Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes.

Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with steroid-sensitive NS in relapse and remission, 10 healthy controls, and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry, and Western blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. Gene Ontology classification analysis and Ingenuity Pathway Analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho-p38 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro.NEW & NOTEWORTHY Up to now, the role of extracellular vesicles (EVs) in the pathogenesis of steroid-sensitive nephrotic syndrome (NS) has not been studied. Here, we found that relapse NS EVs contain significantly increased active RAC1, induce enhanced podocyte motility, and increase expression of RAC-GTP and phospho-p38 expression in vitro. These results suggest that plasma EVs are biologically active molecules in the pathogenesis of NS.

MRI Spectrum of Brain Involvement in Sphingosine-1-Phosphate Lyase Insufficiency Syndrome.

SGPL1 encodes sphingosine-1-phosphate lyase, the final enzyme of sphingolipid metabolism. In 2017, a condition featuring steroid-resistant nephrotic syndrome and/or adrenal insufficiency associated with pathogenic SGPL1 variants was reported. In addition to the main features of the disease, patients often exhibit a range of neurologic deficits. In a handful of cases, brain imaging results were described. However, high-quality imaging results and a systematic analysis of brain MR imaging findings associated with the condition are lacking. In this study, MR images from 4 new patients and additional published case reports were reviewed by a pediatric neuroradiologist. Analysis reveals recurring patterns of features in affected patients, including isolated callosal dysgenesis and prominent involvement of the globus pallidus, thalamus, and dentate nucleus, with progressive atrophy and worsening of brain lesions. MR imaging findings of abnormal deep gray nuclei, microcephaly, or callosal dysgenesis in an infant or young child exhibiting other typical clinical features of sphingosine-1-phosphate lyase insufficiency syndrome should trigger prompt genetic testing for SGPL1 mutations.

Plasma kallikrein activates the epithelial sodium channel in vitro but is not essential for volume retention in nephrotic mice.

AIM: Recent work has demonstrated that activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases causes sodium retention in nephrotic syndrome. The aim of this study was to elucidate a potential role of plasma kallikrein (PKLK) as a candidate serine protease in this context. METHODS: We analysed PKLK in the urine of patients with chronic kidney disease (CKD, n = 171) and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in PKLK-deficient mice (klkb1-/- ) with experimental nephrotic syndrome induced by doxorubicin injection. RESULTS: In patients with CKD, we found that PKLK is excreted in the urine up to a concentration of 2 µg mL-1 which was correlated with albuminuria (r = .71) and overhydration as assessed by bioimpedance spectroscopy (r = .44). PKLK increased ENaC-mediated whole-cell currents, which was associated with the appearance of a 67 kDa γ-ENaC cleavage product at the cell surface consistent with proteolytic activation. Mutating a putative prostasin cleavage site in γ-ENaC prevented channel stimulation by PKLK. In a mouse model for nephrotic syndrome, active PKLK was present in nephrotic urine of klkb1+/+ but not of klkb1-/- mice. However, klkb1-/- mice were not protected from ENaC activation and sodium retention compared to nephrotic klkb1+/+ mice. CONCLUSION: Plasma kallikrein is detected in the urine of proteinuric patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site. However, PKLK is not essential for volume retention in nephrotic mice.

Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome.

