Anti-phospholipase A2 receptor antibodies directly induced podocyte damage in vitro

BackgroundThe pathogenesis of primary membranous nephropathy (MN) involves the antibodies against antigens on the cell surface of podocytes, with the majority of M-type phospholipase A2 receptor (PLA2R), and a profound podocyte dysfunction. The effects of anti-PLA2R antibodies directly to the podocytes remain unclear.MethodsAnti-PLA2R antibodies from patients with PLA2R-associated MN were affinity-purified using a column coupled with recombinant human PLA2R protein. Their effects on conditionally immortalized human podocytes were assessed by apoptosis assays, cellular calcium detection, wound healing assay, and immunofluorescent staining. Proteomics analysis was performed by LC-MS/MS and on PANTHER database.ResultsThe stimulation by anti-PLA2R antibodies could induce early-stage apoptosis of podocytes (MFI of Annexin V = 104.3 ± 19.2 vs. 36.7 ± 7.6, p = 0.004). The increase of calcium concentration in podocytes (MFI = 3309.3 ± 363.6 vs. 1776.3 ± 212.7, p = 0.015) might attribute to the endoplasmic reticulum calcium efflux. The expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) was also increased (MFI = 134.4 ± 9.8 vs. 105.3 ± 10.1, p = 0.011). Proteomics results suggested that anti-PLA2R antibody treatment led to damage on cellular structure, and produced functional disorders on protein binding, actin filament binding, and microtubule motor activity. The staining of F-actin on foot process was reduced (MFI = 27.3 ± 2.8 vs. 47.5 ± 1.0, p = 0.001) and the motility and adherence capacity of podocytes were reduced (number of migrated cells = 44.7 ± 3.1 vs. 53.3 ± 4.9, p = 0.001) after incubation with anti-PLA2R antibodies.ConclusionThese data indicate that anti-PLA2R antibodies may directly induce podocyte damage independent of the complement system, which expands the mechanism of anti-PLA2R antibodies on MN.     

The p.R229Q variant of the NPHS2 (podocin) gene in focal segmental glomerulosclerosis and steroid-resistant nephrotic syndrome: a meta-analysis

While many previous studies have reported an association between the p.R229Q variant of the NPHS2 gene and focal segmental glomerulosclerosis (FSGS) or steroid-resistant nephrotic syndrome (SRNS), a conclusive relationship has not been defined. In this study, we performed a meta-analysis of the published data to investigate the impact of the p.R229Q polymorphism on FSGS and SRNS patients. Despite significant heterogeneity within some of the comparisons, the results revealed significantly higher risks of SRNS in individuals homozygous for the variant allele (OR 7.411, 95 % confidence interval 1.876–29.436, p = 0.004) compared to homozygous non-variant individuals. However, the carrier rate of the p.R229Q variant was not significantly different between SRNS patients and steroid-sensitive nephrotic syndrome patients. No statistically significant differences in the p.R229Q carrier rate were observed between FSGS patients and controls or FSGS patients and patients with different pathology classifications. No notable differences in the p.R229Q carrier rate were found between patients and controls in any group with early-onset disease (onset age < 18). In conclusion, our meta-analysis suggests that for adult-onset disease (onset age > 18), the homozygous variant could be a potential predictor of hereditary nephrotic syndrome and that the p.R229Q allele cannot currently be considered a risk factor for predicting FSGS.     

ADCK4-Associated Glomerulopathy Causes Adolescence-Onset FSGS

Hereditary defects of coenzyme Q₁₀ biosynthesis cause steroid-resistant nephrotic syndrome (SRNS) as part of multiorgan involvement but may also contribute to isolated SRNS. Here, we report 26 patients from 12 families with recessive mutations in ADCK4. Mutation detection rate was 1.9% among 534 consecutively screened cases. Patients with ADCK4 mutations showed a largely renal-limited phenotype, with three subjects exhibiting occasional seizures, one subject exhibiting mild mental retardation, and one subject exhibiting retinitis pigmentosa. ADCK4 nephropathy presented during adolescence (median age, 14.1 years) with nephrotic-range proteinuria in 44% of patients and advanced CKD in 46% of patients at time of diagnosis. Renal biopsy specimens uniformly showed FSGS. Whereas 47% and 36% of patients with mutations in WT1 and NPHS2, respectively, progressed to ESRD before 10 years of age, ESRD occurred almost exclusively in the second decade of life in ADCK4 nephropathy. However, CKD progressed much faster during adolescence in ADCK4 than in WT1 and NPHS2 nephropathy, resulting in similar cumulative ESRD rates (>85% for each disorder) in the third decade of life. In conclusion, ADCK4-related glomerulopathy is an important novel differential diagnosis in adolescents with SRNS/FSGS and/or CKD of unknown origin.     

