Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier.

SIGNIFICANCE STATEMENT: A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of ß1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function. BACKGROUND: Impairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown. METHODS: We used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and ß1 integrins. RESULTS: Depletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of ß1 integrins. CONCLUSIONS: These findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function.

Urinary phosphate is associated with cardiovascular disease incidence.

INTRODUCTION: Elevated phosphate (P) in urine may reflect a high intake of inorganic P salts from food additives. Elevated P in plasma is linked to vascular dysfunction and calcification. OBJECTIVE: To explore associations between P in urine as well as in plasma and questionnaire-estimated P intake, and incidence of cardiovascular disease (CVD). METHODS: We used the Swedish Mammography Cohort-Clinical, a population-based cohort study. At baseline (2004-2009), P was measured in urine and plasma in 1625 women. Dietary P was estimated via a food-frequency questionnaire. Incident CVD was ascertained via register-linkage. Associations were assessed using Cox proportional hazards regression. RESULTS: After a median follow-up of 9.4 years, 164 composite CVD cases occurred (63 myocardial infarctions [MIs] and 101 strokes). Median P (percentiles 5-95) in urine and plasma were 2.4 (1.40-3.79) mmol/mmol creatinine and 1.13 (0.92-1.36) mmol/L, respectively, whereas dietary P intake was 1510 (1148-1918) mg/day. No correlations were observed between urinary and plasma P (r = -0.07) or dietary P (r = 0.10). Urinary P was associated with composite CVD and MI. The hazard ratio of CVD comparing extreme tertiles was 1.57 (95% confidence interval 1.05, 2.35; P trend 0.037)-independently of sodium excretion, the estimated glomerular filtration rate, both P and calcium in plasma, and diuretic use. Association with CVD for plasma P was 1.41 (0.96, 2.07; P trend 0.077). CONCLUSION: Higher level of urinary P, likely reflecting a high consumption of highly processed foods, was linked to CVD. Further investigation is needed to evaluate the potential cardiovascular toxicity associated with excessive intake of P beyond nutritional requirements.

The role of dendrin in IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) and its systemic variant IgA vasculitis (IgAV) damage the glomeruli, resulting in proteinuria, hematuria and kidney impairment. Dendrin is a podocyte-specific protein suggested to be involved in the pathogenesis of IgAN. Upon cell injury, dendrin translocates from the slit diaphragm to the nucleus, where it is suggested to induce apoptosis and cytoskeletal changes, resulting in proteinuria and accelerated disease progression in mice. Here we investigated gene and protein expression of dendrin in relation to clinical and histopathological findings to further elucidate its role in IgAN/IgAV. METHODS: Glomerular gene expression was measured using microarray on 30 IgAN/IgAV patients, 5 patients with membranous nephropathy (MN) and 20 deceased kidney donors. Dendrin was spatially evaluated on kidney tissue sections by immunofluorescence (IF) staining (IgAN patients, n = 4; nephrectomized kidneys, n = 3) and semi-quantified by immunogold electron microscopy (IgAN/IgAV patients, n = 21; MN, n = 5; living kidney donors, n = 6). Histopathological grading was performed according to the Oxford and Banff classifications. Clinical data were collected at the time of biopsy and follow-up. RESULTS: Dendrin mRNA levels were higher (P = .01) in IgAN patients compared with MN patients and controls and most prominently in patients with preserved kidney function and fewer chronic histopathological changes. Whereas IF staining did not differ between groups, immunoelectron microscopy revealed that a higher relative nuclear dendrin concentration in IgAN patients was associated with a slower annual progression rate and milder histopathological changes. CONCLUSION: Dendrin messenger RNA levels and relative nuclear protein concentrations are increased and associated with a more benign phenotype and progression in IgAN/IgAV patients.

Serum levels of galactose-deficient IgA are elevated in patients with IgA nephropathy but do not correlate to disease activity or progression.