Volume retention in nephrotic syndrome has been linked to activation of the epithelial sodium channel (ENaC) by proteolysis of its γ-subunit following urinary excretion of serine proteases such as plasmin. Here we tested whether pharmacological inhibition of urinary serine protease activity might protect from ENaC activation and volume retention in nephrotic syndrome. Urine from both nephrotic mice (induced by doxorubicin injection) and nephrotic patients exhibited high aprotinin-sensitive serine protease activity. Treatment of nephrotic mice with the serine protease inhibitor aprotinin by means of subcutaneous sustained-release pellets normalized urinary serine protease activity and prevented sodium retention, as did treatment with the ENaC inhibitor amiloride. In the kidney cortex from nephrotic mice, immunofluorescence revealed increased apical γ-ENaC staining, normalized by aprotinin treatment. In Xenopus laevis oocytes heterologously expressing murine ENaC, aprotinin had no direct inhibitory effect on channel activity but prevented proteolytic channel activation. Thus, our study shows that volume retention in experimental nephrotic syndrome is related to proteolytic ENaC activation by proteasuria and can be prevented by treatment with aprotinin. Hence, inhibition of urinary serine protease activity might become a therapeutic approach to treat patients with nephrotic-range proteinuria.

Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome.

Nie X, Chanley MA, Pengal R, Thomas DB, Agrawal S, Smoyer WE. Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome. Am J Physiol Renal Physiol 314: F602-F613, 2018. First published November 29, 2017; doi: 10.1152/ajprenal.00207.2017 .-The p38 MAPK pathway plays a crucial role in various glomerulopathies, with activation being associated with disease and inhibition being associated with disease amelioration. We hypothesized that the downstream targets of p38 MAPK, MAPK-activated protein kinase 2 and/or 3 (MK2 and/or MK3), play an important role in mediating injury in experimental nephrotic syndrome via their actions on their downstream substrates heat shock protein B1 (HSPB1) and cyclooxygenase-2 (COX-2). To test this hypothesis, the effects of both pharmacological and genetic inhibition of MK2 and MK3 were examined in mouse adriamycin (ADR) and rat puromycin aminonucleoside (PAN) nephropathy models. MK2-/-, MK3-/-, and MK2-/-MK3-/- mice were generated in the Sv129 background and subjected to ADR-induced nephropathy. MK2 and MK3 protein expression was completely abrogated in the respective knockout genotypes, and massive proteinuria and renal histopathological changes developed after ADR treatment. Furthermore, renal cortical HSPB1 was induced in all four genotypes by day 21, but HSPB1 was activated only in the wild-type and MK3-/- mice. Expression of the stress proteins HSPB8 and glucose-regulated protein 78 (GRP78) remained unaltered across all genotypes. Finally, while MK2 and/or MK3-knockout downregulated the proinflammatory enzyme COX-2, ADR significantly induced renal cortical COX-2 only in MK2-/- mice. Additionally, pharmacological MK2 inhibition with PF-318 during PAN-induced nephropathy did not result in significant proteinuria reduction in rats. Together, these data suggest that while the inhibition of MK2 and/or MK3 regulates the renal stress response, our currently available approaches are not yet able to safely and effectively reduce proteinuria in experimental nephrotic syndrome and that other p38MAPK downstream targets should also be considered to improve the future treatment of glomerular disease.

Histone deacetylase-2 expression and activity in children with nephrotic syndrome with different glucocorticoid response.

BACKGROUND: Glucocorticosteroid (GC) is one of the most effective drugs available for the treatment of primary nephrotic syndrome (PNS) in children. However, some patients show little or no response to GC. The purpose of our research was to observe and describe the different levels of histone deacetylase-2 (HDAC2) expression in peripheral blood lymphocytes in children with PNS compared with various responses to the GC treatment, with the primary aim to assess the correlation between HDAC2 and GC resistance in PNS children. METHODS: Forty-eight patients with PNS suffering from their first attack prior to GC treatment were chosen as subjects. They were divided into two groups, those who had steroid-sensitive nephrotic syndrome (SSNS; n = 25) and those with steroid-resistant NS (SRNS; n = 23), according to their response to a 6-week course of oral prednisone. Twenty healthy children from the Physical Examination Center in the hospital served as the control group; Peripheral blood was collected at different time points prior to GC treatment and after regular therapy. RT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA) techniques were adopted to analyze HDAC2 mRNA, protein expression, and activity, respectively, in peripheral blood lymphocytes. The level of interleukin-8 (IL-8) in serum was measured by an ELISA. RESULTS: Prior to GC treatment, HDAC2 expression level and activity were lower in the SRNS group than in the SSNS and control group. A statistically significant difference in HDAC2 expression and activity were observed after GC treatment between these groups, with HDAC2 expression and activity lower in the SRNS group than in the SSNS and control groups. In the SSNS group, the expression and activity of HDAC2 were higher following GC treatment than prior to GC treatment. There was a clear difference in HDAC2 expression and activity of SRNS at the different time points. No statistically significant difference was found between the two groups. The pre-treatment and post-treatment serum IL-8 levels in the SRNS group were significantly higher than those in the SSNS group. HDAC2 from children with PNS before GC treatment and after regular therapy for 6 weeks was negatively correlated with serum IL-8 level. CONCLUSION: The GC effect was influenced by the HDAC2 expression and activity, leading to decreased serum IL-8 levels in children with PNS. HDAC2 seems to be one of the markers of GC resistance in children with PNS.

