Steroid-Resistant Nephrotic Syndrome-Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity.

BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.

Mutations in PRDM15 Are a Novel Cause of Galloway-Mowat Syndrome.

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Whole-Exome Sequencing Enables a Precision Medicine Approach for Kidney Transplant Recipients.

BACKGROUND: Whole-exome sequencing (WES) finds a CKD-related mutation in approximately 20% of patients presenting with CKD before 25 years of age. Although provision of a molecular diagnosis could have important implications for clinical management, evidence is lacking on the diagnostic yield and clinical utility of WES for pediatric renal transplant recipients. METHODS: To determine the diagnostic yield of WES in pediatric kidney transplant recipients, we recruited 104 patients who had received a transplant at Boston Children's Hospital from 2007 through 2017, performed WES, and analyzed results for likely deleterious variants in approximately 400 genes known to cause CKD. RESULTS: By WES, we identified a genetic cause of CKD in 34 out of 104 (32.7%) transplant recipients. The likelihood of detecting a molecular genetic diagnosis was highest for patients with urinary stone disease (three out of three individuals), followed by renal cystic ciliopathies (seven out of nine individuals), steroid-resistant nephrotic syndrome (nine out of 21 individuals), congenital anomalies of the kidney and urinary tract (ten out of 55 individuals), and chronic glomerulonephritis (one out of seven individuals). WES also yielded a molecular diagnosis for four out of nine individuals with ESRD of unknown etiology. The WES-related molecular genetic diagnosis had implications for clinical care for five patients. CONCLUSIONS: Nearly one third of pediatric renal transplant recipients had a genetic cause of their kidney disease identified by WES. Knowledge of this genetic information can help guide management of both transplant patients and potential living related donors.

Panel sequencing distinguishes monogenic forms of nephritis from nephrosis in children.

BACKGROUND: Alport syndrome (AS) and atypical hemolytic-uremic syndrome (aHUS) are rare forms of chronic kidney disease (CKD) that can lead to a severe decline of renal function. Steroid-resistant nephrotic syndrome (SRNS) is more common than AS and aHUS and causes 10% of childhood-onset CKD. In recent years, multiple monogenic causes of AS, aHUS and SRNS have been identified, but their relative prevalence has yet to be studied together in a typical pediatric cohort of children with proteinuria and hematuria. We hypothesized that identification of causative mutations by whole exome sequencing (WES) in known monogenic nephritis and nephrosis genes would allow distinguishing nephritis from nephrosis in a typical pediatric group of patients with both proteinuria and hematuria at any level. METHODS: We therefore conducted an exon sequencing (WES) analysis for 11 AS, aHUS and thrombotic thrombocytopenic purpura-causing genes in an international cohort of 371 patients from 362 families presenting with both proteinuria and hematuria before age 25 years. In parallel, we conducted either WES or high-throughput exon sequencing for 23 SRNS-causing genes in all patients. RESULTS: We detected pathogenic mutations in 18 of the 34 genes analyzed, leading to a molecular diagnosis in 14.1% of families (51 of 362). Disease-causing mutations were detected in 3 AS-causing genes (4.7%), 3 aHUS-causing genes (1.4%) and 12 NS-causing genes (8.0%). We observed a much higher mutation detection rate for monogenic forms of CKD in consanguineous families (35.7% versus 10.1%). CONCLUSIONS: We present the first estimate of relative frequency of inherited AS, aHUS and NS in a typical pediatric cohort with proteinuria and hematuria. Important therapeutic and preventative measures may result from mutational analysis in individuals with proteinuria and hematuria.

Defects of CRB2 cause steroid-resistant nephrotic syndrome.

Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.

