Anti-Neuron-Derived Neurotrophic Factor Antibodies in Secondary Membranous Nephropathy Caused by Syphilis: A Case Report.

We present the case of a 61-year-old man who developed nephrotic syndrome as a result of syphilis-associated secondary membranous nephropathy (MN). The patient showed nephrotic syndrome remission following antibiotic treatment for syphilis alone. Pathologically, the target antigen of immune complexes accumulated on glomerular basement membranes (GBM) in secondary MN caused by syphilis has been reported to be neuron-derived neurotrophic factor (NDNF). His renal histopathology was consistent with secondary MN caused by syphilis, with a full-house pattern on immunofluorescence microscopy, in addition to NDNF deposits that colocalized with IgG deposits granularly on the GBM. However, to date, there is no serological evidence for the involvement of NDNF in the GBM. In the present study, we found that anti-NDNF autoantibodies in the acute-phase serum disappeared in the convalescent-phase serum of a patient who recovered from syphilis and nephrotic syndrome after antibiotic therapy alone. This result supports the hypothesis that treatment of syphilis with antibiotics suppresses NDNF's antigenicity. In summary, we found new serological evidence emphasizing that NDNF is an etiological antigen in secondary MN caused by syphilis.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

Predicting dry weight change in Hemodialysis patients using machine learning.

BACKGROUND: Machine Learning has been increasingly used in the medical field, including managing patients undergoing hemodialysis. The random forest classifier is a Machine Learning method that can generate high accuracy and interpretability in the data analysis of various diseases. We attempted to apply Machine Learning to adjust dry weight, the appropriate volume status of patients undergoing hemodialysis, which requires a complex decision-making process considering multiple indicators and the patient's physical conditions. METHODS: All medical data and 69,375 dialysis records of 314 Asian patients undergoing hemodialysis at a single dialysis center in Japan between July 2018 and April 2020 were collected from the electronic medical record system. Using the random forest classifier, we developed models to predict the probabilities of adjusting the dry weight at each dialysis session. RESULTS: The areas under the receiver-operating-characteristic curves of the models for adjusting the dry weight upward and downward were 0.70 and 0.74, respectively. The average probability of upward adjustment of the dry weight had sharp a peak around the actual change over time, while the average probability of downward adjustment of the dry weight formed a gradual peak. Feature importance analysis revealed that median blood pressure decline was a strong predictor for adjusting the dry weight upward. In contrast, elevated serum levels of C-reactive protein and hypoalbuminemia were important indicators for adjusting the dry weight downward. CONCLUSIONS: The random forest classifier should provide a helpful guide to predict the optimal changes to the dry weight with relative accuracy and may be useful in clinical practice.

Intestinal Bacterial Translocation Contributes to Diabetic Kidney Disease.

BACKGROUND: In recent years, many studies have focused on the intestinal environment to elucidate pathogenesis of various diseases, including kidney diseases. Impairment of the intestinal barrier function, the "leaky gut," reportedly contributes to pathologic processes in some disorders. Mitochondrial antiviral signaling protein (MAVS), a component of innate immunity, maintains intestinal integrity. The effects of disrupted intestinal homeostasis associated with MAVS signaling in diabetic kidney disease remains unclear. METHODS: To evaluate the contribution of intestinal barrier impairment to kidney injury under diabetic conditions, we induced diabetic kidney disease in wild-type and MAVS knockout mice through unilateral nephrectomy and streptozotocin treatment. We then assessed effects on the kidney, intestinal injuries, and bacterial translocation. RESULTS: MAVS knockout diabetic mice showed more severe glomerular and tubular injuries compared with wild-type diabetic mice. Owing to impaired intestinal integrity, the presence of intestine-derived Klebsiella oxytoca and elevated IL-17 were detected in the circulation and kidneys of diabetic mice, especially in diabetic MAVS knockout mice. Stimulation of tubular epithelial cells with K. oxytoca activated MAVS pathways and the phosphorylation of Stat3 and ERK1/2, leading to the production of kidney injury molecule-1 (KIM-1). Nevertheless, MAVS inhibition induced inflammation in the intestinal epithelial cells and KIM-1 production in tubular epithelial cells under K. oxytoca supernatant or IL-17 stimulation. Treatment with neutralizing anti-IL-17 antibody treatment had renoprotective effects. In contrast, LPS administration accelerated kidney injury in the murine diabetic kidney disease model. CONCLUSIONS: Impaired MAVS signaling both in the kidney and intestine contributes to the disrupted homeostasis, leading to diabetic kidney disease progression. Controlling intestinal homeostasis may offer a novel therapeutic approach for this condition.