NOX-mediated impairment of PDGF-induced DNA synthesis in peripheral blood lymphocytes of children with idiopathic nephrotic syndrome.

BackgroundCellular oxidative stress, inflammatory responses, and immunogenic events are involved in pathogenesis of idiopathic nephrotic syndrome (INS); however, the exact mechanism remains unknown. We examined NADPH oxidase (NOX) activity and platelet-derived growth factor (PDGF)-induced DNA synthesis in peripheral blood lymphocytes (PBL) of patients with INS.MethodsPBL from 15 patients with INS and 15 age- and gender-matched controls were isolated, and enzyme activities of NOX, catalase, and superoxide dismutase (SOD) were measured along with the assay of malondialdehyde levels and bromo-deoxyuridine incorporation. Protein expression of NOX-1 was measured using western blot analysis.ResultsPatients with INS had significantly (P<0.01) higher NOX activity and increased protein expression of NOX-1 in PBL as compared with controls. Catalase and SOD activities were markedly lower with lipid peroxide levels significantly (P<0.01) increased in patients with INS. Ex vivo DNA synthesis in PDGF-stimulated PBL was significantly (P<0.01) reduced in patients with INS; however, diphenyliodonium, an inhibitor of NOX, markedly corrected impairment in growth factor-induced BrdU incorporation.ConclusionsThese results show that NOX activation might have a role in regulation of lymphocytic activity in patients with INS through the impairment of PDGF mitogenic function and might contribute toward pathogenesis of nephrotic syndrome.

Wolf-Hirschhorn syndrome candidate 1-like 1 epigenetically regulates nephrin gene expression.

Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.

Deficiency of the sphingosine-1-phosphate lyase SGPL1 is associated with congenital nephrotic syndrome and congenital adrenal calcifications.

We identified two unrelated consanguineous families with three children affected by the rare association of congenital nephrotic syndrome (CNS) diagnosed in the first days of life, of hypogonadism, and of prenatally detected adrenal calcifications, associated with congenital adrenal insufficiency in one case. Using exome sequencing and targeted Sanger sequencing, two homozygous truncating mutations, c.1513C>T (p.Arg505*) and c.934delC (p.Leu312Phefs*30), were identified in SGPL1-encoding sphingosine-1-phosphate (S1P) lyase 1. SGPL1 catalyzes the irreversible degradation of endogenous and dietary S1P, the final step of sphingolipid catabolism, and of other phosphorylated long-chain bases. S1P is an intracellular and extracellular signaling molecule involved in angiogenesis, vascular maturation, and immunity. The levels of SGPL1 substrates, S1P, and sphingosine were markedly increased in the patients' blood and fibroblasts, as determined by liquid chromatography-tandem mass spectrometry. Vascular alterations were present in a patient's renal biopsy, in line with changes seen in Sgpl1 knockout mice that are compatible with a developmental defect in vascular maturation. In conclusion, loss of SGPL1 function is associated with CNS, adrenal calcifications, and hypogonadism.

