type of Kidney Disease Called Focal Segmental Glomerulosclerosis, NCT05213624.

The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase ( Uprt ) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU–tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.