Impairment of Proteasome Function in Podocytes Leads to CKD.

BACKGROUND: The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system (APLS) are major intracellular degradation procedures. The importance of the APLS in podocytes is established, but the role of the UPS is not well understood. METHODS: To investigate the role of the UPS in podocytes, mice were generated that had deletion of Rpt3 (Rpt3pdKO), which encodes an essential regulatory subunit required for construction of the 26S proteasome and its deubiquitinating function. RESULTS: Rpt3pdKO mice showed albuminuria and glomerulosclerosis, leading to CKD. Impairment of proteasome function caused accumulation of ubiquitinated proteins and of oxidative modified proteins, and it induced podocyte apoptosis. Although impairment of proteasome function normally induces autophagic activity, the number of autophagosomes was lower in podocytes of Rpt3pdKO mice than in control mice, suggesting the autophagic activity was suppressed in podocytes with impairment of proteasome function. In an in vitro study, antioxidant apocynin and autophagy activator rapamycin suppressed podocyte apoptosis induced by proteasome inhibition. Moreover, rapamycin ameliorated the glomerular injury in the Rpt3pdKO mice. The accumulation of ubiquitinated proteins and of oxidative modified proteins, which were detected in the podocytes of Rpt3pdKO mice, is a characteristic feature of aging. An aging marker was increased in the podocytes of Rpt3pdKO mice, suggesting that impairment of proteasome function promoted signs of aging in podocytes. CONCLUSIONS: Impairment of proteasome function in podocytes led to CKD, and antioxidants and autophagy activators can be therapeutic agents for age-dependent CKD.

Proteolytic cleavage of Podocin by Matriptase exacerbates podocyte injury.

Podocyte injury is a critical step toward the progression of renal disease and is often associated with a loss of slit diaphragm proteins, including Podocin. Although there is a possibility that the extracellular domain of these slit diaphragm proteins can be a target for a pathological proteolysis, the precise mechanism driving the phenomenon remains unknown. Here we show that Matriptase, a membrane-anchored protein, was activated at podocytes in CKD patients and mice, whereas Matriptase inhibitors slowed the progression of mouse kidney disease. The mechanism could be accounted for by an imbalance favoring Matriptase over its cognate inhibitor, hepatocyte growth factor activator inhibitor type 1 (HAI-1), because conditional depletion of HAI-1 in podocytes accelerated podocyte injury in mouse model. Matriptase was capable of cleaving Podocin, but such a reaction was blocked by either HAI-1 or dominant-negative Matriptase. Furthermore, the N terminus of Podocin, as a consequence of Matriptase cleavage of Podocin, translocated to nucleoli, suggesting that the N terminus of Podocin might be involved in the process of podocyte injury. Given these observations, we propose that the proteolytic cleavage of Podocin by Matriptase could potentially cause podocyte injury and that targeting Matriptase could be a novel therapeutic strategy for CKD patients.

MAGI-2 orchestrates the localization of backbone proteins in the slit diaphragm of podocytes.

Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. The slit diaphragm between the foot processes of podocytes functions as a final filtration barrier to prevent serum protein leakage into urine. The slit-diaphragm consists mainly of Nephrin and Neph1, and localization of these backbone proteins is essential to maintaining the integrity of the glomerular filtration barrier. However, the mechanisms that regulate the localization of these backbone proteins have remained elusive. Here, we focused on the role of membrane-associated guanylate kinase inverted 2 (MAGI-2) in order to investigate mechanisms that orchestrate localization of slit-diaphragm backbone proteins. MAGI-2 downregulation coincided with a reduced expression of slit-diaphragm backbone proteins in human kidneys glomerular disease such as focal segmental glomerulosclerosis or IgA nephropathy. Podocyte-specific deficiency of MAGI-2 in mice abrogated localization of Nephrin and Neph1 independently of other scaffold proteins. Although a deficiency of zonula occuldens-1 downregulated the endogenous Neph1 expression, MAGI-2 recovered Neph1 expression at the cellular edge in cultured podocytes. Additionally, overexpression of MAGI-2 preserved Nephrin localization to intercellular junctions. Co-immunoprecipitation and pull-down assays also revealed the importance of the PDZ domains of MAGI-2 for the interaction between MAGI-2 and slit diaphragm backbone proteins in podocytes. Thus, localization and stabilization of Nephrin and Neph1 in intercellular junctions is regulated mainly via the PDZ domains of MAGI-2 together with other slit-diaphragm scaffold proteins. Hence, these findings may elucidate a mechanism by which the backbone proteins are maintained.

Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes.

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.

Protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes.

To examine the cell-protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes, we analyzed the renal phenotype of tamoxifen (TM)-inducible podocyte-specific Atg5-deficient (iPodo-Atg5-/-) mice with experimental endothelial dysfunction. In both control and iPodo-Atg5-/- mice, high fat diet (HFD) feeding induced glomerular endothelial damage characterized by decreased urinary nitric oxide (NO) excretion, collapsed endothelial fenestrae, and reduced endothelial glycocalyx. HFD-fed control mice showed slight albuminuria and nearly normal podocyte morphology. In contrast, HFD-fed iPodo-Atg5-/- mice developed massive albuminuria accompanied by severe podocyte injury that was observed predominantly in podocytes adjacent to damaged endothelial cells by scanning electron microscopy. Although podocyte-specific autophagy deficiency did not affect endothelial NO synthase deficiency-associated albuminuria, it markedly exacerbated albuminuria and severe podocyte morphological damage when the damage was induced by intravenous neuraminidase injection to remove glycocalyx from the endothelial surface. Furthermore, endoplasmic reticulum stress was accelerated in podocytes of iPodo-Atg5-/- mice stimulated with neuraminidase, and treatment with molecular chaperone tauroursodeoxycholic acid improved neuraminidase-induced severe albuminuria and podocyte injury. In conclusion, podocyte autophagy plays a renoprotective role against diabetes-related structural endothelial damage, providing an additional insight into the pathogenesis of massive proteinuria in diabetic nephropathy.

Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton. CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

Molecular assay for an intronic variant in NUP93 that causes steroid resistant nephrotic syndrome.

Advances in molecular genetics have revealed that approximately 30% of cases with steroid-resistant nephrotic syndrome (SRNS) are caused by single-gene mutations. More than 50 genes are responsible for SRNS. One such gene is the nucleoporin, 93-KD (NUP93). Thus far, few studies have reported mutations of NUP93 in SRNS. Here, we describe an NUP93 biallelic mutation in a 9-year-old boy with focal segmental glomerular sclerosis (FSGS). Notably, one mutation comprised an intronic variant; we conducted in vivo and in vitro analysis to characterize this variant. We found two heterozygous mutations in NUP93: c.2137-18G>A in intron 19 and a novel nonsense mutation c.727A>T (p.Lys243*) in exon 8. We conducted RNA sequencing and in vitro splicing assays by using minigene construction, combined with protein expression analysis to determine the pathogenicity of the intronic variant. Both RNA sequencing and in vitro splicing assay showed exon 20-skipping by the intronic variant. In protein expression analysis, aberrant subcellular localization with small punctate vesicles in the cytoplasm was observed for the intronic variant. Taken together, we concluded that c.2137-18G>A was linked to pathogenicity due to aberrant splicing. NUP93 variants are quite rare; however, we have shown that even intronic variants in NUP93 can cause SRNS. This study provides a fundamental approach to validate the intronic variant, as well as new insights regarding the clinical spectrum of SRNS caused by rare gene variants.

MAGI-2 and scaffold proteins in glomerulopathy.

In many cells and tissues, including the glomerular filtration barrier, scaffold proteins are critical in optimizing signal transduction by enhancing structural stability and functionality of their ligands. Recently, mutations in scaffold protein membrane-associated guanylate kinase inverted 2 (MAGI-2) encoding gene were identified among the etiology of steroid-resistant nephrotic syndrome. MAGI-2 interacts with core proteins of multiple pathways, such as transforming growth factor-ß signaling, planar cell polarity pathway, and Wnt/ß-catenin signaling in podocyte and slit diaphragm. Through the interaction with its ligand, MAGI-2 modulates the regulation of apoptosis, cytoskeletal reorganization, and glomerular development. This review aims to summarize recent findings on the role of MAGI-2 and some other scaffold proteins, such as nephrin and synaptopodin, in the underlying mechanisms of glomerulopathy.

Glomerulosclerosis Induced by Deficiency of Membrane-Associated Guanylate Kinase Inverted 2 in Kidney Podocytes.