Protective Role of the M-Sec–Tunneling Nanotube System in Podocytes

BackgroundPodocyte dysfunction and loss are major determinants in the development of proteinuria. FSGS is one of the most common causes of proteinuria, but the mechanisms leading to podocyte injury or conferring protection against FSGS remain poorly understood. The cytosolic protein M-Sec has been involved in the formation of tunneling nanotubes (TNTs), membrane channels that transiently connect cells and allow intercellular organelle transfer. Whether podocytes express M-Sec is unknown and the potential relevance of the M-Sec–TNT system in FSGS has not been explored.MethodsWe studied the role of the M-Sec–TNT system in cultured podocytes exposed to Adriamycin and in BALB/c M-Sec knockout mice. We also assessed M-Sec expression in both kidney biopsies from patients with FSGS and in experimental FSGS (Adriamycin-induced nephropathy).ResultsPodocytes can form TNTs in a M-Sec–dependent manner. Consistent with the notion that the M-Sec–TNT system is cytoprotective, podocytes overexpressed M-Sec in both human and experimental FSGS. Moreover, M-Sec deletion resulted in podocyte injury, with mitochondrial abnormalities and development of progressive FSGS. In vitro, M-Sec deletion abolished TNT-mediated mitochondria transfer between podocytes and altered mitochondrial bioenergetics. Re-expression of M-Sec reestablishes TNT formation and mitochondria exchange, rescued mitochondrial function, and partially reverted podocyte injury.ConclusionsThese findings indicate that the M-Sec–TNT system plays an important protective role in the glomeruli by rescuing podocytes via mitochondrial horizontal transfer. M-Sec may represent a promising therapeutic target in FSGS, and evidence that podocytes can be rescued via TNT-mediated horizontal transfer may open new avenues of research.     

COQ2 nephropathy: a newly described inherited mitochondriopathy with primary renal involvement

Primary coenzyme Q(10) (CoQ(10)) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Rarely, glomerular involvement has been reported. The COQ2 gene encodes the para-hydroxybenzoate-polyprenyl-transferase enzyme of the CoQ(10) synthesis pathway. We identified two patients with early-onset glomerular lesions that harbored mutations in the COQ2 gene. The first patient presented with steroid-resistant nephrotic syndrome at the age of 18 months as a result of collapsing glomerulopathy, with no extrarenal symptoms. The second patient presented at five days of life with oliguria, had severe extracapillary proliferation on renal biopsy, rapidly developed end-stage renal disease, and died at the age of 6 months after a course complicated by progressive epileptic encephalopathy. Ultrastructural examination of renal specimens from these cases, as well as from two previously reported patients, showed an increased number of dysmorphic mitochondria in glomerular cells. Biochemical analyses demonstrated decreased activities of respiratory chain complexes [II+III] and decreased CoQ(10) concentrations in skeletal muscle and renal cortex. In conclusion, we suggest that inherited COQ2 mutations cause a primary glomerular disease with renal lesions that vary in severity and are not necessarily associated with neurological signs. COQ2 nephropathy should be suspected when electron microscopy shows an increased number of abnormal mitochondria in podocytes and other glomerular cells.     

