The urokinase plasminogen activator receptor (uPAR) is a membrane-bound protein found on the surface of immune cells. Through the action of proteases, uPAR is cleaved to produce several circulating proteins in the bloodstream, including the soluble form suPAR and the fragments D1 and D2D3. Initially studied in the context of infectious diseases and cancer, recent research has revealed roles for suPAR and its related proteins as mediators linking innate immunity to the pathogenesis of kidney and cardiovascular diseases, as well as insulin-dependent diabetes. While these proteins have long been recognized as prognostic biomarkers, growing clinical, experimental, and genetic evidence highlights their active involvement in the onset and progression of these diverse conditions. This Review examines suPAR's evolution from its discovery as a modulator of innate immunity to its current status as a key driver in chronic kidney and cardiovascular diseases. Furthermore, we explore the molecular mechanisms through which suPAR and D2D3 contribute to multiorgan damage, emphasizing emerging opportunities for therapeutic interventions across interconnected organ systems.

In genetic disease, an accurate expression landscape of disease genes and faithful animal models can facilitate genetic diagnoses and therapeutic advances respectively. Previously, we found that variants in NOS1AP, the gene that encodes nitric oxide synthase 1 adaptor protein, cause monogenic nephrotic syndrome. Here, we determine that an intergenic splice product of NOS1AP/Nos1ap and neighboring C1orf226/Gm7694, which prevents NOS1AP from binding to nitric oxide synthase 1, is the predominant isoform in mammalian kidney transcriptional and proteomic data. Gm7694-/- mice, whose allele exclusively disrupts the intergenic product, develop nephrotic syndrome phenotypes. In two male human subjects with nephrotic syndrome, we identify causative NOS1AP splice variants, including one predicted to abrogate intergenic splicing but initially misclassified as benign based on the canonical transcript. Finally, by modifying genetic background, we generate a faithful mouse model of NOS1AP-associated monogenic nephrotic syndrome that responds to anti-proteinuric treatment.

ABSTRACT In genetic disease, an accurate expression landscape of disease genes and faithful animal models will enable precise genetic diagnoses and therapeutic discoveries, respectively. We previously discovered that variants in NOS1AP , encoding nitric oxide synthase 1 (NOS1) adaptor protein, cause monogenic nephrotic syndrome (NS). Here, we determined that an intergenic splice product of N OS1AP / Nos1ap and neighboring C1orf226/Gm7694 , which precludes NOS1 binding, is the predominant isoform in mammalian kidney transcriptional and proteomic data. Gm7694 -/- mice, whose allele exclusively disrupts the intergenic product, developed NS phenotypes. In two human NS subjects, we identified causative NOS1AP splice variants, including one predicted to abrogate intergenic splicing but initially misclassified as benign based on the canonical transcript. Finally, by modifying genetic background, we generated a faithful mouse model of NOS1AP -associated NS, which responded to anti-proteinuric treatment. This study highlights the importance of intergenic splicing and a potential treatment avenue in a mendelian disorder.

1The University of Texas Medical Branch at Galveston, Galveston, Texas 2Harvard Medical School aCorresponding author: [email protected]

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic β cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored β cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired β cell proliferation and inhibited the bioenergetics of β cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

Soluble urokinase-type plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. Here we report the presence of C-terminal suPAR fragment, D2D3, in patients with diabetic nephropathy. D2D3-positive human sera inhibited glucose-stimulated insulin release in human islets and were associated with patients requiring insulin therapy. D2D3 transgenic mice presented kidney disease and diabetes marked by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic β-cell mass, and high fasting glucose. D2D3 fragment dysregulated glucose-induced cytoskeletal dynamics, impaired maturation and trafficking of insulin granules, and inhibited bioenergetics of β-cells in culture. An anti-uPAR antibody restored β-cell function in D2D3 transgenic mice. We show that the D2D3 fragment injures the kidney and pancreas, offering a unique dual therapeutic approach for kidney diseases and insulin-dependent diabetes. Summary Proteolytic suPAR fragment, D2D3, simultaneously injures two organs, the kidney and pancreas, thus causing a dual organ disease.

Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin’s role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin’s crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.

Drug-induced kidney injury frequently leads to aborted clinical trials and drug withdrawals. Sufficiently sensitive sensors capable of detecting mild signs of chemical insult in cell-based screening assays are critical to identifying and eliminating potential toxins in the preclinical stage. Oxidative stress is a common early manifestation of chemical toxicity, and biomolecule carbonylation is an irreversible repercussion of oxidative stress. Here, we present a novel fluorogenic assay using a sensor, TFCH, that responds to biomolecule carbonylation and efficiently detects modest forms of renal injury with much greater sensitivity than standard assays for nephrotoxins. We demonstrate that this sensor can be deployed in live kidney cells and in renal tissue. Our robust assay may help inform preclinical decisions to recall unsafe drug candidates. The application of this sensor in identifying and analyzing diverse pathologies is envisioned.