INTRODUCTION: IgA nephropathy (IgAN) is the most common glomerulonephritis globally. Because of the heterogeneity of the disease prognostic biomarkers are highly needed. AIM: To investigate associations between galactose-deficient IgA1 (Gd-IgA1) concentrations in plasma and urine and disease activity and progression in patients with IgAN. METHODS: Serum and urine samples were collected at the time of kidney biopsy (baseline) in patients with IgAN (n = 40) and analysed for Gd-IgA1. Patients with chronic kidney disease (CKD) without IgAN (n = 21) and healthy controls (n = 19) were examined as controls. In 19 patients with IgAN, analyses of Gd-IgA1 were repeated after a median follow up time of approximately 10 years. RESULTS: Serum Gd-IgA1 and Gd-IgA1:IgA were significantly elevated at the time of kidney biopsy in patients with IgAN compared to patients with non-IgAN CKD and healthy controls (p < 0.001). Urinary Gd-IgA1:creatinine was significantly elevated in patients with IgAN compared to patients with non-IgAN CKD. Neither serum Gd-IgA1, nor serum Gd-IgA1:IgA, correlated significantly to estimated GFR, urine albumin:creatinine (UACR), or blood pressure, at baseline. Serum Gd-IgA1 and Gd-IgA1:IgA at time of biopsy did not correlate significantly to annual changes in eGFR or UACR during follow up. In patients with IgAN, serum Gd-IgA1 decreased significantly over time during approximately 10 years of follow up (Δ-20 ± 85%, p = 0.027). Urinary Gd-IgA1:creatinine showed a strong positive correlation to UACR in patients with IgAN and likely reflected unspecific glomerular barrier injury. CONCLUSION: Although serum Gd-IgA1 and the Gd-IgA1:IgA ratio were significantly elevated in patients with IgAN at the time of kidney biopsy they were not related to disease activity or progression in this patient cohort.

Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A.

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-ß) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.

Localization and Regulation of Polymeric Ig Receptor in Healthy and Diseased Human Kidney.

The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR in vitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-ß1 caused decreased expression. Furthermore, interferon-γ increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.

Novel insights into the disease transcriptome of human diabetic glomeruli and tubulointerstitium.

BACKGROUND: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown. METHODS: RNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30-85) years, chronic kidney disease stages 1-4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30-70) years]. RESULTS: Principal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients. CONCLUSIONS: Here we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.

GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway.

BACKGROUND: Inflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood. METHODS: We performed large-scale gene expression profiling of glomerulus-associated G protein-coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes. RESULTS: We identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-κB signaling. CONCLUSIONS: GPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-κB signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.

Identification of cell and disease specific microRNAs in glomerular pathologies.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR-screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. We performed a combined miR-screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells and human tubular cells. The miR-screening in renal cells was done in untreated conditions and after stimulation with TGF-ß. A merge of the detected regulated miRs led us to identify disease-specific, cell type-specific and cell stress-induced miRs. Most miRs were down-regulated following the stimulation with TGF-ß in all cell types. Up-regulation of miRs after TGF-ß was cell type-specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR-155 was up-regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, a combined urinary and cell miR-screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.

Novel ELISA for thrombospondin type 1 domain-containing 7A autoantibodies in membranous nephropathy.

Autoantibodies against phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain-containing 7A (THSD7A) are emerging as biomarkers to classify membranous nephropathy (MN) and to predict outcome or response to treatment. Anti-THSD7A autoantibodies are detected by Western blot and indirect immunofluorescence test (IIFT). Here, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) optimized for quantitative detection of anti-THSD7A autoantibodies. Among 1012 biopsy-proven MN patients from 6 cohorts, 28 THSD7A-positive patients were identified by ELISA, indicating a prevalence of 2.8%. By screening additional patients, mostly referred because of PLA2R1-unrelated MN, we identified 21 more cases, establishing a cohort of 49 THSD7A-positive patients. Twenty-eight patients (57%) were male, and male patients were older than female patients (67 versus 49 years). Eight patients had a history of malignancy, but only 3 were diagnosed with malignancy within 2 years of MN diagnosis. We compared the results of ELISA, IIFT, Western blot, and biopsy staining, and found a significant correlation between ELISA and IIFT titers. Anti-THSD7A autoantibodies were predominantly IgG4 in all patients. Eight patients were double positive for THSD7A and PLA2R1. Levels of anti-THSD7A autoantibodies correlated with disease activity and with response to treatment. Patients with high titer at baseline had poor clinical outcome. In a subgroup of patients with serial titers, persistently elevated anti-THSD7A autoantibodies were observed in patients who did not respond to treatment or did not achieve remission. We conclude that the novel anti-THSD7A ELISA can be used to identify patients with THSD7A-associated MN and to monitor autoantibody titers during treatment.

Mice exposed to maternal androgen excess and diet-induced obesity have altered phosphorylation of catechol-O-methyltransferase in the placenta and fetal liver.