Analysis of 24 genes reveals a monogenic cause in 11.1% of cases with steroid-resistant nephrotic syndrome at a single center.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of end-stage renal disease (ESRD) among patients manifesting at under 25 years of age. We performed mutation analysis using a high-throughput PCR-based microfluidic technology in 24 single-gene causes of SRNS in a cohort of 72 families, who presented with SRNS before the age of 25 years. METHODS: Within an 18-month interval, we obtained DNA samples, pedigree information, and clinical information from 77 consecutive children with SRNS from 72 different families seen at Boston Children's Hospital (BCH). Mutation analysis was completed by combining high-throughput multiplex PCR with next-generation sequencing. We analyzed the sequences of 18 recessive and 6 dominant genes of SRNS in all 72 families for disease-causing variants. RESULTS: We identified the disease-causing mutation in 8 out of 72 (11.1%) families. Mutations were detected in the six genes: NPHS1 (2 out of 72), WT1 (2 out of 72), NPHS2, MYO1E, TRPC6, and INF2. Median age at onset was 4.1 years in patients without a mutation (range 0.5-18.8), and 3.2 years in those in whom the causative mutation was detected (range 0.1-14.3). Mutations in dominant genes presented with a median onset of 4.5 years (range 3.2-14.3). Mutations in recessive genes presented with a median onset of 0.5 years (range 0.1-3.2). CONCLUSION: Our molecular genetic diagnostic study identified underlying monogenic causes of steroid-resistant nephrotic syndrome in ~11% of patients with SRNS using a cost-effective technique. We delineated some of the therapeutic, diagnostic, and prognostic implications. Our study confirms that genetic testing is indicated in pediatric patients with SRNS.

Mutations in EMP2 cause childhood-onset nephrotic syndrome.

Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.

Spectrum of mutations in Chinese children with steroid-resistant nephrotic syndrome.

BACKGROUND: The aim of this study was to elucidate whether genetic screening test results of pediatric patients with steroid-resistant nephrotic syndrome (SRNS) vary with ethnicity. METHODS: Using high-throughput DNA sequencing, 28 nephrotic syndrome-related genes were analyzed in 110 chil-dren affected by SRNS and 10 children with isolated proteinuria enrolled by 5 centers in China (67 boys, 53 girls). Their age at disease onset ranged from 1 day to 208 months (median, 48.8 months). Patients were excluded if their age at onset of disease was over 18 years or if they were diagnosed as having Alport syndrome. RESULTS: A genetic etiology was identified in 28.3% of our cohort and the likelihood of establishing a genetic diagnosis decreased as the age at onset of nephrotic syndrome increased. The most common mutated genes were ADCK4 (6.67%), NPHS1 (5.83%), WT1 (5.83%), and NPHS2 (3.33%), and the difference in the frequencies of ADCK4 and NPHS2 mutations between this study and a study on monogenic causes of SRNS in the largest international cohort of 1,783 different families was significant. A case of congenital nephrotic syndrome was attributed to a homozygous missense mutation in ADCK4, and a de novo missense mutation in TRPC6 was detected in a case of infantile nephrotic syndrome. CONCLUSIONS: Our results showed that, in the first and the largest multicenter cohort of Chinese pediatric SRNS reported to date, ADCK4 is the most common causative gene, whereas there is a low prevalence of NPHS2 mutations. Our data indicated that the genetic testing results for pediatric SRNS patients vary with different ethnicities, and this information will help to improve management of the disease in clinical practice.

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Genetic testing in steroid-resistant nephrotic syndrome: when and how?

Steroid-resistant nephrotic syndrome (SRNS) represents the second most frequent cause of chronic kidney disease in the first three decades of life. It manifests histologically as focal segmental glomerulosclerosis (FSGS) and carries a 33% risk of relapse in a renal transplant. No efficient treatment exists. Identification of single-gene (monogenic) causes of SRNS has moved the glomerular epithelial cell (podocyte) to the center of its pathogenesis. Recently, mutations in >30 recessive or dominant genes were identified as causing monogenic forms of SRNS, thereby revealing the encoded proteins as essential for glomerular function. These findings helped define protein interaction complexes and functional pathways that could be targeted for treatment of SRNS. Very recently, it was discovered that in the surprisingly high fraction of ∼30% of all individuals who manifest with SRNS before 25 years of age, a causative mutation can be detected in one of the ∼30 different SRNS-causing genes. These findings revealed that SRNS and FSGS are not single disease entities but rather are part of a spectrum of distinct diseases with an identifiable genetic etiology. Mutation analysis should be offered to all individuals who manifest with SRNS before the age of 25 years, because (i) it will provide the patient and families with an unequivocal cause-based diagnosis, (ii) it may uncover a form of SRNS that is amenable to treatment (e.g. coenzyme Q10), (iii) it may allow avoidance of a renal biopsy procedure, (iv) it will further unravel the puzzle of pathogenic pathways of SRNS and (v) it will permit personalized treatment options for SRNS, based on genetic causation in way of 'precision medicine'.

Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

A single-gene cause in 29.5% of cases of steroid-resistant nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of ESRD in the first two decades of life. Effective treatment is lacking. First insights into disease mechanisms came from identification of single-gene causes of SRNS. However, the frequency of single-gene causation and its age distribution in large cohorts are unknown. We performed exon sequencing of NPHS2 and WT1 for 1783 unrelated, international families with SRNS. We then examined all patients by microfluidic multiplex PCR and next-generation sequencing for all 27 genes known to cause SRNS if mutated. We detected a single-gene cause in 29.5% (526 of 1783) of families with SRNS that manifested before 25 years of age. The fraction of families in whom a single-gene cause was identified inversely correlated with age of onset. Within clinically relevant age groups, the fraction of families with detection of the single-gene cause was as follows: onset in the first 3 months of life (69.4%), between 4 and 12 months old (49.7%), between 1 and 6 years old (25.3%), between 7 and 12 years old (17.8%), and between 13 and 18 years old (10.8%). For PLCE1, specific mutations correlated with age of onset. Notably, 1% of individuals carried mutations in genes that function within the coenzyme Q10 biosynthesis pathway, suggesting that SRNS may be treatable in these individuals. Our study results should facilitate molecular genetic diagnostics of SRNS, etiologic classification for therapeutic studies, generation of genotype-phenotype correlations, and the identification of individuals in whom a targeted treatment for SRNS may be available.

Epithelial cell states associated with kidney and allograft injury.

The kidney epithelium, with its intricate arrangement of highly specialized cell types, constitutes the functional core of the organ. Loss of kidney epithelium is linked to the loss of functional nephrons and a subsequent decline in kidney function. In kidney transplantation, epithelial injury signatures observed during post-transplantation surveillance are strong predictors of adverse kidney allograft outcomes. However, epithelial injury is currently neither monitored clinically nor addressed therapeutically after kidney transplantation. Several factors can contribute to allograft epithelial injury, including allograft rejection, drug toxicity, recurrent infections and postrenal obstruction. The injury mechanisms that underlie allograft injury overlap partially with those associated with acute kidney injury (AKI) and chronic kidney disease (CKD) in the native kidney. Studies using advanced transcriptomic analyses of single cells from kidney or urine have identified a role for kidney injury-induced epithelial cell states in exacerbating and sustaining damage in AKI and CKD. These epithelial cell states and their associated expression signatures are also observed in transplanted kidney allografts, suggesting that the identification and characterization of transcriptomic epithelial cell states in kidney allografts may have potential clinical implications for diagnosis and therapy.

Coping strategies and depressiveness in primary systemic vasculitis--what is their impact on health-related quality of life?

OBJECTIVE: To investigate the influence of disease-related coping strategies and depressiveness on health-related quality of life (HRQOL) in primary systemic vasculitis (PSV) patients. METHODS: One hundred and twenty-two patients with definite diagnosis of PSV were examined in a cross-sectional study. HRQOL (SF-36), depressiveness (BDI), illness perception (B-IPQ) and coping strategies (FKV-LIS) were measured using validated instruments. Additional disease-related and demographic data were retrieved from the patients' records. RESULTS: HRQOL in PSV patients was reduced compared with the SF-36 norm sample. Specific organ manifestation, size of vessel involvement and disease activity were not related to HRQOL. Linear regression modelling revealed a questionable relationship of emotional to physical HRQOL (P = 0.003, potential suppression effect of BDI), whereas both domains were influenced by depressiveness (P ≤ 0.001). Physical HRQOL was additionally related to fatigue and widowed marital status, while emotional HRQOL was associated with a depressive coping style. CONCLUSION: HRQOL is impaired in PSV as compared with the general population. Current depressiveness strongly affects physical as well as mental HRQOL. Cognitive intervention strategies should be established in order to improve quality of life in PSV patients.

Circulating endothelial cells as potential diagnostic biomarkers in primary central nervous system vasculitis.

OBJECTIVE: Histological evidence is considered the only proof of primary central nervous system vasculitis (PCNSV). However, brain biopsy is often omitted or delayed because of the invasiveness and possible complications of the procedure. Circulating endothelial cells (CEC) were shown to be elevated in patients with active antineutrophil cytoplasmic antibody-associated vasculitis. We hypothesise that CEC are also elevated in patients with active PCNSV and may contribute to the diagnosis. METHODS: CEC were assessed in 18 patients, 3 of whom had biopsy-proven PCNSV and 15 clinical, cerebrospinal fluid and imaging data, highly suggestive of PCNSV. In 3 of these 15 patients CEC assessment was performed after initiation of successful immunosuppressive therapy. CEC numbers of all patients were compared to those of 16 healthy volunteers and 123 subjects with cerebrovascular risk factors and/or ischaemic stroke, who had been studied in our group before. CEC were assessed by immunomagnetic isolation from peripheral blood. RESULTS: In patients with proven and suspected active PCNSV, CEC were extremely elevated (>400 cells/ml in most of the patients) and significantly higher than in healthy and disease controls (p≤0.01 for each group). CEC significantly decreased with immunosuppressive treatment. CONCLUSIONS: For the first time it is shown that CEC are significantly elevated in patients with active PCNSV in contrast to other pathologies associated with brain infarction and correlate with disease activity. Sensitivity and specificity of the method for diagnosing PCNSV and the use of the method for treatment monitoring should be addressed in future prospective studies with a larger patient group.

Treatment of autoimmunity: The impact of disease-modifying therapies in multiple sclerosis and comorbid autoimmune disorders.

More than 10 disease-modifying therapies (DMT) are approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for the treatment of multiple sclerosis (MS) and new therapeutic options are on the horizon. Due to different underlying therapeutic mechanisms, a more individualized selection of DMTs in MS is possible, taking into account the patient's current situation. Therefore, concomitant treatment of various comorbid conditions, including autoimmune mediated disorders such as rheumatoid arthritis, should be considered in MS patients. Because the pathomechanisms of autoimmunity partially overlap, DMT could also treat concomitant inflammatory diseases and simplify the patient's treatment. In contrast, the exacerbation and even new occurrence of several autoimmune diseases have been reported as a result of immunomodulatory treatment of MS. To simplify treatment and avoid disease exacerbation, knowledge of the beneficial and adverse effects of DMT in other autoimmune disorders is critical. Therefore, we conducted a literature search and described the beneficial and adverse effects of approved and currently studied DMT in a large number of comorbid autoimmune diseases, including rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel diseases, cutaneous disorders including psoriasis, Sjögren´s syndrome, systemic lupus erythematosus, systemic vasculitis, autoimmune hepatitis, and ocular autoimmune disorders. Our review aims to facilitate the selection of an appropriate DMT in patients with MS and comorbid autoimmune diseases.

Arteriolar vascular smooth muscle cell differentiation in benign nephrosclerosis.

BACKGROUND: Benign nephrosclerosis (bN) is the most prevalent form of hypertensive damage in kidney biopsies. It is defined by early hyalinosis and later fibrosis of renal arterioles. Despite its high prevalence, very little is known about the contribution of arteriolar vascular smooth muscle cells (VSMCs) to bN. We examined classical and novel candidate markers of the normal contractile and the pro-fibrotic secretory phenotype of VSMCs in arterioles in bN. METHODS: Sixty-three renal tissue specimens with bN and eight control specimens were examined by immunohistochemistry for the contractile markers caldesmon, alpha-smooth muscle actin (alpha-SMA), JunB, smoothelin and the secretory marker S100A4 and by double stains for caldesmon or smoothelin with S100A4. RESULTS: Smoothelin immunostaining showed an inverse correlation with hyalinosis and fibrosis scores, while S100A4 correlated with fibrosis scores only. Neither caldesmon, alpha-SMA nor JunB correlated with hyalinosis or fibrosis scores. Cells in the arteriolar wall were exclusively positive either for caldesmon/smoothelin or S100A4. CONCLUSIONS: This is the first systematic analysis of VSMC differentiation in bN. The results suggest that smoothelin is the most sensitive marker for the contractile phenotype and that S100A4 could be a novel marker for the secretory phenotype in vivo. The other markers did not seem to differentiate these phenotypes in bN. Thus, VSMC phenotype markers should be defined in the context of the vessel segment and disease under examination. S100A4 could not only be a marker of pro-fibrotic secretory VSMCs in bN but also an important mediator of arteriolar fibrosis.

Unilateral nephrectomy causes an abrupt increase in inflammatory mediators and a simultaneous decrease in plasma ADMA: a study in living kidney donors.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases (NOS). Reducing inducible NOS activity in acute inflammation seems to be desirable. In vitro data show that ADMA increases in response to inflammatory mediators, yet the effect of acute inflammation in vivo is scarcely studied. The aim of the study was to evaluate ADMA plasma levels before, during, and after the acute (nonbacterial) inflammatory-like state. Plasma ADMA, l-arginine, C-reactive protein, and IL-6 were determined in 24 healthy subjects undergoing living related kidney donation before as well as 1, 6, 12, 24, 72, and 168 h thereafter. Six hours after nephrectomy, ADMA levels decreased compared with baseline (0.488 ± 0.075 vs. 0.560 ± 0.060 µmol/l, P < 0.05). This difference became even more marked 24 h after the operation (0.478 ± 0.083 µmol/l, P < 0.01 vs. baseline), when the proinflammatory cytokine IL-6 peaked. Seven days after unilateral nephrectomy, ADMA levels were elevated above baseline (0.63 ± 0.05 µmol/l, P < 0.001 vs. baseline). l-Arginine levels decreased already 1 h after nephrectomy (97.5 ± 22.5 µmol/l, P < 0.01 vs. baseline) and paralleled the change in ADMA thereafter. At the end of the observation period when inflammation markers were regressing, l-arginine levels were significantly elevated above baseline (160.6 ± 25.1 µmol/l, P < 0.001 vs. baseline). In summary, this is the first study showing that both ADMA and l-arginine decrease temporarily after unilateral nephrectomy coinciding with the increase in inflammatory mediators. The l-arginine/ADMA ratio, a surrogate for NO production capacity, was only altered for <24 h.

ADAMTS13--marker of contractile phenotype of arterial smooth muscle cells lost in benign nephrosclerosis.

BACKGROUND: Hypertensive nephrosclerosis alone and in combination with other renal diseases is a leading cause of terminal renal insufficiency. Histologic lesions manifest as benign nephrosclerosis (bN) with arteriolar hyalinosis and later fibrosis. Procoagulant micromilieus have been implicated in fibrosis. Hyalinosis is considered to consist of plasma insudation possibly containing procoagulant factors like von Willebrand factor (VWF). Therefore, it is hypothesized that VWF cleaving protease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif, 13) is normally expressed by arteriolar vascular smooth muscle cells (VSMCs) and diminished in bN and that this reduction contributes to fibrosis in bN. METHODS: ADAMTS13 expression was examined by immunohistochemistry and quantitative real-time polymerase chain reaction in VSMCs of various human organs. Fifty-four specimens with and seven without bN were immunostained for ADAMTS13, VWF, CD61 and VSMC differentiation markers in arteriolar walls. RESULTS: Expression of ADAMTS13 is confirmed in VSMCs. In bN, ADAMTS13 immunostaining of arterial VSMCs correlated inversely with fibrotic but not hyalinotic lesions. Smooth muscle myosin heavy chain showed an inverse correlation with hyalinotic, as opposed to fibrotic lesions of bN. Smoothelin showed an inverse correlation with both hyalinotic and fibrotic lesions of bN. VWF was absent in normal controls and hyalinotic lesions, but present exclusively in fibrotic lesions in 7/54 (13%) bN cases. CD61 was absent in all arteriolar walls. CONCLUSIONS: The present results establish ADAMTS13 as a novel marker of contractile VSMCs that is retained in early hyalinotic bN but partially lost later in fibrotic bN. Loss of ADAMTS13 and accumulation of VWF in fibrotic but not hyalinotic arteriolar walls could further propagate fibrosis in bN.