Glycosaminoglycan excretion in children with nephrotic syndrome

Although most childhood nephrotic syndromes respond to steroid treatment, steroid resistant nephrotic syndrome (SRNS) is also common and is particularly difficult to treat. This study investigated the role of glycosaminoglycans (GAG) in the pathogenesis and clinical course of nephrotic syndrome in children. Thirty-four children (21 males and 13 females, mean age 3.7±1.6 years) with steroid-sensitive nephrotic syndrome and 20 children with steroid-resistant nephrotic syndrome (12 males and 8 females, mean age 10.9±3.8 years; of the twenty, four had primary SRNS (FSGS) and the others had secondary SRNS) were included the study. Mean urine levels of GAG relative to creatinine (U_GAG/U_Cr) in patients with SRNS (n=20, 113.01±78.46 mg g^–1 Cr) and in patients experiencing the nephrotic period of steroid-sensitive nephrotic syndrome (n=34, 132.15±101.55 mg g^–1 Cr) were both significantly higher than mean U_GAG/U_Cr for control subjects (n=30, 51.83±47.66 mg g^–1 Cr) (P<0.01 for both). Patients excreted significantly more GAG during the nephrotic period of steroid-sensitive nephrotic syndrome than during remission (132.15±101.55 vs 39.11±42.73 mg g^–1 Cr, respectively; P<0.01). There was, however, no significant difference between U_GAG/U_Cr for patients with steroid-resistant nephrotic syndrome and U_GAG/U_Cr in the nephrotic period of steroid-sensitive nephrotic syndrome. Urine GAG excretion correlated significantly with the severity of proteinuria. The results suggest that GAG play a significant role in the pathogenesis of nephrotic syndrome but that GAG excretion is not a marker for response to steroid treatment in pediatric patients with this condition.     

Comparison of the prophylactic antithrombotic effect of indobufen and warfarin in patients with nephrotic syndrome: a randomized controlled trial

PurposeThe risk of thromboembolic events is elevated in patients with nephrotic syndrome, and warfarin use has been associated with an increased risk of bleeding. Indobufen, a selective cyclooxygenase-1 inhibitor, is currently being evaluated for the prevention of thromboembolic events in nephrotic syndrome. This study aimed to compare the efficacy and safety of indobufen with that of warfarin in patients with nephrotic syndrome.Materials and methodsThis multicenter, randomized, three-arm, open-label, parallel controlled trial involved a total of 180 adult patients with nephrotic syndrome from four centers in China. Patients were randomly assigned to receive 100 mg indobufen (bid), 200 mg indobufen (bid), and 3 mg warfarin (qd) daily for 12 weeks. The primary endpoints included thromboembolic and bleeding events, while laboratory results and adverse events constituted secondary endpoints.ResultsNo thromboembolic events occurred in the high-/low-dose indobufen and warfarin groups. Moreover, the use of a low dose of indobufen significantly reduced the risk of minor bleeding events compared with warfarin use (2% versus 18%, p < .05). Finally, adverse events were more frequent in warfarin-treated patients.ConclusionsThis study found that indobufen therapy provided equivalent effects in preventing thromboembolic events compared with warfarin therapy, while low dose of indobufen was associated with a reduced risk of bleeding events, thus it should be recommended for the prevention of thromboembolic events in clinical practice in patients with nephrotic syndrome.Trial registration numberChiCTR-IPR-17013428.     

Mutations in the Gene That Encodes the F-Actin Binding Protein Anillin Cause FSGS

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1–associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm–associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.FSGS is a clinicopathologic entity that is characterized by nephrotic syndrome (NS), focally sclerotic glomeruli, and effacement of podocyte foot processes and is a common cause of ESRD.¹ The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided evidence that disruption of podocyte structure and function is central to its pathogenesis. The highly specialized apparatus of the glomerular filtration barrier (GFB) is composed of the podocyte and slit diaphragm, the glomerular basement membrane, and specialized fenestrated endothelium. The podocyte and slit diaphragm play a central role in maintaining the structural and functional integrity of the GFB. This is evidenced by the number of genetic mutations found in familial FSGS and NS; many of these gene products contribute to signaling at the podocyte slit diaphragm or localize to the podocyte cytoskeleton.^2–8 However, the entire repertoire of genes and proteins that are important in maintaining the functional integrity of the GFB remains unknown. It is estimated that >90% of the genetic causes of hereditary FSGS and NS remain unknown⁹; thus, the study of large kindreds remains an invaluable tool for unraveling the complexity of the molecular interactions that are responsible for maintaining the integrity of the podocyte and GFB.We identified a pedigree from the United States with autosomal dominant (AD) FSGS. Linkage analysis was carried out and we obtained suggestive multipoint logarithm of odds (LOD) scores of 1.7, 1.7, and 1.8 on chromosomes 2p, 5p, and 7p respectively. Whole-exome sequencing identified a deleterious heterozygous mutation R431C in ANLN, the gene encoding the F-actin binding protein anillin. We found another variant G618C that segregates with disease in a second family with FSGS. Anillin is important in cytokinesis and it interacts with key proteins such as CD2AP and mDia2 during cell division.^10–13 In addition, it regulates cell growth by its interaction with the promigratory and prosurvival phosphoinositide 3 kinase/AKT (protein kinase B) pathway.^10–13 Overexpression of the mutant anillin in immortalized human podocytes causes increased podocyte motility compared with wild-type overexpression. In contrast with intact anillin, the mutant displays a significantly reduced binding affinity to CD2AP. These findings reemphasize the importance of podocyte cell integrity, cell survival pathways, and cell migration in the pathogenesis of FSGS.     

Variable renal phenotype in a family with an INF2 mutation

Recent advances in the genetics of glomerular diseases have identified several causative genes of nephrotic syndrome and/or glomerular proteinuria. In 2010, the INF2 gene, which encodes a member of the formin family of actin-regulating proteins, was identified as a novel causative gene of the autosomal dominant form of focal segmental glomerulosclerosis (FSGS). Here, we describe an additional familial case of FSGS associated with INF2 mutations. In the family, two siblings and their father had a heterozygous p.E220K mutation on INF2. This mutation manifested in these three individuals as incidentally detected proteinuria without overt nephrotic syndrome, but at different ages of 7, 9, and 30 years, respectively. Two siblings had nephrotic range proteinuria, and one developed end-stage renal disease 5 years later. Conversely, their father had a modest degree of proteinuria, and maintained normal renal function until age 47. A renal biopsy of one of the siblings revealed FSGS with irregular podocyte foot process morphology and focal glomerular basement membrane changes. This is the second paper describing a familial case of FSGS associated with INF2 mutations as well as intrafamilial phenotype variability.     

Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ₃ integrin-dependent migration in vitro. Furthermore, αVβ₃ integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ₃ integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ₃ integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target.     

Acute kidney injury in idiopathic nephrotic syndrome of childhood is a major risk factor for the development of chronic kidney disease

Background: Acute kidney injury (AKI) is an important complication of idiopathic nephrotic syndrome (INS) and is associated with adverse outcomes, especially the development of chronic kidney disease (CKD). We aimed to determine the clinical profile of children with INS who developed AKI and its short-term outcome.

Material and methods: This prospective study was conducted from March 2014 to October 2015. A total of 119 children of INS (age: 2–18 years) fulfilling the pediatric RIFLE criteria for the diagnosis of AKI were enrolled and followed up for 3 months to determine the outcome. Factors predisposing to CKD were studied.

Results: The mean age at presentation was 8.8?±?3.59 years and males were 74 (62.2%). At presentation, 61 (51.3%) children were in Risk category, 43 (36.1%) in Injury category, and 15 (12.6%) in Failure category. Most of them (41.2%) had steroid-resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS) on histopathology (33.6%).

Infections were the major predisposing factor for AKI in 67 (56.3%) cases. Drug toxicity was the next common, found in 52 (43.7%) children. A total of 65 (54.6%) children recovered from AKI, while 54 (45.4%) did not. CKD developed in 49 (41.2%) non-recovered cases and 5 (4.2%) children succumbed to acute illness. SRNS, cyclosporine use, FSGS on histology, and drug toxicity were significant factors associated with the development of CKD.

Conclusion: AKI associated with INS is a reversible condition in most cases but it can progress to CKD, especially among those who have SRNS, FSGS, and drug toxicity.     

NPHS2 homozygous p.R229Q variant: potential modifier instead of causal effect in focal segmental glomerulosclerosis

Background

The pathogenicity of the NPHS2 homozygous p.R229Q variant in steroid-resistant nephrotic syndrome (SRNS) is doubtful. While it has been reported in unaffected controls, it is enriched in patients with SRNS, suggesting pathogenicity.

Case-Diagnosis/Treatment

A family with three members homozygous for the NPHS2 p.R229Q variant is presented: a 37-year-old patient who was diagnosed with proteinuria at age 7 months, focal segmental glomerulosclerosis (FSGS) at age 20 years, and end-stage renal disease (ESRD) at age 33 years, his 59 year-old father and his 40 year-old brother, both unaffected with no proteinuria. The affected son also harbors a heterozygous de novo, truncating PAX2 mutation (c.76dupG, p.V26Gfs*28), which can explain his chronic renal failure but which is rarely associated with FSGS.

Conclusions

This family provides further evidence that homozygous p.R229Q in itself may not cause FSGS. Nevertheless, the rare association of FSGS to a PAX2 mutation may reflect the modifier effect of p.R229Q in the homozygous state. Such a modifier effect can also explain its enrichment in SRNS patients. Patients with homozygous p.R229Q should be screened for the causative mutation in a second gene.     

Follow-up of steroid-resistant nephrotic syndrome: tubular proteinuria and enzymuria

The aim of this study was to examine the compromise of proximal tubule cells in steroid-resistant nephrotic syndrome patients with a histologic diagnosis of focal segmental glomerulosclerosis (FSGS) through assessment of the urinary levels of β₂-microglobulin (β₂M) and N-acetyl-β-d-glucosaminidase (NAG) during active disease and remission over a follow-up period of 3 years. We studied 34 children with nephrotic syndrome: 12 with steroid-resistant nephrotic syndrome (SRNS) and massive proteinuria, 7 with steroid-dependent nephrotic syndrome (SDNS) and 15 with steroid-sensitive nephrotic syndrome (SSNS). Of the SSNS patients, 8 children were in remission (RM) and 7 were in relapse (RL). Seven healthy children were included as controls. Urinary β₂M, measured by enzyme-linked immunosorbent assay, was significantly increased in the SRNS group as compared to the SDNS group (P<0.01), SSNS in remission (P<0.01), and controls (P<0.01). There were no differences between the SRNS group and SSNS in relapse. Analysis of urinary N-acetyl-β-d-glucosaminidase (U-NAG) by colorimetric assay showed significantly higher values in the SRNS group of patients than in SDNS, SSNS, and control groups. A positive correlation between U-NAG and proteinuria was demonstrated (r=0. 73, P<0.01). The SRNS group of patients (n=12, 11 with a histologic diagnosis of FSGS and one with diffuse mesangial proliferation) was treated with the same protocol of i.v. methylprednisone and oral cyclophosphamide. Long-term follow-up showed a progressive decrease in U-β₂M and U-NAG excretion to control values in the 3rd year, except in one patient who did not respond to the treatment. In the FSGS patients, evaluation of the contribution of structural interstitial histological abnormalities, including each of the histological parameters considered in interstitial scarring to the functional tubule abnormalities assessed by β₂M and NAG excretion, was performed by multiple regression analysis. The r ² values for β₂M and NAG were 53.99%, P=0.19, and 57.90%, P=0.14, respectively; neither was significant. We conclude that: (1) proximal tubule cell dysfunction, partially affected by massive albuminuria, may account for the higher values of β₂M and NAG excretion in the SRNS patients and (2) urine β₂M and NAG levels are not helpful in identifying histological evidence of structural tubulointerstitial damage in children with steroid-resistant nephrotic syndrome. Received: 28 December 1999 / Revised: 20 July 2000 / Accepted: 21 July 2000     

Anti-PLA2R antibody measured by ELISA predicts the risk of vein thrombosis in patients with primary membranous nephropathy

BackgroundPrimary membranous nephropathy (PMN) is associated with the highest risk for developing venous thrombosis compared with other nephrotic diseases. The aim of the study was to assess the predictive value of the pathognomonic anti-phospholipase A2 receptor (PLA2R) antibody with regard to incidence of venous thrombosis in PMN.MethodsA total of 365 in-hospital patients diagnosed with PMN were enrolled in the study. Anti-PLA2R antibody was detected by commercial enzyme-linked immunosorbent assay. Multivariate logistic regression was used to detect the independent risk factors for venous thrombosis.ResultsThirty-seven patients (10.14%) had venous thrombosis. Patients with venous thrombosis had higher levels of cholesterol (CHOL), low-density lipoprotein (LDL), and D-dimer than those without venous thrombosis (p < .05). Patients with venous thrombosis had significantly lower levels of albumin (23.95 ± 5.53 vs. 26.18 ± 6.59 g/L, p = .049). No significant differences were found in proteinuria, serum creatinine, estimated glomerular filtration rate, platelets, and fibrinogen between patients with and without thrombosis. Anti-PLA2R antibody levels in patients with venous thrombosis were significantly higher than in patients without it (p = .002). In the univariate logistic regression, Ln anti-PLA2R antibody (OR: 1.340; p = .004), albumin (OR: 0.945; p = .050), CHOL (OR: 1.191; p = .006), and LDL (OR: 1.271, p = .006) were associated with venous thrombosis. Ln anti-PLA2R antibody (OR = 1.269; 95%CI: 1.032–1.561), and LDL (OR = 1.213; 95%CI: 1.017–1.448) were the independent risk factors for venous thrombosis (p < .05) in multivariate analysis.ConclusionsAnti-PLA2R antibody was the independent risk factor for venous thrombosis in PMN. Larger prospective studies were warranted to verify the results in future.     

NPHS2 mutations in sporadic steroid-resistant nephrotic syndrome in Japanese children

Podocin is an integral membrane protein encoded by NPHS2, which is mapped to 1q25-31 and is exclusively expressed in glomerular podocytes. NPHS2 mutations are responsible for autosomal recessive familial steroid-resistant nephrotic syndrome (SRNS) with minor glomerular abnormalities or focal segmental glomerulosclerosis (FSGS), which is characterized by early childhood onset (age less than 6 years) and rapid progression to chronic renal insufficiency. This gene mutation is also responsible for an adolescent/adult onset form of autosomal recessive familial FSGS with heavy proteinuria. It has been demonstrated that sporadic SRNS and heavy proteinuria are also due to NPHS2 gene mutations. We isolated genomic DNA from 36 Japanese children with chronic renal insufficiency caused by SRNS or heavy proteinuria, and analyzed all eight exons and exon-intron boundaries of NPHS2 using the polymerase chain reaction and direct sequencing. The age at onset of disease was 3.9+/-0.5 years. There were 29 patients with SRNS and 7 with heavy proteinuria without nephrotic syndrome at the onset, but all patients developed chronic renal insufficiency 4.6+/-0.8 years after the onset. A new homozygous missense variant of NPHS2, G34E (G101A) in exon 1, was detected in 1 of 36 patients. However, this homozygous variant was also found in 1 of 44 normal controls, suggesting that the mutation is a polymorphism. Two silent variants (T954C and A1038G) in exon 8 of this gene were also identified in some of the patients and normal controls, indicating that the silent variants are also polymorphisms. There was no significant difference in the genotypic and allelic frequencies of T954C and A1038G polymorphisms between the patients and normal controls. In conclusion, NPHS2 gene mutations are not a major cause of chronic renal insufficiency caused by sporadic SRNS or heavy proteinuria in Japanese children.     

Circulating suPAR in Two Cohorts of Primary FSGS

Overexpression of soluble urokinase receptor (suPAR) causes pathology in animal models similar to primary FSGS, and one recent study demonstrated elevated levels of serum suPAR in patients with the disease. Here, we analyzed circulating suPAR levels in two cohorts of children and adults with biopsy-proven primary FSGS: 70 patients from the North America–based FSGS clinical trial (CT) and 94 patients from PodoNet, the Europe-based consortium studying steroid-resistant nephrotic syndrome. Circulating suPAR levels were elevated in 84.3% and 55.3% of patients with FSGS patients in the CT and PodoNet cohorts, respectively, compared with 6% of controls (P<0.0001); inflammation did not account for this difference. Multiple regression analysis suggested that lower suPAR levels associated with higher estimated GFR, male sex, and treatment with mycophenolate mofetil. In the CT cohort, there was a positive association between the relative reduction of suPAR after 26 weeks of treatment and reduction of proteinuria, with higher odds for complete remission (P=0.04). In the PodoNet cohort, patients with an NPHS2 mutation had higher suPAR levels than those without a mutation. In conclusion, suPAR levels are elevated in geographically and ethnically diverse patients with FSGS and do not reflect a nonspecific proinflammatory milieu. The associations between a change in circulating suPAR with different therapeutic regimens and with remission support the role of suPAR in the pathogenesis of FSGS.FSGS is an important cause of ESRD in children and adults.^1–3 It can occur as a primary disorder, consequent to genetic mutations in specific podocyte proteins, secondary to a wide range of medical problems, or as a consequence of adaptive changes to reduced kidney mass.^4,5 Intensive research for >50 years has focused mainly on genetic, immunologic, and systemic causes of FSGS but the cause of primary disease remains elusive.In recent years, there has been a great deal of investigation into the role of permeability factors in the pathogenesis of proteinuria and the development of primary FSGS. Proposed candidate molecules include hemopexin, vascular endothelial growth factor, and cardiotrophin-like cytokine-1.^6–8 However, the detection of increased levels of most circulating permeability factors is generally not specific for a given glomerular disorder, and the activity or expression of these factors may be increased in patients with FSGS and minimal change nephrotic syndrome, two disorders with markedly disparate responses to therapy and long-term prognosis.⁹Soluble urokinase receptor (suPAR), the soluble form of urokinase plasminogen-type activator receptor, has been reported to be elevated in a number of diseases including cancer and infection in a nonspecific manner. However, its underlying mechanism of action in these conditions and its clinical value either in diagnosis or prognosis is still far from clear.¹⁰ Recently, Wei et al. reported the molecular identity of a putative permeability factor in FSGS.¹¹ Approximately two-thirds of patients with primary FSGS had increased suPAR. Moreover, the circulating suPAR concentration was mostly normal in other forms of primary glomerular diseases including minimal change nephrotic syndrome, idiopathic membranous nephropathy, or preeclampsia.¹¹ The investigators related the abnormal suPAR level to the pathogenesis of FSGS based on both in vitro and in vivo studies, which demonstrated suPAR binding to and, more importantly, activation of β3 integrin on podocytes. This interaction led to alterations in the morphology (foot process effacement) and function of podocytes, which resulted in proteinuria and initiation of FSGS.¹¹The prevalence of elevated circulating suPAR levels in patients with primary FSGS has not been systematically evaluated in any other large patient cohort. In addition, it is unclear if the circulating suPAR is modulated by immunosuppressive therapy. Therefore, in this report, we detail suPAR levels in two discrete well described cohorts of children and young adults with biopsy-proven primary FSGS—the North America–based FSGS clinical trial (CT), and the Europe-based consortium for study of steroid-resistant nephrotic syndrome (PodoNet).     

Analysis of recessive CD2AP and ACTN4 mutations in steroid-resistant nephrotic syndrome

Mutations in podocyte genes have been identified in patients with steroid-resistant nephrotic syndrome (SRNS). Point mutations in the ACTN4 gene cause an autosomal dominant form of human focal segmental glomerular sclerosis (FSGS); however, reports of CD2AP mutations remain scarce. Based on the phenotype of Actn4 and Cd2ap null mice, we aimed to define the role of recessive CD2AP and ACTN4 mutations in a cohort of children with SRNS for which NPHS1, NPHS2, and PLCE1 mutations had been previously excluded. CD2AP and ACTN4 mutational analysis was performed in 42 children from 35 unrelated families. The median age of disease onset was 20 (range 0–102) months. Sixteen patients reached end-stage kidney disease at a median age of 84 (range 4–161) months. Renal histology showed FSGS lesions and minimal glomerular changes in 49% and 20% of patients, respectively. Microsatellite marker analysis excluded linkage to the CD2AP locus in 26 families and to the ACTN4 locus in 31 families. No disease-causing mutations were identified in the remaining families. Recessive CD2AP and ACTN4 mutations are rare in children with SRNS. The absence of mutations in this study suggests that there are other genetic causes of SRNS that still need to be identified.     

Effect of galactose on glomerular permeability and proteinuria in steroid-resistant nephrotic syndrome

Background

Idiopathic steroid-resistant nephrotic syndrome (SRNS) has been associated with the presence of a circulating focal sclerosis permeability factor (FSPF) thought to damage the glomerular barrier and increase permeability to albumin. Galactose binds and inactivates FSPF in vitro, but its effect in vivo is uncertain.

