MANF stimulates autophagy and restores mitochondrial homeostasis to treat autosomal dominant tubulointerstitial kidney disease in mice.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD), a leading hereditary kidney disease. There are no targeted therapies. In our generated mouse model recapitulating human ADTKD-UMOD carrying a leading UMOD mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are impaired, leading to cGAS-STING activation and tubular injury. Moreover, we demonstrate that inducible tubular overexpression of mesencephalic astrocyte-derived neurotrophic factor (MANF), a secreted endoplasmic reticulum protein, after the onset of disease stimulates autophagy/mitophagy, clears mutant UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, thus protecting kidney function in our ADTKD mouse model. Conversely, genetic ablation of MANF in the mutant thick ascending limb tubular cells worsens autophagy suppression and kidney fibrosis. Together, we have discovered MANF as a biotherapeutic protein and elucidated previously unknown mechanisms of MANF in the regulation of organelle homeostasis, which may have broad therapeutic applications to treat various proteinopathies.

MANF stimulates autophagy and restores mitochondrial homeostasis to treat toxic proteinopathy.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD- UMOD ), one of the leading hereditary kidney diseases, and Alzheimer’s disease etc. There are no targeted therapies. ADTKD is also a genetic form of renal fibrosis and chronic kidney disease, which affects 500 million people worldwide. For the first time, in our newly generated mouse model recapitulating human ADTKD- UMOD carrying a leading UMOD deletion mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are severely impaired, leading to cGAS- STING activation and tubular injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel endoplasmic reticulum stress-regulated secreted protein. We provide the first study that inducible tubular overexpression of MANF after the onset of disease stimulates autophagy/mitophagy and clearance of the misfolded UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, resulting in protection of kidney function. Conversely, genetic ablation of endogenous MANF upregulated in the mutant mouse and human tubular cells worsens autophagy suppression and kidney fibrosis. Together, we discover MANF as a novel biotherapeutic protein and elucidate previously unknown mechanisms of MANF in regulating organelle homeostasis to treat ADTKD, which may have broad therapeutic application to treat various proteinopathies.

Targeting Endoplasmic Reticulum for Novel Therapeutics and Monitoring in Acute Kidney Injury.

BACKGROUND: Endoplasmic reticulum (ER) stress response is a conservative mechanism involving a complex network of different molecular branches to determine cell fate through specific transcription factors and downstream executors. Emerging evidence shows that ER stress is implicated in the occurrence and progression of acute kidney injury (AKI) in different animal models and human patients. However, there is still a lack of therapeutics targeting the ER in AKI. SUMMARY: Several therapeutic chemicals, including a compound that induces activating transcription factor 6 (ATF6) and chemical chaperones, have been developed to target the ER in the treatment of AKI. Meanwhile, ER stress-inducible secreted proteins, mesencephalic astrocyte-derived neurotrophic factor (MANF), and cysteine-rich with EGF-like domains 2 (CRELD2) could serve as potential ER stress biomarkers in the early diagnosis and treatment response monitoring of human patients with AKI. KEY MESSAGES: Experimental and clinical evidence suggests the critical role of ER in the pathogenesis and progression of AKI, and ER is a novel target in AKI therapy.

Blocking CHOP-dependent TXNIP shuttling to mitochondria attenuates albuminuria and mitigates kidney injury in nephrotic syndrome.

Albuminuria is a hallmark of glomerular disease of various etiologies. It is not only a symptom of glomerular disease but also a cause leading to glomerulosclerosis, interstitial fibrosis, and eventually, a decline in kidney function. The molecular mechanism underlying albuminuria-induced kidney injury remains poorly defined. In our genetic model of nephrotic syndrome (NS), we have identified CHOP (C/EBP homologous protein)-TXNIP (thioredoxin-interacting protein) as critical molecular linkers between albuminuria-induced ER dysfunction and mitochondria dyshomeostasis. TXNIP is a ubiquitously expressed redox protein that binds to and inhibits antioxidant enzyme, cytosolic thioredoxin 1 (Trx1), and mitochondrial Trx2. However, very little is known about the regulation and function of TXNIP in NS. By utilizing Chop-/- and Txnip-/- mice as well as 68Ga-Galuminox, our molecular imaging probe for detection of mitochondrial reactive oxygen species (ROS) in vivo, we demonstrate that CHOP up-regulation induced by albuminuria drives TXNIP shuttling from nucleus to mitochondria, where it is required for the induction of mitochondrial ROS. The increased ROS accumulation in mitochondria oxidizes Trx2, thus liberating TXNIP to associate with mitochondrial nod-like receptor protein 3 (NLRP3) to activate inflammasome, as well as releasing mitochondrial apoptosis signal-regulating kinase 1 (ASK1) to induce mitochondria-dependent apoptosis. Importantly, inhibition of TXNIP translocation and mitochondrial ROS overproduction by CHOP deletion suppresses NLRP3 inflammasome activation and p-ASK1-dependent mitochondria apoptosis in NS. Thus, targeting TXNIP represents a promising therapeutic strategy for the treatment of NS.

Ultrabright plasmonic fluor nanolabel-enabled detection of a urinary ER stress biomarker in autosomal dominant tubulointerstitial kidney disease.

Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is the most common nonpolycystic genetic kidney disease, but it remains unrecognized due to its clinical heterogeneity and lack of screening test. Moreover, the fact that the clinical feature is a poor predictor of disease outcome further highlights the need for the development of mechanistic biomarkers in ADTKD. However, low abundant urinary proteins secreted by thick ascending limb cells, where UMOD is synthesized, have posed a challenge for the detection of biomarkers in ADTKD-UMOD. In the CRISPR/Cas9-generated murine model and patients with ADTKD-UMOD, we found that immunoglobulin heavy chain-binding protein (BiP), an endoplasmic reticulum chaperone, was exclusively upregulated by mutant UMOD in the thick ascending limb and easily detected by Western blot analysis in the urine at an early stage of disease. However, even the most sensitive ELISA failed to detect urinary BiP in affected individuals. We therefore developed an ultrasensitive, plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA) to quantify urinary BiP concentration by harnessing the newly invented ultrabright fluorescent nanoconstruct, termed "plasmonic Fluor." p-FLISA demonstrated that urinary BiP excretion was significantly elevated in patients with ADTKD-UMOD compared with unaffected controls, which may have potential utility in risk stratification, disease activity monitoring, disease progression prediction, and guidance of endoplasmic reticulum-targeted therapies in ADTKD.NEW & NOTEWORTHY Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is an underdiagnosed cause of chronic kidney disease (CKD). Lack of ultrasensitive bioanalytical tools has hindered the discovery of low abundant urinary biomarkers in ADTKD. Here, we developed an ultrasensitive plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA). p-FLISA demonstrated that secreted immunoglobulin heavy chain-binding protein is an early urinary endoplasmic reticulum stress biomarker in ADTKD-UMOD, which will be valuable in monitoring disease progression and the treatment response in ADTKD.

Endoplasmic Reticulum-Associated Biomarkers for Molecular Phenotyping of Rare Kidney Disease.

The endoplasmic reticulum (ER) is the central site for folding, post-translational modifications, and transport of secretory and membrane proteins. An imbalance between the load of misfolded proteins and the folding capacity of the ER causes ER stress and an unfolded protein response. Emerging evidence has shown that ER stress or the derangement of ER proteostasis contributes to the development and progression of a variety of glomerular and tubular diseases. This review gives a comprehensive summary of studies that have elucidated the role of the three ER stress signaling pathways, including inositol-requiring enzyme 1 (IRE1), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 (ATF6) signaling in the pathogenesis of kidney disease. In addition, we highlight the recent discovery of ER-associated biomarkers, including MANF, ERdj3, ERdj4, CRELD2, PDIA3, and angiogenin. The implementation of these novel biomarkers may accelerate early diagnosis and therapeutic intervention in rare kidney disease.

Endoplasmic Reticulum Calcium Homeostasis in Kidney Disease: Pathogenesis and Therapeutic Targets.

Calcium (Ca2+) homeostasis is a crucial determinant of cellular function and survival. Endoplasmic reticulum (ER) acts as the largest intracellular Ca2+ store that maintains Ca2+ homeostasis through the ER Ca2+ uptake pump, sarco/ER Ca2+ ATPase, ER Ca2+ release channels, inositol 1,4,5-trisphosphate receptor channel, ryanodine receptor, and Ca2+-binding proteins inside of the ER lumen. Alterations in ER homeostasis trigger ER Ca2+ depletion and ER stress, which have been associated with the development of a variety of diseases. In addition, recent studies have highlighted the role of ER Ca2+ imbalance caused by dysfunction of sarco/ER Ca2+ ATPase, ryanodine receptor, and inositol 1,4,5-trisphosphate receptor channel in various kidney diseases. Despite progress in the understanding of the importance of these ER Ca2+ channels, pumps, and binding proteins in the pathogenesis of kidney disease, treatment is still lacking. This mini-review is focused on: i) Ca2+ homeostasis in the ER, ii) ER Ca2+ dyshomeostasis and apoptosis, and iii) altered ER Ca2+ homeostasis in kidney disease, including podocytopathy, diabetic nephropathy, albuminuria, autosomal dominant polycystic kidney disease, and ischemia/reperfusion-induced acute kidney injury.

Genetic and Clinical Predictors of Age of ESKD in Individuals With Autosomal Dominant Tubulointerstitial Kidney Disease Due to UMOD Mutations.

INTRODUCTION: Autosomal dominant tubulo-interstitial kidney disease due to UMOD mutations (ADTKD-UMOD) is a rare condition associated with high variability in the age of end-stage kidney disease (ESKD). The minor allele of rs4293393, located in the promoter of the UMOD gene, is present in 19% of the population and downregulates uromodulin production by approximately 50% and might affect the age of ESKD. The goal of this study was to better understand the genetic and clinical characteristics of ADTKD-UMOD and to perform a Mendelian randomization study to determine if the minor allele of rs4293393 was associated with better kidney survival. METHODS: An international group of collaborators collected clinical and genetic data on 722 affected individuals from 249 families with 125 mutations, including 28 new mutations. The median age of ESKD was 47 years. Men were at a much higher risk of progression to ESKD (hazard ratio 1.78, P < 0.001). RESULTS: The allele frequency of the minor rs4293393 allele was only 11.6% versus the 19% expected (P < 0.01), resulting in Hardy-Weinberg disequilibrium and precluding a Mendelian randomization experiment. An in vitro score reflecting the severity of the trafficking defect of uromodulin mutants was found to be a promising predictor of the age of ESKD. CONCLUSION: We report the clinical characteristics associated with 125 UMOD mutations. Male gender and a new in vitro score predict age of ESKD.

Discovery of endoplasmic reticulum calcium stabilizers to rescue ER-stressed podocytes in nephrotic syndrome.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.

Endoplasmic reticulum stress and monogenic kidney diseases in precision nephrology.

The advent of next-generation sequencing (NGS) in recent years has led to a rapid discovery of novel or rare genetic variants in human kidney cell genes, which is transforming the risk assessment, diagnosis, and treatment of kidney disease. Mutations may lead to protein misfolding, disruption of protein trafficking, and endoplasmic reticulum (ER) retention. An imbalance between the load of misfolded proteins and the folding capacity of the ER causes ER stress and unfolded protein response. Mutations in nephrin (NPHS1), podocin (NPHS2), laminin ß2 (LAMB2), and α-actinin-4 (ACTN4) have been shown to induce ER stress in HEK293 cells and podocytes in hereditary nephrotic syndromes; various founder mutations in collagen IV α chains (COL4A) have been demonstrated to activate podocyte ER stress in collagen IV nephropathies; and mutations in uromodulin (UMOD) have been reported to trigger tubular ER stress in autosomal dominant tubulointerstitial kidney disease. Meanwhile, ER resident protein SEC63 may modify disease severity in autosomal dominant polycystic kidney disease. These findings underscore the importance of ER stress in the pathogenesis of monogenic kidney disease. Recently, we have identified mesencephalic astrocyte-derived neurotrophic factor (MANF) and cysteine-rich with EGF-like domains 2 (CRELD2) as urinary ER stress biomarkers in ER stress-mediated kidney diseases.

Elevated urinary CRELD2 is associated with endoplasmic reticulum stress-mediated kidney disease.

ER stress has emerged as a signaling platform underlying the pathogenesis of various kidney diseases. Thus, there is an urgent need to develop ER stress biomarkers in the incipient stages of ER stress-mediated kidney disease, when a kidney biopsy is not yet clinically indicated, for early therapeutic intervention. Cysteine-rich with EGF-like domains 2 (CRELD2) is a newly identified protein that is induced and secreted under ER stress. For the first time to our knowledge, we demonstrate that CRELD2 can serve as a sensitive urinary biomarker for detecting ER stress in podocytes or renal tubular cells in murine models of podocyte ER stress-induced nephrotic syndrome and tunicamycin- or ischemia-reperfusion-induced acute kidney injury (AKI), respectively. Most importantly, urinary CRELD2 elevation occurs in patients with autosomal dominant tubulointerstitial kidney disease caused by UMOD mutations, a prototypical tubular ER stress disease. In addition, in pediatric patients undergoing cardiac surgery, detectable urine levels of CRELD2 within postoperative 6 hours strongly associate with severe AKI after surgery. In conclusion, our study has identified CRELD2 as a potentially novel urinary ER stress biomarker with potential utility in early diagnosis, risk stratification, treatment response monitoring, and directing of ER-targeted therapies in selected patient subgroups in the emerging era of precision nephrology.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), a new player in endoplasmic reticulum diseases: structure, biology, and therapeutic roles.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), a newly identified 18-kDa soluble protein, localizes to the luminal endoplasmic reticulum (ER), whose stress can stimulate MANF expression and secretion. In Drosophila and zebrafish, MANF regulates dopaminergic neuron development. In contrast, in mice, MANF deficiency leads to diabetes and activation of the unfolded protein response. Recent studies in rodent models have demonstrated that MANF mitigates diabetes, exerts neurotrophic function in neurodegenerative disease, protects cardiomyocytes and neurons in myocardial infarction and cerebral ischemia, respectively, and promotes immune cell phenotype switch from proinflammatory macrophages to prorepair anti-inflammatory macrophages. The cytoprotective mechanisms of MANF on ER stress are currently under active investigation. In addition, for the first time, we have discovered that MANF can potentially serve as a urinary ER stress biomarker in ER stress-mediated kidney disease. These studies have underscored the diagnostic and therapeutic importance of MANF in ER diseases.

A rare case of renal thrombotic microangiopathy associated with Castleman's disease.

BACKGROUND: Castleman's disease (CD) is an uncommon, heterogeneous lympho-proliferative disorder leading to high circulating levels of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). Renal involvement has been only described in a limited number of small studies. Herein, we report a rare case of renal thrombotic microangiopathy (TMA) associated with CD and investigate the podocyte expression of VEGF in the renal biopsy prior to initiation of treatment. CASE PRESENTATION: An 18-year-old male presented with fever, diarrhea, diffuse lymphadenopathy, ascites and acute kidney injury. Laboratory tests for hemolytic uremic syndrome and thrombotic thrombocytopenic purpura were negative. The kidney biopsy showed TMA. An excisional lymph node biopsy was consistent with CD, plasma cell variant. Immunofluorescence staining showed suppressed podocyte VEGF expression. Chemotherapy that inhibits production of inflammatory mediators including IL-6 and VEGF led to complete recovery of renal function. CONCLUSIONS: Our case illustrates a rare renal histological feature of CD. IL-6 and VEGF are postulated to suppress glomerular VEGF expression, thereby causing renal TMA. Therapy directed against these inflammatory mediators may have important therapeutic implications.

Tubular Overexpression of Angiopoietin-1 Attenuates Renal Fibrosis.

Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor ß1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans.

Mesencephalic Astrocyte-Derived Neurotrophic Factor as a Urine Biomarker for Endoplasmic Reticulum Stress-Related Kidney Diseases.

Endoplasmic reticulum (ER) stress and disrupted proteostasis contribute to the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop noninvasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when a kidney biopsy is not yet clinically indicated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) localizes to the ER lumen and is secreted in response to ER stress in several cell types. Here, using mouse models of human nephrotic syndrome caused by mutant laminin ß2 protein-induced podocyte ER stress and AKI triggered by tunicamycin- or ischemia-reperfusion-induced tubular ER stress, we examined MANF as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells. ER stress upregulated MANF expression in podocytes and tubular cells. Notably, urinary MANF excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histologic manifestations of the corresponding disease. Thus, MANF can potentially serve as a urine diagnostic or prognostic biomarker in ER stress-related kidney diseases to help stratify disease risk, predict disease progression, monitor treatment response, and identify subgroups of patients who can be treated with ER stress modulators in a highly targeted manner.

Focal segmental glomerulosclerosis: molecular genetics and targeted therapies.

Recent advances show that human focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy caused by podocyte-specific gene mutations including NPHS1, NPHS2, WT-1, LAMB2, CD2AP, TRPC6, ACTN4 and INF2. This review focuses on genes discovered in the investigation of complex FSGS pathomechanisms that may have implications for the current FSGS classification scheme. It also recounts recent recommendations for clinical management of FSGS based on translational studies and clinical trials. The advent of next-generation sequencing promises to provide nephrologists with rapid and novel approaches for the diagnosis and treatment of FSGS. A stratified and targeted approach based on the underlying molecular defects is evolving.

Laminin ß2 gene missense mutation produces endoplasmic reticulum stress in podocytes.

Mutations in the laminin ß2 gene (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. LAMB2 is a component of the laminin-521 (α5ß2γ1) trimer, an important constituent of the glomerular basement membrane (GBM). The C321R-LAMB2 missense mutation leads to congenital nephrotic syndrome but only mild extrarenal symptoms; the mechanisms underlying the development of proteinuria with this mutation are unclear. We generated three transgenic mouse lines, in which rat C321R-LAMB2 replaced mouse LAMB2 in the GBM. During the first postnatal month, expression of C321R-LAMB2 attenuated the severe proteinuria exhibited by Lamb2(-/-) mice in a dose-dependent fashion; proteinuria eventually increased, however, leading to renal failure. The C321R mutation caused defective secretion of laminin-521 from podocytes to the GBM accompanied by podocyte endoplasmic reticulum (ER) stress, likely resulting from protein misfolding. Moreover, ER stress preceded the onset of significant proteinuria and was manifested by induction of the ER-initiated apoptotic signal C/EBP homologous protein (CHOP), ER distention, and podocyte injury. Treatment of cells expressing C321R-LAMB2 with the chemical chaperone taurodeoxycholic acid (TUDCA), which can facilitate protein folding and trafficking, greatly increased the secretion of the mutant LAMB2. Taken together, these results suggest that the mild variant of Pierson syndrome caused by the C321R-LAMB2 mutation may be a prototypical ER storage disease, which may benefit from treatment approaches that target the handling of misfolded proteins.

Glomerular basement membrane and related glomerular disease.

The glomerular basement membrane (GBM) is lined by fenestrated endothelium from the capillary-lumen side and by interdigitating foot processes of the podocytes from the urinary- space side. These three layers of the glomerular capillary wall constitute the functional unit of the glomerular filtration barrier. The GBM is assembled through an interweaving of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations in genes encoding LAMB2, COL4A3, COL4A4, and COL4A5 cause glomerular disease in humans as well as in mice. In addition, laminin α5 mutation in podocytes leads to proteinuria and renal failure in mice. Moreover, more neoepitopes in Goodpasture's disease and for the first time alloepitopes in Alport post-transplantation nephritis have been located in the collagen α5(IV) NC1 domain. These discoveries underscore the importance of the GBM in establishing and maintaining the integrity of the glomerular filtration barrier.

An unusual cause of membranous glomerulonephritis in a patient with HIV.

A 68-year old Caucasian male with a past medical history of human immunodeficiency virus (HIV) infection presented with acute oliguric renal failure and maculopapular rash. Renal biopsy demonstrated extensive foot process effacement as well as confluent small subepithelial electron-dense deposits, which is diagnostic of membranous glomerulonephritis. Subsequent serological tests showed venereal disease research laboratory test was positive in both serum and cerebral spinal fluid. Following penicillin treatment, the patient's creatinine returned to baseline 4 weeks later. Secondary membranous glomerulonephritis caused by syphilis in patients with HIV is discussed.

A missense LAMB2 mutation causes congenital nephrotic syndrome by impairing laminin secretion.

Laminin ß2 is a component of laminin-521, which is an important constituent of the glomerular basement membrane (GBM). Null mutations in laminin ß2 (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. In contrast, patients with LAMB2 missense mutations, such as R246Q, can have less severe extrarenal defects but still exhibit congenital nephrotic syndrome. To investigate how such missense mutations in LAMB2 cause proteinuria, we generated three transgenic lines of mice in which R246Q-mutant rat laminin ß2 replaced the wild-type mouse laminin ß2 in the GBM. These transgenic mice developed much less severe proteinuria than their nontransgenic Lamb2-deficient littermates; the level of proteinuria correlated inversely with R246Q-LAMB2 expression. At the onset of proteinuria, expression and localization of proteins associated with the slit diaphragm and foot processes were normal, and there were no obvious ultrastructural abnormalities. Low transgene expressors developed heavy proteinuria, foot process effacement, GBM thickening, and renal failure by 3 months, but high expressors developed only mild proteinuria by 9 months. In vitro studies demonstrated that the R246Q mutation results in impaired secretion of laminin. Taken together, these results suggest that the R246Q mutation causes nephrotic syndrome by impairing secretion of laminin-521 from podocytes into the GBM; however, increased expression of the mutant protein is able to overcome this secretion defect and improve glomerular permselectivity.