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a component of the slit diaphragm (SD) of glomerular podocytes. Here, we investigated the podocyte-specific function of MAGI-2 using newly generated podocyte-specific MAGI-2-knockout (MAGI-2-KO) mice. Compared with podocytes from wild-type mice, podocytes from MAGI-2-KO mice exhibited SD disruption, morphologic abnormalities of foot processes, and podocyte apoptosis leading to podocyte loss. These pathologic changes manifested as massive albuminuria by 8 weeks of age and glomerulosclerosis and significantly higher plasma creatinine levels at 12 weeks of age; all MAGI-2-KO mice died by 20 weeks of age. Loss of MAGI-2 in podocytes associated with decreased expression and nuclear translocation of dendrin, which is also a component of the SD complex. Dendrin translocates from the SD to the nucleus of injured podocytes, promoting apoptosis. Our coimmunoprecipitation and in vitro reconstitution studies showed that dendrin is phosphorylated by Fyn and dephosphorylated by PTP1B, and that Fyn-induced phosphorylation prevents Nedd4-2-mediated ubiquitination of dendrin. Under physiologic conditions in vivo, phosphorylated dendrin localized at the SDs; in the absence of MAGI-2, dephosphorylated dendrin accumulated in the nucleus. Furthermore, induction of experimental GN in rats led to the downregulation of MAGI-2 expression and the nuclear accumulation of dendrin in podocytes. In summary, MAGI-2 and Fyn protect dendrin from Nedd4-2-mediated ubiquitination and from nuclear translocation, thereby maintaining the physiologic homeostasis of podocytes, and the lack of MAGI-2 in podocytes results in FSGS.

USP40 gene knockdown disrupts glomerular permeability in zebrafish.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

Re-expression of Sall1 in podocytes protects against adriamycin-induced nephrosis.

The highly conserved spalt (sal) gene family members encode proteins characterized by multiple double zinc finger motifs of the C2H2 type. Humans and mice each have four known Sal-like genes (SALL1-4 in humans and Sall1-4 in mice). Sall1 is known to have a crucial role in kidney development. To explore the significance of Sall1 in differentiated podocytes, we investigated podocyte-specific Sall1-deficient mice (Sall1 KOp°d°/p°d°) using a podocin-Cre/loxP system and siRNA Sall1 knockdown (Sall1 KD) podocytes. Under physiological conditions, Sall1 KOp°d°/p°d° mice exhibited no proteinuria during their lifetime, but foot-process effacement was detected in some of the podocytes. To elucidate the role of Sall1 in injured podocytes, we used an adriamycin (ADR)-induced model of nephrosis and glomerulosclerosis. Surprisingly, the expression of Sall1 was elevated in control mice on day 14 after ADR injection. On day 28 after ADR injection, Sall1 KOp°d°/p°d° mice exhibited significantly higher levels of proteinuria and higher numbers of sclerotic glomeruli. Differentiated Sall1 KD podocytes showed a loss of synaptopodin, suppressed stress fiber formation, and, ultimately, impaired directed cell migration. In addition, the loss of Sall1 increased the number of apoptotic podocytes following ADR treatment. These results indicated that Sall1 has a protective role in podocytes; thus, we investigated the endoplasmic reticulum stress marker GRP78. GRP78 expression was higher in ADR-treated Sall1 KOp°d°/p°d° mice than in control mice. Sall1 appeared to influence the expression of GRP78 in injured podocytes. These results suggest that Sall1 is associated with actin reorganization, endoplasmic reticulum stress, and apoptosis in injured podocytes. These protective aspects of Sall1 re-expression in injured podocytes may have the potential to reduce apoptosis and possibly glomerulosclerosis.

The role of Notch signaling in kidney podocytes.

The Notch signaling pathway is a basic cell-to-cell communication mechanism. This pathway is activated by the interaction between Notch receptors and the ligands of adjacent cells. Once activated, Notch receptors are cleaved and the intracellular domains translocate into the nucleus, where the transcription of target genes starts. In the mammalian kidney, Notch receptors are activated during nephrogenesis. Afterwards, in the mature glomeruli, the Notch pathway becomes silent. However, many researchers have reported the activation of Notch receptors in mature podocytes under pathological conditions. In this review, we discuss the role of Notch signaling in podocytes.

Cathepsin D in Podocytes Is Important in the Pathogenesis of Proteinuria and CKD.