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome.

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism.

Effects of Shenkangling intervention on the MAPK pathway in rats with doxorubicin-induced nephropathy.

Shenkangling plays a role of Yishenhuoxue effect for the treatment of children with nephrotic syndrome. The aim of this study was to investigate the effects of Shenkangling intervention on the mitogen-activated protein kinase (MAPK) pathway in rats with Adriamycin-induced nephropathy (AN) and its underlying mechanism of action. Nephrosis was induced in healthy Sprague-Dawley rats by doxorubicin and the rats were untreated or treated with prednisone, simvastatin, Shenkangling, or a combination thereof. Using real-time PCR, the mRNA expression levels of Chemokine (C-X-C motif) ligand 16 (CXCL16), A Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), and ADAM17 in the renal tissues of these rats were found to be decreased by the various treatments compared to those in the untreated doxorubicin-induced nephrosis rats. To quantify the activation of the MAPK pathway, western blotting was used to detect the phosphorylation levels of MAPK pathway-associated proteins (p38, ERK1/2, SAPK/JNK) and nuclear factor (NF)-κB p65, which were reduced by the various treatments compared to those in the untreated doxorubicin-induced rats. Serum levels of transforming growth factor (TGF)-ß1, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, quantified by ELISA, were decreased by the various treatments compared to the levels in the untreated doxorubicin-induced nephrosis rats. The rats treated with prednisone, simvastatin, and Shenkangling showed the best outcome. The Chinese medicine Shenkangling that is known for nourishing the kidney and promoting blood circulation reduced urinary protein levels, increased serum albumin levels, and reduced cholesterol levels by reducing the release of CXCL16, ADAM10, ADAM17, TGF-ß1, TNF-α, IL-1ß, IL- 6, and other inflammatory mediators and inhibiting the activation of the MAPK signaling pathway, thereby effectively improving the state of nephropathy in AN rats. These results indicate that Shenkangling can be used clinically to treat nephropathy.

Reciprocal Regulation of 11ß-HSDs May Predict Steroid Sensitivity in Childhood Nephrotic Syndrome.

Childhood nephrotic syndrome, in which steroid-dependence occurs concurrently with steroid-resistance, requires aggressive therapy to prevent relapse. Predictive biomarkers that can be used to stratify treatment are urgently needed. Here we report that reciprocal regulation of the glucocorticoid metabolizing enzymes, 11ß-hydroxysteroid dehydrogenase types 1 and 2, is associated with steroid-responsiveness and disease remission in childhood nephrotic syndrome, potentially providing a marker to identify patients in which aggressive therapy is required.

The Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Nephrotic Syndrome-Associated Hypercholesterolemia.

BACKGROUND: In nephrotic syndrome, damage to the podocytes of the kidney produces severe hypercholesterolemia for which novel treatments are urgently needed. PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as an important regulator of plasma cholesterol levels and therapeutic target. Here, we tested the role of PCSK9 in mediating the hypercholesterolemia of nephrotic syndrome. METHODS: PCSK9 and plasma lipids were studied in nephrotic syndrome patients before and after remission of disease, mice with genetic ablation of the podocyte (Podocyte Apoptosis Through Targeted Activation of Caspase-8, Pod-ATTAC mice) and mice treated with nephrotoxic serum (NTS), which triggers immune-mediated podocyte damage. In addition, mice with hepatic deletion of Pcsk9 were treated with NTS to determine the contribution of PCSK9 to the dyslipidemia of nephrotic syndrome. RESULTS: Patients with nephrotic syndrome showed a decrease in plasma cholesterol and plasma PCSK9 on remission of their disease (P<0.05, n=47-50). Conversely, Pod-ATTAC mice and NTS-treated mice showed hypercholesterolemia and a 7- to 24-fold induction in plasma PCSK9. The induction of plasma PCSK9 appeared to be attributable to increased secretion of PCSK9 from the hepatocyte coupled with decreased clearance. Interestingly, knockout of Pcsk9ameliorated the effects of NTS on plasma lipids. Thus, in the presence of NTS, mice lacking hepatic Pcsk9 showed a 40% to 50% decrease in plasma cholesterol and triglycerides. Moreover, the ability of NTS treatment to increase the percentage of low-density lipoprotein-associated cholesterol (from 9% in vehicle-treated Flox mice to 47% after NTS treatment), was lost in mice with hepatic deletion of Pcsk9 (5% in both the presence and absence of NTS). CONCLUSIONS: Podocyte damage triggers marked inductions in plasma PCSK9, and knockout of Pcsk9 ameliorates dyslipidemia in a mouse model of nephrotic syndrome. These data suggest that PCSK9 inhibitors may be beneficial in patients with nephrotic syndrome-associated hypercholesterolemia.