Background Transient receptor potential channel 5 (TRPC5) is a non-selective cationic ion channel expressed in brain, kidney and other organs where its activation underlies podocyte injury in chronic kidney diseases. Specifically, it has been suggested that a podocyte TRPC5 plasma membrane relocation and channel activation following injury results from activation of Rac-1, propagating podocyte dysfunction and proteinuria. However, previous TRPC5 transgenic mouse studies had questioned a pathogenic role for TRPC5 in podocytes. This investigation was designed to specifically evaluate podocyte Rac-1 activation in the context of functional TRPC5 or a TRPC5 pore mutant to assess effects on proteinuria. Materials and Methods We employed single cell patch-clamp studies of cultured podocytes and studied proteinuria in transgenic mouse models to characterize the effects of TRPC5 following podocyte Rac-1 activation. Results Inhibition of TRPC5 by small molecules reportedly ameliorated proteinuria in murine models of proteinuric kidney diseases. In order to directly examine TRPC5 function following Rac-1-induced podocyte injury, we analyzed TRPC5 inhibition in podocyte specific Rac-1 (active) transgenic mice. In addition, we generated a double-transgenic mouse constitutively overexpressing either TRPC5 (TRPC5 WT ) or a TRPC5 dominant-negative pore mutant (TRPC5 DN ) in concert with podocyte specific and inducible activation of active Rac-1 (Rac-1 Dtg ). In electrophysiological experiments, active TRPC5 was detected in primary podocytes overexpressing TRPC5 but not in podocytes with endogenous TRPC5 expression, nor with Rac-1 overexpressing podocytes. TRPC5 inhibition did not change proteinuria in mice with active podocyte Rac-1, nor did an increase or loss of TRPC5 activity affected podocyte injury in Rac-1 Dtg animals. Administration of TRPC5 inhibitors, ML204 and AC1903, did not alleviate podocyte Rac-1 induced proteinuria. Conclusion TRPC5 inhibition did not modify podocyte Rac-1 induced proteinuria in mice. Significance Statement TRPC5 is a calcium conducting ion channel involved in a plethora of biological functions in the brain, kidney and other organs. In proteinuric kidney diseases, others proposed a model that links activation of small GTPase Rac-1 in podocytes to activation of TRPC5 channels propagating cellular injury and eventually leading to progressive kidney disease. To test this hypothesis, we have developed a novel transgenic mouse model that employs podocyte Rac-1 activation in the presence or absence of a functional TRPC5 channel. Our data shows that transgenic mice with activated Rac-1 in podocytes did not enhance endogenous TRPC5 expression or its activity. Furthermore, TRPC5 blockade or activation did not modify Rac-1 induced proteinuria in mice.

A common manifestation of drug toxicity is kidney injury or nephrotoxicity. Nephrotoxicity frequently leads to termination of clinical trials and drug withdrawals, which jeopardize biomedical progress. Efficient preclinical screening platforms capable of detecting mild signs of kidney damage, which may elicit considerable toxic response in vivo, are essential. A common manifestation of chemical toxicity is oxidative modification of cellular biomolecules. Therefore, we have developed a facile biomolecule carbonyl detection assay that well surpasses the sensitivity of the standard assays in identifying modest forms of renal injury. Using a novel fluorogenic sensor, TFCH, we have demonstrated the applicability of the assay in live kidney cells and in renal tissue. This robust assay can help inform preclinical decisions to recall unsafe drug candidates. Application of this assay in identifying and analyzing diverse pathologies is envisioned.

Background Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. Methods We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. Results The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. Conclusions High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.) Soluble urokinase plasminogen activator receptor was measured in three patient cohorts at risk for acute kidney injury. Experimental models were used to assess suPAR as a therapeutic target. High suPAR levels were associated with acute kidney injury in both clinical and experimental settings.

The selectivity of the glomerular filter is established by physical, chemical, and signaling interplay among its three core constituents: glomerular endothelial cells, the glomerular basement membrane, and podocytes. Functional impairment or injury of any of these three components can lead to proteinuria. Podocytes are injured in many forms of human and experimental glomerular disease, including minimal change disease, focal segmental glomerulosclerosis, and diabetes mellitus. One of the earliest signs of podocyte injury is loss of their distinct structure, which is driven by dysregulated dynamics of the actin cytoskeleton. The status of the actin cytoskeleton in podocytes depends on a set of actin binding proteins, nucleators and inhibitors of actin polymerization, and regulatory GTPases. Mutations that alter protein function in each category have been implicated in glomerular diseases in humans and animal models. In addition, a growing body of studies suggest that pharmacological modifications of the actin cytoskeleton have the potential to become novel therapeutics for podocyte-dependent chronic kidney diseases. This review presents an overview of the essential proteins that establish actin cytoskeleton in podocytes and studies demonstrating the feasibility of drugging actin cytoskeleton in kidney diseases.

The selectivity of the glomerular filter is established by physical, chemical, and signaling interplay among its three core constituents: glomerular endothelial cells, the glomerular basement membrane, and podocytes. Functional impairment or injury of any of these three components can lead to proteinuria. Podocytes are injured in many forms of human and experimental glomerular disease, including minimal change disease, focal segmental glomerulosclerosis, and diabetes mellitus. One of the earliest signs of podocyte injury is loss of their distinct structure, which is driven by dysregulated dynamics of the actin cytoskeleton. The status of the actin cytoskeleton in podocytes depends on a set of actin binding proteins, nucleators and inhibitors of actin polymerization, and regulatory GTPases. Mutations that alter protein function in each category have been implicated in glomerular diseases in humans and animal models. In addition, a growing body of studies suggest that pharmacological modifications of the actin cytoskeleton have the potential to become novel therapeutics for podocyte-dependent chronic kidney diseases. This review presents an overview of the essential proteins that establish actin cytoskeleton in podocytes and studies demonstrating the feasibility of drugging actin cytoskeleton in kidney diseases.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding β3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin β3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for β3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases.