Mammalian Kidney Research Articles

Hedgehog-dependent and hedgehog-independent roles for growth arrest specific 1 in mammalian kidney morphogenesis.

Growth arrest specific 1 (GAS1) is a key regulator of mammalian embryogenesis, best known for its role in hedgehog (HH) signaling, but with additional described roles in the FGF, RET, and NOTCH pathways. Previous work indicated a later role for GAS1 in kidney development through FGF pathway modulation. Here, we demonstrate that GAS1 is essential for both mesonephrogenesis and metanephrogenesis - most notably, Gas1 deletion in mice results in renal agenesis in a genetic background-dependent fashion. Mechanistically, GAS1 promotes mesonephrogenesis in a HH-dependent fashion, performing a unique co-receptor function, while promoting metanephrogenesis in a HH-independent fashion, acting as a putative secreted RET co-receptor. Our data indicate that Gas1 deletion leads to renal agenesis through a transient reduction in metanephric mesenchyme proliferation - a phenotype that can be rescued by exogenous RET pathway stimulation. Overall, this study indicates that GAS1 contributes to early kidney development through the integration of multiple different signaling pathways.

Carbonic anhydrase, water dissociation and physiology of Kidney.

Carbonic anhydrase (CA) is a zinc metalloenzyme widely distributed throughout the tissues of the body. In the kidney, this enzyme is thought to play a pivotal role in urinary acidification and bicarbonate reabsorption. Two distinct isozymes of carbonic anhydrase have now been identified in the mammalian kidney. A soluble cytoplasmic form, similar if not identical to human erythrocyte carbonic anhydrase C, accounts for the bulk of the renal carbonic anhydrase activity.

Comparative single-cell analyses identify shared and divergent features of human and mouse kidney development.

The mammalian kidney maintains fluid homeostasis through diverse epithelial cell types generated from nephron and ureteric progenitor cells. To extend a developmental understanding of the kidney's epithelial networks, we compared chromatin organization (single-nuclear assay for transposase-accessible chromatin sequencing [ATAC-seq]; 112,864 nuclei) and gene expression (single-cell/nuclear RNA sequencing [RNA-seq]; 109,477 cells/nuclei) in the developing human (10.6-17.6weeks; n= 10) and mouse (post-natal day [P]0; n= 10) kidney, supplementing analysis with published mouse datasets from earlier stages. Single-cell/nuclear datasets were analyzed at a species level, and then nephron and ureteric cellular lineages were extracted and integrated into a common, cross-species, multimodal dataset. Comparative computational analyses identified conserved and divergent features of chromatin organization and linked gene activity, identifying species-specific and cell-type-specific regulatory programs. In situ validation of human-enriched gene activity points to human-specific signaling interactions in kidney development. Further, human-specific enhancer regions were linked to kidney diseases through genome-wide association studies (GWASs), highlighting the potential for clinical insight from developmental modeling.

The scaffold protein IQGAP1 promotes reorientation of epithelial cell polarity at the two-cell stage for cystogenesis.

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen-harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two-cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM-facing and cell-cell-contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM-abutting periphery and subsequent apical vesicle delivery to a cell-cell contact site for lumen formation. Here, we show that downstream of the ECM-signal-transducer β1-integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin-Darby canine kidney (MDCK) cells at a later two-cell stage in three-dimensional culture. Rac1-GTP facilitates IQGAP1 interaction with the Rac-specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1-GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin-mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two-cell stage via its interaction with IQGAP1.

Actin cytoskeleton and associated myosin motors within the renal epithelium.

This review highlights the complexity of renal epithelial cell membrane architectures and organelles through careful review of ultrastructural and physiological studies published over the past several decades. We also showcase the vital roles played by the actin cytoskeleton and actin-associated myosin motor proteins in regulating cell type-specific physiological functions within the cells of the renal epithelium. The purpose of this review is to provide a fresh conceptual framework to explain the structure-function relationships that exist between the actin cytoskeleton, organelle structure, and cargo transport within the mammalian kidney. With recent advances in technologies to visualize the actin cytoskeleton and associated proteins within intact kidneys, it has become increasingly imperative to reimagine the functional roles of these proteins in situ to provide a rationale for their unique, cell type-specific functions that are necessary to establish and maintain complex physiological processes.

Development of Recombinant Antibodies and Its Application in Immunomagnetic Separation-Based Rapid Detection of Vibrio cholerae in Aquatic Environments.

Cholera caused by Vibrio cholerae remains a major public health concern in many countries. The greatest obstacle to detection of V. cholerae contamination in drinking water or aquatic environments mainly relates to sample preparation steps, especially the enrichment step. In this study, immunomagnetic separation methods were developed based on sequence-defined recombinant antibodies (rAbs) against V. cholerae, then used for the specific and efficient enrichment of V. cholerae in water samples. Using the variable region genes of the anti-V. cholerae monoclonal antibodies (mAbs) 5F2, the full-length IgG rAbs (R5F2) were produced using mammalian human embryonic kidney 293T cells. Two antibodies, 5F2 and R5F2, were used to prepare immunomagnetic beads (IMBs), and their capture efficiencies (CEs) were evaluated. The results showed that 0.4 mg of 5F2-IMBs and R5F2-IMBs exhibited good CEs (96.0% and 75.9%, respectively) against V. cholerae within 40 min. The IMBs could still effectively capture V. cholerae in large-volume reaction systems (5 ml to 25 ml). The CEs of 5F2-IMBs and R5F2-IMBs ranged from 90.2% to 70.7% and 65.1% to 44.2%, respectively. Furthermore, 5F2-IMBs and R5F2-IMBs did not show significant cross-reactivity with other bacteria and exhibited high specificity. When R5F2-IMS was used in combination with quantitative real-time PCR, the detection limit was approximately 5 colony-forming units/25 ml after enrichment for 4 h. Our results suggest that the rAbs produced herein could provide useful alternatives to traditional hybridoma-based antibodies for accurate detection of V. cholerae in food safety and environmental monitoring.

Cadherin adhesion complexes direct cell aggregation in the epithelial transition of Wnt-induced nephron progenitor cells.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a β-catenin (Ctnnb1)-driven, transcriptional nephrogenic program and the mesenchymal to epithelial transition (MET) of NPCs. Using an in vitro mouse NPC culture model, we observed that activation of the Wnt pathway results in the aggregation of induced NPCs, which is an initiating step in the MET program. Genetic removal showed aggregation was dependent on β-catenin. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+ dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in NPCs, and the β-catenin directed upregulation of Cdh3 and Cdh4 accompanying the MET of induced NPCs. Mutational analysis of β-catenin supported a role for a Lef/Tcf-β-catenin-mediated transcriptional response in the cell aggregation process. Genetic removal of all four cadherins, and independent removal of α-catenin or of β-catenin-α-catenin interactions, abolished aggregation, but not the inductive response to Wnt pathway activation. These findings, and data in an accompanying article highlight the role of β-catenin in linking transcriptional programs to the morphogenesis of NPCs in mammalian nephrogenesis.

Paracellular barriers: Advances in assessing their contribution to renal epithelial function

Regulation of salt and water balance occupies a dominant role in the physiology of many animals and often relies on the function of the renal system. In the mammalian kidney, epithelial ion and water transport requires high degree of coordination between the transcellular and paracellular pathways, the latter being defined by the intercellular tight junctions (TJs). TJs seal the paracellular pathway in a highly specialized manner, either by forming a barrier against the passage of solutes and/or water or by allowing the passage of ions and/or water through them. This functional TJ plasticity is now known to be provided by the members of the claudin family of tetraspan proteins. Unlike mammalian nephron, the renal structures of insects, the Malpighian tubules, lack TJs and instead have smooth septate junctions (sSJs) as paracellular barrier forming junctions. Many questions regarding the molecular and functional properties of sSJs remain open but research on model species have begun to inform our understanding. The goal of this commentary is to highlight key concepts and most recent findings that have emerged from the molecular and functional dissection of paracellular barriers in the mammalian and insect renal epithelia.

Nephrotoxicity of Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) on Mammalian Kidney: Wistar Rat as a Model Assessment

Naturally derived cyanotoxins, cylindrospermopsin (CYN), and microcystin-LR (MC-LR) have shown hepatotoxic and nephrotoxic effects in several studies. The present study aimed to determine the possible nephrotoxicity of MC-LR and CYN on mammalian kidneys using male Wistar rats as an animal model. Potential nephrotoxicity was evaluated at different doses of CYN (0.175 μg.kg-1, 0.140 μg.kg-1, 0.105 μg.kg-1) and MC-LR (0.105 μg.kg-1, 0.070 μg.kg-1, 0.035 μg.kg-1) was observed. Water samples from dug wells contaminated with CYN (0.161 μg.kg-1) and MC-LR (0.091 μg.kg-1) from the Padaviya area in Anuradhapura, Sri Lanka were used as environmental samples. The control groups were treated with distilled water. The exposure time of rats to the toxin was 90 days. Evaluation of urinary creatinine, serum creatinine, and Kidney Injury Molecule-1 (KIM-1) were estimated using standard protocols. A significant increase in serum creatinine levels was observed in all CYN and MC-LR treated groups (p<0.05) after 7 and 42 days of exposure, respectively, compared to control. It was found a decrease of urine creatinine when rats were treated with different concentrations of CYN and MC-LR (p<0.05) after 7 days compared to the control. The highest KIM-1 concentrations were recorded at 0.175 μg.kg-1 of CYN and 0.105 μg.kg-1 of MC-LR. The concentrations of KIM-1 in the control groups for CYN-treated and MC-LR-treated were not detected. Luminal protein, nuclear pyknosis, mild tubular epithelial swelling, vascular congestion, and interstitial inflammation in CYN and MC-LR treated groups were common. No predominant changes were observed in the control groups treated with CYN and MC-LR. The results of the present study confirm that the consumption of CYN and MC-LR-contaminated water may lead to kidney injury in Wistar rats.

With No Lysine (K) Kinases and Sodium Transporter Function in Solute Exchange with Implications for BP Regulation as Elucidated through Drosophila.

Like other multicellular organisms, the fruit fly Drosophila melanogaster must maintain homeostasis of the internal milieu, including the maintenance of constant ion and water concentrations. In mammals, the with no lysine (K) (WNK)-Ste20-proline/alanine rich kinase/oxidative stress response 1 kinase cascade is an important regulator of epithelial ion transport in the kidney. This pathway regulates SLC12 family cotransporters, including sodium-potassium-2-chloride, sodium chloride, and potassium chloride cotransporters. The WNK-Ste20-proline/alanine rich kinase/oxidative stress response 1 kinase cascade also regulates epithelial ion transport via regulation of the Drosophila sodium-potassium-2-chloride cotransporter in the Malpighian tubule, the renal epithelium of the fly. Studies in Drosophila have contributed to the understanding of multiple regulators of WNK pathway signaling, including intracellular chloride and potassium, the scaffold protein Mo25, hypertonic stress, hydrostatic pressure, and macromolecular crowding. These will be discussed together, with implications for mammalian kidney function and BP control.

Roles of myosin 1e and the actin cytoskeleton in kidney functions and familial kidney disease.

Mammalian kidneys are responsible for removing metabolic waste and maintaining fluid and electrolyte homeostasis via selective filtration. One of the proteins closely linked to selective renal filtration is myosin 1e (Myo1e), an actin-dependent molecular motor found in the specialized kidney epithelial cells involved in the assembly and maintenance of the renal filter. Point mutations in the gene encoding Myo1e, MYO1E, have been linked to familial kidney disease, and Myo1e knockout in mice leads to the disruption of selective filtration. In this review, we discuss the role of the actin cytoskeleton in renal filtration, the known and hypothesized functions of Myo1e, and the possible explanations for the impact of MYO1E mutations on renal function.

Cellular Mechanisms for pH Regulation and Energy Metabolism in Sharks and Rays

Similar to mammalian kidney distal tubule cells, specialized shark and ray gill epithelial cells maintain blood pH homeostasis. These shark and ray gill cells are either enriched with the Na+/K+-ATPase (NKA) and responsible for acid secretion or enriched for the vacuolar H+-ATPase (VHA) and responsible for base secretion. Moreover, both cell types contain abundant mitochondria and glycogen, and glycogen content decreases in active acid-base regulatory cells in vivo. Additional experiments with isolated gill cells in vitro show that glycogen coincides with the subcellular localization of NKA and VHA. Under control conditions, glycogen and NKA are at the cell membrane of acid-secreting cells while glycogen and VHA are in the cytoplasm of base-secreting cells. During exposure to alkalosis, NKA and glycogen localization remains unchanged in acid-secreting cells; however, both VHA and glycogen translocate to the cell membrane of base-secreting cells. Interestingly, this process is prevented by pharmacological inhibition of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC). This suggests a novel mechanism, a coupling of glycogen metabolism with acid-base regulation that sustains ATPase function during sudden periods of increased energy demand. Since VHA, sAC, and glycogen are evolutionarily widespread, this mechanism is likely to apply broadly to other organisms and systems, perhaps including chronic kidney disease. APS Porter Predoctoral Fellowship and National Science Foundation Postdoctoral Research Fellowship in Biology #1709911 to JR, National Science Foundation Grant IOS 1354181 to MT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multimodal characterization of sexual dimorphism in the mammalian kidney

Multimodal characterization of sexual dimorphism in the mammalian kidney

Toxins profiles, toxicological properties, and histological alteration potentiality of Trimeresurus erythrurus venom: In vitro and in vivo experiments

ObjectivesTrimeresurus erythrurus (Red-tailed bamboo pit viper) contributes significantly to snakebite envenoming cases in Bangladesh, India, Myanmar, and Nepal. However, the absence of specific antivenom and challenging socio-economic conditions in these regions necessitate the development of symptom-based medication to mitigate the severity of T. erythrurus envenoming. Therefore, documenting the effects of T. erythrurus venom on mammalian organs can establish a foundation for further research on symptom-based medication. MethodsWe profiled T. erythrurus venom toxins using SDS-PAGE. Additionally, we assessed the venom's toxicological properties through lethality, hemorrhagic, edematogenic, hemolytic, and phospholytic assays. Furthermore, we examined the histological alterations induced by crude venom on various organs of mice. ResultsSDS-PAGE profiling confirmed the presence of Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs), Phospholipase A2 (PLA2), L-amino Acid Oxidases (LAAOs), Myotoxins, and Disintegrins as toxic components in T. erythrurus venom. The venom exhibited a Median Lethal Dose (LD50) of 1.131 mg/kg, Minimum Hemorrhagic Dose (MHD) of 0.21 mg/kg, Minimum Edematogenic Dose (MED) of 0.17 mg/kg, and Median Hemolytic Dose (HD50) of 2.527 mg/L. Moreover, the venom induced significant pathological changes in mammalian skin, skeletal muscle, lung, heart, kidney, and intestine. ConclusionsThis study elucidates the major toxins, critical toxicological indices, and histopathological effects of T. erythrurus venom on mammalian organs. These findings provide valuable insights on the development of effective treatment strategies for pathophysiological complications that could manifest post T. erythrurus envenoming.

The synthetic peptide, PnPP-15, derived from the PnTx2-6 toxin of the spider Phoneutria nigriventer, induces peripheral antinociception involving neprilysin, opioid, and cannabinoid systems

BackgroundThe PnTx2–6 toxin is isolated from the Phoneutria nigriventer spider venom. The synthetic peptide PnPP-19 is derived from this toxin and induces antinociception. When topically applied, it is cleaved by proteases generating a 15 amino acid residues peptide, PnPP-15. PnPP-15 showed an antinociceptive effect in neuropathic pain developed by streptozotocin-induced diabetic mice. The present work aimed to investigate if PnPP-15 is also effective against the inflammatory pain induced by PGE2. We also evaluated its cytotoxicity in vitro and studied its possible action mechanism in the pain pathway. MethodsThe nociceptive threshold was assessed using the mechanical paw pressure test. Paw tissue was collected for protein extraction and western blot analyses. The FRET enzymatic assay measured neprilysin activity. The two-electrode voltage clamp technique measured evoked electrical currents in Xenopus laevis oocytes. Cytotoxicity was measured using multiple cell lines. ResultsPnPP-15 has peripheral antinociceptive activity via µ-opioid and CB1 cannabinoid receptors and inhibits neprilysin. In addition, it induced direct activation of mu-opioid receptors expressed in Xenopus laevis oocytes. PnPP-15 was not cytotoxic against mammalian kidney, liver, heart, nerve, or lung cells. ConclusionThis work demonstrated the antinociceptive effect of PnPP-15 in an inflammatory pain model, suggesting its use as a possible tool for developing new analgesic drugs.

SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.

The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), injured proximal tubular epithelial cells activate SOX9 for self-restoration. Using a multimodal approach for a head-to-head comparison of injury-induced SOX9 lineages, we identified a dynamic SOX9 switch in repairing epithelia. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate, which were identified as a SOX9on-on Cadherin6pos cell state. These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses. Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.

A comparative modeling study of the mitochondrial function of the proximal tubule and thick ascending limb cells in the rat kidney.

To reabsorb >99% of the glomerular filtrate, the metabolic demand of the kidney is high. Interestingly, renal blood flow distribution exhibits marked inhomogeneity, with typical tissue oxygen tension (Po2) of 50-60 mmHg in the well-perfused cortex and 10-20 mmHg in the inner medulla. Cellular fluid composition and acidity also varies substantially. To understand how different renal epithelial cells adapt to their local environment, we have developed and applied computational models of mitochondrial function of proximal convoluted tubule cell (baseline Po2 = 50 mmHg, cytoplasmic pH = 7.20) and medullary thick ascending limb (mTAL) cell (baseline Po2 = 10 mmHg, cytoplasmic pH = 6.85). The models predict key cellular quantities, including ATP generation, P/O (phosphate/oxygen) ratio, proton motive force, electrical potential gradient, oxygen consumption, the redox state of key electron carriers, and ATP consumption. Model simulations predict that close to their respective baseline conditions, the proximal tubule and mTAL mitochondria exhibit qualitatively similar behaviors. Nonetheless, because the mTAL mitochondrion has adapted to a much lower Po2, it can sustain a sufficiently high ATP production at Po2 as low as 4-5 mmHg, whereas the proximal tubule mitochondria would not. Also, because the mTAL cytosol is already acidic under baseline conditions, the proton motive force (pmf) exhibits higher sensitivity to further acidification. Among the different pathways that lead to oxidative phosphorylation impairment, the models predict that both the proximal tubule and mTAL mitochondria are most sensitive to reductions in Complex III activity.NEW & NOTEWORTHY Tissue fluid composition varies substantially within the mammalian kidney. The renal cortex is well perfused and pH neutral, whereas some medullary regions are hypoxic and acidic. How do these environments affect the mitochondrial function of proximal convoluted tubule and medullary thick ascending limb cells, which reside in the cortex and medulla, respectively? This computational modeling study demonstrates that these mitochondria can adapt to their contrasting environments and exhibit different sensitivities to perturbations to local environments.

The Screening and Mechanism of Influenza-Virus Sensitive MDCK Cell Lines for Influenza Vaccine Production.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin-Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

Viromes of Tabanids from Russia.

Advances in sequencing technologies and bioinformatics have greatly enhanced our knowledge of virus biodiversity. Currently, the viromes of hematophagous invertebrates, such as mosquitoes and ixodid ticks, are being actively studied. Tabanidae (Diptera) are a widespread family, with members mostly known for their persistent hematophagous behavior. They transmit viral, bacterial, and other pathogens, both biologically and mechanically. However, tabanid viromes remain severely understudied. In this study, we used high-throughput sequencing to describe the viromes of several species in the Hybomitra, Tabanus, Chrysops, and Haematopota genera, which were collected in two distant parts of Russia: the Primorye Territory and Ryazan Region. We assembled fourteen full coding genomes of novel viruses, four partial coding genomes, as well as several fragmented viral sequences, which presumably belong to another twelve new viruses. All the discovered viruses were tested for their ability to replicate in mammalian porcine embryo kidney (PEK), tick HAE/CTVM8, and mosquito C6/36 cell lines. In total, 16 viruses were detected in at least one cell culture after three passages (for PEK and C6/36) or 3 weeks of persistence in HAE/CTVM8. However, in the majority of cases, qPCR showed a decline in virus load over time.

Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In chronic kidney disease, affecting hundreds of millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a supra-structure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark, and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another, and from the mammalian Col-IVα345 scaffold and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.