Role of calcineurin (CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm.

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles ofCNremained in each type of the cells are not well understood yet. ACNinhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. ACNIis assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest thatCNIhas a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized thatCNIhas an effect on theSD In this study, we analyzed the expression ofCNin physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of theSD We observed thatCNis expressed at theSDin normal rat and human kidney sections and has an interaction with nephrin. The staining ofCNat theSDwas reduced in the nephrotic model, whileCNactivity in glomeruli was increased. We also observed that the treatment with tacrolimus, aCNI, in this nephrotic model suppressed the redistribution ofCN, nephrin, and otherSDcomponents and ameliorated proteinuria. These observations suggested that the redistribution and the activation ofCNmay participate in the development of theSDinjury.

Antibodies to m-type phospholipase A2 receptor in children with idiopathic membranous nephropathy.

Idiopathic membranous nephropathy (IMN), the commonest cause of adult nephrotic syndrome (NS), accounts for only a minority of paediatric NS. Antibodies to m-type phospholipase A2 receptor (PLA2R) are seen in two-thirds of adult IMN cases. PLA2R staining in glomerular deposits is observed in 74% and 45% of adult and paediatric IMN cases, respectively. However, there are no reports of anti-PLA2R in paediatric IMN. We evaluated anti-PLA2R levels and PLA2R in gloemrular deposits in paediatric IMN seen at our center. Five cases were enrolled, all the cases stained for PLA2R in glomeruli and three (60%) had antibodies to PLA2R antigen. There was a parellel reduction in proteinuria and anti-PLA2R titer. The present report suggests that PLA2R has a contributory role in the pathogenesis of paediatric IMN.

Nephroprotective effect of heparanase in experimental nephrotic syndrome.

BACKGROUND: Heparanase, an endoglycosidase that cleaves heparan sulfate (HS), is involved in various biologic processes. Recently, an association between heparanase and glomerular injury was suggested. The present study examines the involvement of heparanase in the pathogenesis of Adriamycin-induced nephrotic syndrome (ADR-NS) in a mouse model. METHODS: BALB/c wild-type (wt) mice and heparanase overexpressing transgenic mice (hpa-TG) were tail-vein injected with either Adriamycin (ADR, 10 mg/kg) or vehicle. Albuminuria was investigated at days 0, 7, and 14 thereafter. Mice were sacrificed at day 15, and kidneys were harvested for various analyses: structure and ultrastructure alterations, podocyte proteins expression, and heparanase enzymatic activity. RESULTS: ADR-injected wt mice developed severe albuminuria, while ADR-hpa-TG mice showed only a mild elevation in urinary albumin excretion. In parallel, light microscopy of stained cross sections of kidneys from ADR-injected wt mice, but not hpa-TG mice, showed mild to severe glomerular and tubular damage. Western blot and immunofluorescence analyses revealed significant reduction in nephrin and podocin protein expression in ADR-wt mice, but not in ADR-hpa-TG mice. These results were substantiated by electron-microscopy findings showing massive foot process effacement in injected ADR-wt mice, in contrast to largely preserved integrity of podocyte architecture in ADR-hpa-TG mice. CONCLUSIONS: Our results suggest that heparanase may play a nephroprotective role in ADR-NS, most likely independently of HS degradation. Moreover, hpa-TG mice comprise an invaluable in vivo platform to investigate the interplay between heparanase and glomerular injury.

Role of PCSK9 and IDOL in the pathogenesis of acquired LDL receptor deficiency and hypercholesterolemia in nephrotic syndrome.

BACKGROUND: Nephrotic syndrome (NS) leads to elevation of serum total and LDL cholesterol. This is largely due to impaired LDL clearance, which is caused by hepatic LDL receptor (LDLR) deficiency despite normal LDLR mRNA expression, pointing to a post-transcriptional process. The mechanism(s) by which NS causes LDLR deficiency is not known. By promoting degradation of LDLR, Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) and inducible degrader of the LDL receptor (IDOL) play a major role in post-translational regulation of LDLR. We, therefore, tested the hypothesis that LDLR deficiency despite its normal gene expression in NS may be due to upregulation of hepatic PCSK9 and IDOL. METHODS: LDLR, IDOL and PCSK9 expressions and nuclear translocation of liver X receptor (LXR) that regulates IDOL expression were determined in the liver of rats with puromycin-induced NS and control (CTL) rats. RESULTS: Compared with the CTLs, the NS rats showed marked elevation of serum total and LDL cholesterol and a significant reduction in hepatic LDLR protein expression. This was accompanied by marked upregulation of hepatic PCSK9 and IDOL expressions and heightened LXR activation. CONCLUSIONS: LDLR deficiency, hypercholesterolemia and elevated plasma LDL in NS are associated with upregulation of PCSK9 and IDOL. Interventions targeting these pathways may be effective in the management of hypercholesterolemia and the associated cardiovascular and other complications of NS.

ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling.

Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.

Inhibition of calcium(2+)/calmodulin-dependent protein kinase type IV ameliorates experimental nephrotic syndrome.

OBJECTIVE: Evidence has demonstrated that Ca(2+)/calmodulin-dependent protein kinase type IV (CaMKIV) contributes to altered cytokine production by promoting the production of inflammatory cytokines. This study aimed to explore the protective role and underlying mechanisms of CaMKIV inhibition in experimental nephrotic syndrome. METHODS: BALB/c mice received single intravenous injections of adriamycin (10 mg/kg) then were sacrificed at two, four and six weeks. In the second study, treatment with KN-93, a CaMKIV inhibitor, or vehicle administered via intraperitoneal injection was started five days after adriamycin injection. Functional and pathologic parameters, the presence of inflammatory infiltration and the expressions of pro-inflammatory cytokines were assessed. RESULTS: The CaMKIV protein expression levels were upregulated in the mice with adriamycin nephropathy, which was significantly inhibited by KN-93 (p<0.01). As compared with the vehicle-treated controls, KN-93 treatment resulted in marked suppression of proteinuria and serum creatinine at week 6 (p<0.01), but not at two weeks after induction of the disease. KN-93 inhibited glomerulosclerosis and the development of tubulointerstitial lesions. The renal alpha-smooth muscle actin (α-SMA) expression was also significantly suppressed by KN-93 treatment at week 6 (p<0.01). Moreover, KN-93 inhibited the renal monocyte chemoattractant protein-1 (MCP-1) expression, paralleled by a reduction in the interstitial infiltration of macrophages and T-cells (p<0.01). CONCLUSION: Our findings suggest that activation of CaMKIV signaling is involved in the progression of glomerular diseases with a proteinuric state. Our data therefore justify the development of small molecule CaMKIV inhibitors for the treatment of clinical nephrotic syndrome.