Studies have revealed many analogies between podocytes and neurons, and these analogies may be key to elucidating the pathogenesis of podocyte injury. Cathepsin D (CD) is a representative aspartic proteinase in lysosomes. Central nervous system neurons in CD-deficient mice exhibit a form of lysosomal storage disease with a phenotype resembling neuronal ceroid lipofuscinoses. In the kidney, the role of CD in podocytes has not been fully explored. Herein, we generated podocyte-specific CD-knockout mice that developed proteinuria at 5 months of age and ESRD by 20-22 months of age. Immunohistochemical analysis of these mice showed apoptotic podocyte death followed by proteinuria and glomerulosclerosis with aging. Using electron microscopy, we identified, in podocytes, granular osmiophilic deposits (GRODs), autophagosome/autolysosome-like bodies, and fingerprint profiles, typical hallmarks of CD-deficient neurons. CD deficiency in podocytes also led to the cessation of autolysosomal degradation and accumulation of proteins indicative of autophagy impairment and the mitochondrial ATP synthase subunit c accumulation in the GRODs, again similar to changes reported in CD-deficient neurons. Furthermore, both podocin and nephrin, two essential components of the slit diaphragm, translocated to Rab7- and lysosome-associated membrane glycoprotein 1-positive amphisomes/autolysosomes that accumulated in podocyte cell bodies in podocyte-specific CD-knockout mice. We hypothesize that defective lysosomal activity resulting in foot process effacement caused this accumulation of podocin and nephrin. Overall, our results suggest that loss of CD in podocytes causes autophagy impairment, triggering the accumulation of toxic subunit c-positive lipofuscins as well as slit diaphragm proteins followed by apoptotic cell death.

Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits.

BACKGROUND: Glomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant disease characterized by massive fibronectin deposits, leading to end-stage renal failure. Although mutations within the heparin-binding domains of the fibronectin 1 gene (FN1) have been associated with GFND, no mutations have been reported within the integrin-binding domains. METHODS: In this study, FN1 mutational analysis was conducted in 12 families with GFND. Biochemical and functional features of mutated proteins were examined using recombinant fibronectin fragments encompassing both the integrin- and heparin-binding domains. RESULTS: We report six FN1 mutations from 12 families with GFND, including five that are novel (p.Pro969Leu, p.Pro1472del, p.Trp1925Cys, p.Lys1953_Ile1961del, and p.Leu1974Pro). p.Pro1472del is localized in the integrin-binding domain of fibronectin, while the others are in heparin-binding domains. We detected p.Tyr973Cys, p.Pro1472del, and p.Leu1974Pro mutations in multiple families, and haplotype analysis implied that p.Pro1472del and p.Leu1974Pro are founder mutations. The protein encoded by the novel integrin-binding domain mutation p.Pro1472del showed decreased cell binding ability via the integrin-binding site. Most affected patients developed urine abnormalities during the first or second decade of life, and some mutation carriers were completely asymptomatic. CONCLUSIONS: This is the second large-scale analysis of GFND families and the first report of an integrin-binding domain mutation. These findings may help determine the pathogenesis of GFND.

Maternal exposure to high-fat and high-fructose diet evokes hypoadiponectinemia and kidney injury in rat offspring.

BACKGROUND: Maternal exposure to overnutrition during fetal development contributes to metabolic and renal damage in offspring. Adiponectin plays a protective role against obesity-related renal injury. However, role of adiponectin in renal injury of offspring exposed to maternal overnutrition remains unknown. We addressed the issue. METHODS: Female Sprague-Dawley rats were fed either a standard (N) or a high-fat and high-fructose (HFF)-diet for 6 weeks before mating, and kept each diet during the gestation and lactation period. After 4 weeks postpartum, all the offspring were fed N diet, and followed by 12 weeks. Kidney weight, urinary albumin excretion, blood pressure, and blood chemistry, including adiponectin and malondialdehyde, a marker of oxidative stress, were evaluated in the offspring. RESULTS: Compared with N-offspring, serum adiponectin levels of 1-day- and 4-week-old HFF-offspring were significantly lower, the latter of which was inversely associated with malondialdehyde. Kidney weight was significantly decreased in 1-day-old HFF-offspring, whereas increased in 4-week-old HFF-offspring. Urinary albumin excretion levels of HFF-offspring at 8, 12, and 16-week old were significantly higher than those of N-offspring at the same age, whose levels at 16-week old were inversely correlated with plasma adiponectin. Compared with N-offspring, HFF-offspring at 16-week old exhibited glomerulosclerosis, hyperglycemia, and high mean blood pressure associated with reduced podocin and increased transforming growth factor-ß1 expression in the kidneys. CONCLUSIONS: Our present study suggests that exposure to maternal HFF-diet during fetal and early postnatal development induces hypoadiponectinemia in offspring, which might cause renal injury and metabolic derangements later in life.