BACKGROUND/OBJECTIVES: Maternal obesity together with androgen excess in mice negatively affects placental function and maternal and fetal liver function as demonstrated by increased triglyceride content with dysfunctional expression of enzymes and transcription factors involved in de novo lipogenesis and fat storage. To identify changes in molecular pathways that might promote diseases in adulthood, we performed a global proteomic analysis using a liquid-chromatography/mass-spectrometry system to investigate total and phosphorylated proteins in the placenta and fetal liver in a mouse model that combines maternal obesity with maternal androgen excess. METHODS: After ten weeks on a control diet (CD) or high fat/high sugar-diet, dams were mated with males fed the CD. Between gestational day (GD) 16.5 and GD 18.5, mice were injected with vehicle or dihydrotestosterone (DHT) and sacrificed at GD 18.5 prior to dissection of the placentas and fetal livers. Four pools of female placentas and fetal livers were subjected to a global proteomic analysis. Total and phosphorylated proteins were filtered by ANOVA q < 0.05, and this was followed by two-way ANOVA to determine the effect of maternal obesity and/or androgen exposure. RESULTS: In placenta, phosphorylated ATP-citrate synthase was decreased due to maternal obesity, and phosphorylated catechol-O-methyltransferase (COMT) was differentially expressed due to the interaction between maternal diet and DHT exposure. In fetal liver, five total proteins and 48 proteins phosphorylated in one or more sites, were differentially expressed due to maternal obesity or androgen excess. In fetal liver, phosphorylated COMT expression was higher in fetus exposed to maternal obesity. CONCLUSION: These results suggest a common regulatory mechanism of catecholamine metabolism in the placenta and the fetal liver as demonstrated by higher phosphorylated COMT expression in the placenta and fetal liver from animals exposed to diet-induced maternal obesity and lower expression of phosphorylated COMT in animals exposed to maternal androgen excess.

A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell-derived kidney model for drug discovery.

Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.

Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy.

IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type-specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell-positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell-positive standard genes. Additionally, patient clinical parameters (serum creatinine values and eGFRs) significantly correlated with Z scores derived from the expression profile of mesangial cell-positive standard genes. Among patients grouped according to Oxford MEST score, patients with segmental glomerulosclerosis had a significantly higher mesangial cell-positive standard gene Z score than patients without segmental glomerulosclerosis. By investigating mesangial cell proteomics and glomerular transcriptomics, we identified 22 common pathways induced in mesangial cells by gd-IgA, most of which mediate inflammation. The genes, proteins, and corresponding pathways identified provide novel insights into the pathophysiologic mechanisms leading to IgAN.

Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases.

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

New insights into crosstalk in the kidney.

PURPOSE OF REVIEW: The kidney is a highly complex organ and renal function depends on many factors, both extrinsic to the kidney and intrinsic. The kidney responds both to systemic hormonal and neuronal signals and to autocrine and paracrine factors produced within the renal tissue. Recently, there has been an increased emphasis on crosstalk in and between different compartments in the kidney. RECENT FINDINGS: Crosstalk in the kidney between different cellular compartments has added new and important understanding of renal function and the development of kidney disease. SUMMARY: Most of the literature cited concern glomerular crosstalk but also tubular and interstitial crosstalk are being reviewed. Mechanistic insight into the communication between the cells may help us find new targets for treating kidney disease.

Long-term clinical outcome for patients poisoned by the fungal nephrotoxin orellanine.

BACKGROUND: Accidental intake of mushrooms of the Cortinarius species (deadly webcap) may cause irreversible renal damage and the need for dialysis or transplantation. The species is found in forests of Northern Europe, Scandinavia and North America and may be mistaken for other edible mushrooms. The highly selective nephrotoxic compound of the mushroom is called orellanine. Very little is known about the long-term effects of the nephrotoxin. METHODS: We identified patients who ingested deadly webcap in the period of 1979 to 2012. Informed consent and medical records were obtained for 28 of the 39 cases that occurred during the 34-year period. A case control group was also studied based on sex, age and initiation of dialysis or transplantation. RESULTS: The average age at time of the accidental intake was 40 ± 3 (n = 28) years. 64% of patients were male, and 22 of 28 patients developed acute kidney injury requiring dialysis. Serum creatinine peaked at 1 329 ± 133 µmol/l, and serum urea was 31 ± 3.5 mmol/l. No signs of acute damage were present in any other organ. The average time of follow-up was 16.9 ± 2.1 years (1.24-34.3 years, n = 28). 15 patients were transplanted and 3 also had a second graft. At follow-up, 23 patients were alive, and five had died at ages of 67 ± 5 (range 54-84). The outcome was similar in the case control group with 6 deaths in 20 patients. CONCLUSION: We conclude that the long-term prognosis for patients poisoned by deadly webcap who lost their renal function is not different compared to other patients in active uremic care.

Melanocortin 1 receptor agonist protects podocytes through catalase and RhoA activation.

Drugs containing adrenocorticotropic hormone have been used as therapy for patients with nephrotic syndrome. We have previously shown that adrenocorticotropic hormone and a selective agonist for the melanocortin 1 receptor (MC1R) exert beneficial actions in experimental membranous nephropathy with reduced proteinuria, reduced oxidative stress, and improved glomerular morphology and function. Our hypothesis is that MC1R activation in podocytes elicits beneficial effects by promoting stress fibers and maintaining podocyte viability. To test the hypothesis, we cultured podocytes and used highly specific agonists for MC1R. Podocytes were subjected to the nephrotic-inducing agent puromycin aminonucleoside, and downstream effects of MC1R activation on podocyte survival, antioxidant defense, and cytoskeleton dynamics were studied. To increase the response and enhance intracellular signals, podocytes were transduced to overexpress MC1R. We showed that puromycin promotes MC1R expression in podocytes and that activation of MC1R promotes an increase of catalase activity and reduces oxidative stress, which results in the dephosphorylation of p190RhoGAP and formation of stress fibers through RhoA. In addition, MC1R agonists protect against apoptosis. Together, these mechanisms protect the podocyte against puromycin. Our findings strongly support the hypothesis that selective MC1R-activating agonists protect podocytes and may therefore be useful to treat patients with nephrotic syndromes commonly considered as podocytopathies.

Overexpression of TGF-ß Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans.

BACKGROUND: TGF-ß is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-ß. Since miR-143-3p (miR-143) is described as a TGF-ß inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. METHODS: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-ß. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. RESULTS: We detected a time dependent, TGF-ß inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. CONCLUSION: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

Mesangial cells from patients with IgA nephropathy have increased susceptibility to galactose-deficient IgA1.

BACKGROUND: IgA nephropathy (IgAN) is the most common glomerulonephritis in the world, affecting close to a million people. Circulating galactose-deficient IgA (gd-IgA), present in patients with IgAN, form immune complex deposits in the glomerular mesangium causing local proliferation and matrix expansion. Intriguing though, individuals having gd-IgA deposits in the kidneys do not necessarily have signs of glomerular disease. Recurrence of IgAN only occurs in less than half of transplanted patients with IgAN, indicating that gd-IgA is not the only factor driving the disease. We hypothesize that, in addition to IgA complexes, patients with IgAN possess a subtype of mesangial cells highly susceptible to gd-IgA induced cell proliferation. METHODS: To test the hypothesis, we designed a technique to culture primary mesangial cells from renal biopsies obtained from IgAN patients and controls. The cell response to gd-IgA treatment was then measured both on gene and protein level and the proliferation rate of the cells in response to PDGF was investigated. RESULTS: When treated with gd-IgA, mesangial cells from patients with IgAN express and release more PDGF compared to controls. In addition, the mesangial cells from patients with IgAN were more responsive to treatment with PDGF resulting in an increased proliferation rate of the cells compared to control. Mesangial cells cultured from patients with IgAN expressed and released more IL-6 than controls and had a higher expression of matrix genes. Both mesangial cells derived from patients with IgAN and controls increased their expressed TGFß1 and CCL5 when treated with gd-IgA. CONCLUSION: We conclude that mesangial cells derived from IgAN patients have a mesangioproliferative phenotype with increased reactivity to IgA and that these cellular intrinsic properties may be important for the development of IgA nephropathy.

Proteomic analysis shows decreased type I fibers and ectopic fat accumulation in skeletal muscle from women with PCOS.

Background: Polycystic ovary syndrome's (PCOS) main feature is hyperandrogenism, which is linked to a higher risk of metabolic disorders. Gene expression analyses in adipose tissue and skeletal muscle reveal dysregulated metabolic pathways in women with PCOS, but these differences do not necessarily lead to changes in protein levels and biological function. Methods: To advance our understanding of the molecular alterations in PCOS, we performed global proteomic and phosphorylation site analysis using tandem mass spectrometry, and analyzed gene expression and methylation. Adipose tissue and skeletal muscle were collected at baseline from 10 women with and without PCOS, and in women with PCOS after 5 weeks of treatment with electrical stimulation. Results: Perilipin-1, a protein that typically coats the surface of lipid droplets in adipocytes, was increased whereas proteins involved in muscle contraction and type I muscle fiber function were downregulated in PCOS muscle. Proteins in the thick and thin filaments had many altered phosphorylation sites, indicating differences in protein activity and function. A mouse model was used to corroborate that androgen exposure leads to a shift in muscle fiber type in controls but not in skeletal muscle-specific androgen receptor knockout mice. The upregulated proteins in muscle post treatment were enriched in pathways involved in extracellular matrix organization and wound healing, which may reflect a protective adaptation to repeated contractions and tissue damage due to needling. A similar, albeit less pronounced, upregulation in extracellular matrix organization pathways was also seen in adipose tissue. Conclusions: Our results suggest that hyperandrogenic women with PCOS have higher levels of extra-myocellular lipids and fewer oxidative insulin-sensitive type I muscle fibers. These could be key factors leading to insulin resistance in PCOS muscle while electric stimulation-induced tissue remodeling may be protective. Funding: Swedish Research Council (2020-02485, 2022-00550, 2020-01463), Novo Nordisk Foundation (NNF22OC0072904), and IngaBritt and Arne Lundberg Foundation. Clinical trial number NTC01457209.