Methods

A prospective clinical trial was conducted to investigate the effect of oral galactose on FSPF and proteinuria in children with SRNS. Seven pediatric subjects with idiopathic SRNS and positive FSPF activity (>0.5) were treated with oral galactose (0.2 gm/kg/dose twice daily) for 16 weeks. Post-treatment FSPF and proteinuria were measured.

Results

Focal sclerosis permeability factor activity of the seven subjects decreased from 0.69?±?0.11 to 0.35?±?0.21 (p?=?0.009) in response to galactose. The two subjects with post-transplant recurrence of focal segmental glomerulosclerosis (FSGS) demonstrated the most significant improvement in FSPF (p?=?0.006). Despite this decrease in FSPF, the pre- and post-treatment urine protein:creatinine ratio remained unchanged and no subject achieved remission.

Conclusions

Galactose decreases FSPF in children with SRNS, with the most significant improvement in those with post-transplant FSGS recurrence, but it fails to improve proteinuria. At the present time there is no evidence to support the use of galactose in children with FSGS, either pre- or post-transplant. Future studies to investigate the role of galactose as preemptive therapy to decrease the risk of post-transplant FSGS recurrence may be useful.     

Methylprednisolone treatment of patients with steroid-resistant nephrotic syndrome

Treatment with a combination of pulse methylprednisolone (MP) and an alkylating agent has been reported to induce long-term remission of proteinuria in patients with steroid-resistant nephrotic syndrome (SRNS). We have treated 13 patients with SRNS with a course of pulse MP. There were 8 black patients and 5 white; 10 had a biopsy diagnosis of focal segmental glomerulosclerosis (FSGS) and 3 nil lesion. Initially 5 patients responded and 2 partially responded. Of the responding patients, 5 relapsed while treated with alternate-week MP therapy. Of these relapsing patients, 3 received a second course of MP plus chlorambucil; 2 responded. The patients were observed for a mean of 47 months (range 4–64 months). When last seen only the 3 patients with a biopsy diagnosis of nil lesion were protein free. There were no complications of steroid therapy. Six patients currently have end-stage renal disease and 2 have renal insufficiency. All of the 6 patients with no response to treatment were black. These data suggest that a course of pulse MP therapy alone induces short-term remission of the nephrotic syndrome in some white patients with FSGS, but in almost no blacks. Patients who relapse may respond to retreatment, but addition of an alkylating agent does not appear to induce longterm remission in patients with FSGS.     

Patients receiving hemodialysis do not lose SARS-CoV-2 antibodies more rapidly than non-renal controls: a prospective cohort study

BackgroundPatients with end-stage kidney disease receiving maintenance hemodialysis (HD) are at increased risk for mortality after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compared with the general population. However, it is currently unknown whether the long-term SARS-CoV-2 humoral and cellular immune responses in patients receiving HD are comparable to individuals with normal kidney function.MethodThe prospective cohort study included 24 patients treated with maintenance HD and 27 non-renal controls with confirmed history of coronavirus disease (COVID-19). In all participants the levels of specific IgG were quantified at three timepoints: 10, 18, and 26 weeks from disease onset. In a subgroup of patients, specific T-cell responses were evaluated.ResultsThe seropositivity rate declined in controls over time and was 85% and 70.4% at weeks 18 and 26, respectively. All HD patients remained seropositive over the study period. Seropositivity rate at week 26 was greater among patients receiving HD: RR = 1.4 [95%CI: 1.17–1.94] (reciprocal of RR = 0.7 [95% CI: 0.52–0.86]), p = 0.0064. In both groups, IgG levels decreased from week 10 to week 26, but antibodies vanished more rapidly in controls than in HD group (ANOVA p = 0.0012). The magnitude of T-cell response was significantly lower in controls than in HD patients at weeks 10 (p = 0.019) and 26 (p = 0.0098) after COVID-19 diagnosis, but not at week 18.ConclusionCompared with non-renal adults, patients receiving HD maintain significant long-term humoral and cellular immune responses following natural COVID-19.