Exosomal and Plasma Non-Coding RNA Signature Associated with Urinary Albumin Excretion in Hypertension

Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, plays important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature associated with early renal damage and its related molecular pathways. Three individual libraries (plasma and urinary exosomes, and total plasma) were prepared from each hypertensive patient (with or without albuminuria) for ncRNA sequencing analysis. Next, an RNA-based transcriptional regulatory network was constructed. The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNAs). We identified a common 24 ncRNA molecular signature related to hypertension-associated urinary albumin excretion, of which lncRNAs were the most representative. In addition, the transcriptional regulatory network showed five lncRNAs (LINC02614, BAALC-AS1, FAM230B, LOC100505824 and LINC01484) and the miR-301a-3p to play a significant role in network organization and targeting critical pathways regulating filtration barrier integrity and tubule reabsorption. Our study found an ncRNA profile associated with albuminuria, independent of biofluid origin (urine or plasma, circulating or in exosomes) that identifies a handful of potential targets, which may be utilized to study mechanisms of albuminuria and cardiovascular damage.

A Tight Control of Non-Canonical TGF-β Pathways and MicroRNAs Downregulates Nephronectin in Podocytes

Nephronectin (NPNT) is an extracellular matrix protein in the glomerular basement membrane that is produced by podocytes and is important for the integrity of the glomerular filtration barrier. Upregulated transforming growth factor β (TGF-β) and altered NPNT are seen in different glomerular diseases. TGF-β downregulates NPNT and upregulates NPNT-targeting microRNAs (miRs). However, the pathways involved were previously unknown. By using selective inhibitors of the canonical, SMAD-dependent, and non-canonical TGF-β pathways, we investigated NPNT transcription, translation, secretion, and regulation through miRs in podocytes. TGF-β decreased NPNT mRNA and protein in cultured human podocytes. TGF-β-dependent regulation of NPNT was meditated through intracellular signaling pathways. Under baseline conditions, non-canonical pathways predominantly regulated NPNT post-transcriptionally. Podocyte NPNT secretion, however, was not dependent on canonical or non-canonical TGF-β pathways. The canonical TGF-β pathway was also dispensable for NPNT regulation after TGF-β stimulation, as TGF-β was still able to downregulate NPNT in the presence of SMAD inhibitors. In contrast, in the presence of different non-canonical pathway inhibitors, TGF-β stimulation did not further decrease NPNT expression. Moreover, distinct non-canonical TGF-β pathways mediated TGF-β-induced upregulation of NPNT-targeting miR-378a-3p. Thus, we conclude that post-transcriptional fine-tuning of NPNT expression in podocytes is mediated predominantly through non-canonical TGF-β pathways.

Evaluation of Clinical and Follow-up Results of Patients with Congenital Nephrotic Syndrome

Congenital nephrotic syndrome (CNS) is characterized by severe proteinuria, hypoalbuminemia, and edema within the first three months of life. Congenital nephrotic syndrome can occur due to perinatal infections or mutation of genes encoding structural or regulatory proteins of the glomerular filtration barrier. Treatment includes albumin infusions, nephrectomy, dialysis, and transplantation. In this study, we aimed to evaluate the demographic, clinical, and follow-up results of patients with CNS followed up in our center between 2010 and 2020. Demographic, clinical, laboratory values of 8 patients diagnosed with CNS between 2010 and 2020, kidney biopsy results, genetic examinations, and follow-up results were retrospectively evaluated. A total of 8 patients (4 girls) were included in this study. The median age at diagnosis was 36 days (3 days-8 months) and the follow-up period was 34 months (7-114 months). There was a history of prematurity and consanguinity in 5 patients. Edema was detected at the admission of all patients. Albumin infusion and captopril therapy were started from the diagnosis. No pathology was seen in the tests for perinatal infection, and ultrasonographic examinations were normal. In the genetic analysis, NPHS1 (Nephrin) homozygous mutation was detected in six patients, and coenzyme Q2 mutation was detected in one patient. Peritoneal dialysis treatment was performed in four patients during the follow-up, and unilateral nephrectomy was completed in one patient. During the follow-up, four of eight patients (three due to sepsis while on dialysis, one on the postoperative after the first day of transplantation) died. Three patients are followed up with kidney transplantation and one with supportive treatment. According to our results, most CNS cases are genetic, and nephrin mutation is the most common cause. Management of complications in CNS is crucial for patient surviva

Neonatal Proteinuria in Calves—A Quantitative Approach

Urine testing is a convenient, non-invasive method of obtaining information about body functions. Depending on the intended purpose, urine testing may be qualitative and/or quantitative. Urine analysis can also include proteins. There are no data in the literature on the occurrence of proteinuria in healthy neonatal calves. The present study was the first that aimed to quantify the hypothesis of proteinuria occurrence in these animals in the first week of life, to assess its intensity and dynamics and to understand the underlying causes of proteinuria in healthy calves. The research was carried out on 15 healthy calves in the first seven days of life. Calves were catheterized to determine minute diuresis. Total protein concentration was determined in blood plasma and urine. Urine proteins were separated by electrophoresis (SDSPAGE) and their concentration and percentage were determined by densitometry using an image archiving and analysis software. The separated proteins were divided into three groups according to molecular weight for albumin, LMW and HMW proteins. The results were standardized per 1 m2 of body surface area and statistically analyzed. Neonatal proteinuria was demonstrated in healthy calves, mainly resulting from the high concentration of LMW proteins in the urine. Their percentages decreased significantly from 84.46% on the first day of calves’ life to 64.02% on day 7. At the same time, a statistically significant increase was observed in the proportion of albumin and high molecular weight proteins in urine total protein. Albumin percentage increased from 9.54% (on day 1) to almost 20% (on day 7), while the proportion of HMW proteins increased from 6.68% to 18.13%, respectively. The concentration of total protein in the urine of newborn calves amounted to 14.64 g/L and decreased statistically significantly during the first 72 h of postnatal life, stabilizing at the level of 3–4 g/L. The mean value of total protein excretion in the first week of life was 4.81 mg/min/m2 (i.e., 6.93 g/24 h/m2). The analysis of protein concentration in the urine and its excretion, as well as changes in urinary excretion of the tested protein fractions, indicated that neonatal proteinuria in healthy neonatal calves was tubular (i.e., main reason is the reduced absorption of proteins in nephrons). In addition, research showed that there was a rapid improvement in resorptive mechanisms in tubular cells. It should be assumed that the filtration barrier in the kidneys of these animals after birth is morphologically prepared to retain high molecular weight proteins. It seems that the increased permeability of the filtration barrier in the glomeruli does not necessarily indicate the immaturity of the kidneys, but may indicate the kidneys’ adaptation to excess protein removal from the body during feeding with high-protein food (colostrum), with an open intestinal barrier enabling protein absorption from the gastrointestinal tract to the blood.

The Pathophysiologic Role of Gelsolin in Chronic Kidney Disease: Focus on Podocytes

Chronic kidney disease (CKD) is normally related to proteinuria, a common finding in a compromised glomerular filtration barrier (GFB). GFB is a structure composed of glomerular endothelial cells, the basement membrane, and the podocytes. CKD with podocyte damage may be associated with actin cytoskeleton reorganization, resulting in podocyte effacement. Gelsolin plays a critical role in several diseases, including cardiovascular diseases and cancer. Our current study aimed to determine the connection between gelsolin and podocyte, and thus the mechanism underlying podocyte injury in CKD. Experiments were carried out on Drosophila to demonstrate whether gelsolin had a physiological role in maintaining podocyte. Furthermore, the survival rate of gelsolin-knocked down Drosophila larvae was extensively reduced after AgNO3 exposure. Secondly, the in vitro podocytes treated with puromycin aminonucleoside (PAN) enhanced the gelsolin protein expression, as well as small GTPase RhoA and Rac1, which also regulated actin dynamic expression incrementally with the PAN concentrations. Thirdly, we further demonstrated in vivo that GSN was highly expressed inside the glomeruli with mitochondrial dysfunction in a CKD mouse model. Our findings suggest that an excess of gelsolin may contribute to podocytes damage in glomeruli.

The podocyte-specific knockout of palladin in mice with a 129 genetic background affects podocyte morphology and the expression of palladin interacting proteins

Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background.

Three-Dimensional Super-Resolved Imaging of Paraffin-Embedded Kidney Samples

Background: Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. Methods: We here apply previously published sample preparation methods together with STED and confocal microscopy for resolving nanoscale podocyte substructure. The protocols are modified and optimized in order to be applied to samples which have been formalin fixed and paraffin-embedded (FFPE). Results: We successfully modify our protocols to allow for deep three-dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. We further show that quantitative analysis can be applied to extract morphometrics of healthy and diseased samples from both mice and humans. Conclusions: The results from this study could increase the feasibility to implement optical kidney imaging protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

MCC Regulator of WNT Signaling Pathway (MCC) Is a Podocyte Essential Gene

Podocytes are an integral part of the glomerular filtration barrier. Many genes are already known to be essential for podocyte survival, structure and function, but there are more podocyte essential genes to be identified. By single-cell RNA-seq of mouse podocytes, we detected the expression of gene encoding MCC regulator of WNT signaling pathway (MCC) in majority of the podocytes and speculated that MCC is essential for podocytes. We confirmed MCC expression in mouse podocytes and further showed its expression in human podocytes. To experimentally prove the essentiality of MCC for podocytes, we knocked down MCC in cultured podocytes and found marked morphological change of cell shape, cytoskeletal F-actin stress fiber disruption, increased apoptosis, and downregulation of podocyte essential genes, CD2AP and WT1, demonstrating that MCC is essential for podocytes. Since MCC has been implicated in cell cycle and β-catenin signaling, we examined the expression of cell cycle related genes and activity of β-catenin in the MCC knockdown podocytes, but did not find significant changes. To further explore the mechanism underlying the role of MCC in podocytes, we performed RNA-sequencing and bioinformatics analysis of MCC knockdown podocytes and found a significant enrichment of the regulated genes in lamellipodia formation. Consistently, we found that MCC is present in lamellipodia and MCC knockdown resulted in loss of lamellipodia in the cells. Lastly, we found that MCC was downregulated in podocytes treated with puromycin aminonucleosides and in glomeruli of diabetic mice and FSGS patients, implicating MCC is involved in the development of podocytopathy and proteinuria. In conclusion, MCC is potentially essential for podocytes and its downregulation may be involved in podocytopathy.

Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease

Background Diseases of the kidney's glomerular filtration barrier are a leading cause of end-stage renal failure. Despite of a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in focal segmental glomerulosclerosis (FSGS) patient cohorts. However, p.R229Q has been proposed to cause disease only when trans-associated to specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results. Methods To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q). We assessed the phenotype using super-resolution microscopy and albuminuria measurements, and evaluated the stability of the mutant protein in cell culture experiments. Results Heterozygous PodR231Q/wildtype mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous PodR231Q/wildtype mice showed a more severe course of disease compared with Podwildtype/wildtype mice. Podocin protein levels were decreased in PodR231Q/wildtype and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation Conclusions Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients