Mahdi Salih

Urinary extracellular vesicles and the kidney: biomarkers and beyond.

Extracellular vesicles have been isolated in various body fluids, including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly pathophysiological state of cells lining the nephron. Because urine is a noninvasive and readily available biofluid, the discovery of uEVs has opened a new field of biomarker research. Their potential use as diagnostic, prognostic, or therapeutic biomarkers for various kidney diseases, including glomerulonephritis, acute kidney injury, tubular disorders, and polycystic kidney disease, is currently being explored. Some challenges, however, remain. These challenges include the need to standardize isolation methods, normalization between samples, and validation of candidate biomarkers. Also, the development of a high-throughput platform to isolate and analyze uEVs, for example, an enzyme-linked immunosorbent assay, is desirable. Here, we review recent studies on uEVs dealing with kidney physiology and pathophysiology. Furthermore, we discuss new and exciting developments regarding vesicles, including their role in cell-to-cell communication and the possibility of using vesicles as a therapy for kidney disorders.

The Phosphorylated Sodium Chloride Cotransporter in Urinary Exosomes Is Superior to Prostasin as a Marker for Aldosteronism

Urinary exosomes are vesicles derived from renal tubular epithelial cells. Exosomes often contain several disease-associated proteins and are thus useful targets for identifying biomarkers of disease. Here, we hypothesized that the phosphorylated (active) form of the sodium chloride cotransporter (pNCC) or prostasin could serve as biomarkers for aldosteronism. We tested this in 2 animal models of aldosteronism (aldosterone infusion or low-sodium diet) and in patients with primary aldosteronism. Urinary exosomes were isolated from 24-hour urine or spot urine using ultracentrifugation. In rats, a normal or a high dose of aldosterone for 2, 3, or 8 days increased pNCC 3-fold in urinary exosomes (P<0.05 for all). A low-sodium diet also increased pNCC in urinary exosomes approximately 1.5-fold after 4 and after 8 days of treatment. The effects of these maneuvers on prostasin in urinary exosomes were less clear, showing a significant 1.5-fold increase only after 2 and 3 days of high-aldosterone infusion. In urinary exosomes of patients with primary aldosteronism, pNCC was 2.6-fold higher (P<0.05) while prostasin was 1.5-fold higher (P=0.07) than in patients with essential hypertension. Urinary exosomal pNCC and, to a lesser extent, prostasin are promising markers for aldosteronism in experimental animals and patients. These markers may be used to assess the biological activity of aldosterone and, potentially, as clinical biomarkers for primary aldosteronism.

Rationale and design of the DIPAK 1 study: A randomized controlled clinical trial assessing the efficacy of lanreotide to halt disease progression in autosomal dominant polycystic kidney disease

BACKGROUND There are limited therapeutic options to slow the progression of autosomal dominant polycystic kidney disease (ADPKD). Recent clinical studies indicate that somatostatin analogues are promising for treating polycystic liver disease and potentially also for the kidney phenotype. We report on the design of the DIPAK 1 (Developing Interventions to Halt Progression of ADPKD 1) Study, which will examine the efficacy of the somatostatin analogue lanreotide on preservation of kidney function in ADPKD. STUDY DESIGN The DIPAK 1 Study is an investigator-driven, randomized, multicenter, controlled, clinical trial. SETTING & PARTICIPANTS We plan to enroll 300 individuals with ADPKD and estimated glomerular filtration rate (eGFR) of 30-60 mL/min/1.73 m(2) who are aged 18-60 years. INTERVENTION Patients will be randomly assigned (1:1) to standard care or lanreotide, 120 mg, subcutaneously every 28 days for 120 weeks, in addition to standard care. OUTCOMES Main study outcome is the slope through serial eGFR measurements starting at week 12 until end of treatment for lanreotide versus standard care. Secondary outcome parameters include change in eGFR from pretreatment versus 12 weeks after treatment cessation, change in kidney volume, change in liver volume, and change in quality of life. MEASUREMENTS Blood and urine will be collected and questionnaires will be filled in following a fixed scheme. Magnetic resonance imaging will be performed for assessment of kidney and liver volume. RESULTS Assuming an average change in eGFR of 5.2 ± 4.3 (SD) mL/min/1.73 m(2) per year in untreated patients, 150 patients are needed in each group to detect a 30% reduction in the rate of kidney function loss between treatment groups with 80% power, 2-sided α = 0.05, and 20% protocol violators and/or dropouts. LIMITATIONS The design is an open randomized controlled trial and measurement of our primary end point does not begin at randomization. CONCLUSIONS The DIPAK 1 Study will show whether subcutaneous administration of lanreotide every 4 weeks attenuates disease progression in patients with ADPKD.

Update on Controls for Isolation and Quantification Methodology of Extracellular Vesicles Derived from Adipose Tissue Mesenchymal Stem Cells

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.

Urinary extracellular vesicles as markers to assess kidney sodium transport.

Purpose of reviewThis article summarizes studies that have analyzed sodium transporters in urinary extracellular vesicles (uEVs) in relation to hypertension. Recent findingsThe majority of kidney sodium transporters are detectable in uEVs. Patients with loss or gain of function mutations in sodium transporter genes have concomitant changes in the abundances of their corresponding proteins in uEVs. The effects of aldosterone on kidney sodium transport, including activation of the sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC), are transferred to uEVs as increases in phosphorylated NCC and the &ggr;-subunit of ENaC. Specific forms of hypertension, including aldosteronism and pseudohypoaldosteronism, are characterized by higher abundances of total or phosphorylated NCC in uEVs. The proteolytic processing of ENaC by urinary proteases is detectable in uEVs as cleaved &ggr;-ENaC, as demonstrated in hypertensive patients with diabetic nephropathy. Analysis of uEVs from patients with essential or salt-sensitive hypertension identified potential candidates for uEV markers of hypertension, including retinoic acid-induced gene 2 protein and hsa-miR-4516. SummaryAnalysis of sodium transporters in uEVs is a promising approach to study renal epithelial transport processes noninvasively in human hypertension. Video abstracthttp://links.lww.com/CONH/A16.

A Missense Mutation in the Extracellular Domain of αENaC Causes Liddle Syndrome

Liddle syndrome is an autosomal dominant form of hypokalemic hypertension due to mutations in the β- or γ-subunit of the epithelial sodium channel (ENaC). Here, we describe a family with Liddle syndrome due to a mutation in αENaC. The proband was referred because of resistant hypokalemic hypertension, suppressed renin and aldosterone, and no mutations in the genes encoding β- or γENaC. Exome sequencing revealed a heterozygous, nonconservative T>C single-nucleotide mutation in αENaC that substituted Cys479 with Arg (C479R). C479 is a highly conserved residue in the extracellular domain of ENaC and likely involved in a disulfide bridge with the partner cysteine C394. In oocytes, the C479R and C394S mutations resulted in similar twofold increases in amiloride-sensitive ENaC current. Quantification of mature cleaved αENaC in membrane fractions showed that the number of channels did not increase with these mutations. Trypsin, which increases open probability of the channel by proteolytic cleavage, resulted in significantly higher currents in the wild type than in C479R or C394S mutants. In summary, a mutation in the extracellular domain of αENaC causes Liddle syndrome by increasing intrinsic channel activity. This mechanism differs from that of the β- and γ-mutations, which result in an increase in channel density at the cell surface. This mutation may explain other cases of patients with resistant hypertension and also provides novel insight into ENaC activation, which is relevant for kidney sodium reabsorption and salt-sensitive hypertension.

Proteomics of Urinary Vesicles Links Plakins and Complement to Polycystic Kidney Disease

Novel therapies in autosomal dominant polycystic kidney disease (ADPKD) signal the need for markers of disease progression or response to therapy. This study aimed to identify disease-associated proteins in urinary extracellular vesicles (uEVs), which include exosomes, in patients with ADPKD. We performed quantitative proteomics on uEVs from healthy controls and patients with ADPKD using a labeled approach and then used a label-free approach with uEVs of different subjects (healthy controls versus patients with ADPKD versus patients with non-ADPKD CKD). In both experiments, 30 proteins were consistently more abundant (by two-fold or greater) in ADPKD-uEVs than in healthy- and CKD-uEVs. Of these proteins, we selected periplakin, envoplakin, villin-1, and complement C3 and C9 for confirmation because they were also significantly overrepresented in pathway analysis and were previously implicated in ADPKD pathogenesis. Immunoblotting confirmed higher abundances of the selected proteins in uEVs from three independent groups of patients with ADPKD. Whereas uEVs of young patients with ADPKD and preserved kidney function already had higher levels of complement, only uEVs of patients with advanced stages of ADPKD had increased levels of villin-1, periplakin, and envoplakin. Furthermore, all five proteins correlated positively with total kidney volume. Analysis in kidney tissue from mice with kidney-specific, tamoxifen-inducible Pkd1 deletion demonstrated higher expression in more severe stages of the disease and correlation with kidney weight for each protein of interest. In summary, proteomic analysis of uEVs identified plakins and complement as disease-associated proteins in ADPKD. These proteins are new candidates for evaluation as biomarkers or targets for therapy in ADPKD.

An Immunoassay for Urinary Extracellular Vesicles

Although nanosized urinary extracellular vesicles (uEVs) are increasingly used for biomarker discovery, their isolation currently relies on time-consuming techniques hindering high-throughput application. To navigate this problem, we designed an immunoassay to isolate, quantify, and normalize uEV proteins. The uEV immunoassay consists of a biotinylated CD9 antibody to isolate uEVs, an antibody against the protein of interest, and two conjugated antibodies to quantify the protein of interest and CD9. As a proof of principle, the immunoassay was developed to analyze the water channel aquaporin-2 (AQP2) and the sodium-chloride cotransporter (NCC). CD9 was used as a capture antibody because immunoprecipitation showed that anti-CD9 antibody, but not anti-CD63 antibody, isolated AQP2 and NCC. CD9 correlated strongly with urine creatinine, allowing CD9 to be used for normalization of spot urines. The uEV immunoassay detected AQP2 and NCC with high sensitivity, low coefficients of variance, and stability in dilution series. After water loading in healthy subjects, the uEV immunoassay detected decreases in AQP2 and NCC equally well as the traditional method using ultracentrifugation and immunoblot. The uEV immunoassay also reliably detected lower and higher AQP2 or NCC levels in uEVs from patients with pathological water or salt reabsorption, respectively. In summary, we report a novel approach to analyze uEVs that circumvents existing isolation and normalization issues, requires small volumes of urine, and detects anticipated changes in physiological responses and clinical disorders.

Urinary renin-angiotensin markers in polycystic kidney disease

In autosomal dominant polycystic kidney disease (ADPKD), activation of the renin-angiotensin aldosterone system (RAAS) may contribute to hypertension and disease progression. Although previous studies have focused on circulating RAAS components, preliminary evidence suggests that APDKD may increase urinary RAAS components. Therefore, our aim was to analyze circulating and urinary RAAS components in ADPKD. We cross-sectionally compared 60 patients with ADPKD with 57 patients with non-ADPKD chronic kidney disease (CKD). The two groups were matched by sex, estimated glomerular filtration rate (eGFR), blood pressure, and RAAS inhibitor use. Despite similar plasma levels of angiotensinogen and renin, urinary angiotensinogen and renin excretion were five- to sixfold higher in ADPKD (P < 0.001). These differences persisted when adjusting for group differences and were present regardless of RAAS inhibitor use. In multivariable analyses, ADPKD, albuminuria, and the respective plasma concentrations were independent predictors for urinary angiotensinogen and renin excretion. In ADPKD, both plasma and urinary renin correlated negatively with eGFR. Total kidney volume correlated with plasma renin and albuminuria but not with urinary renin or angiotensinogen excretions. Albuminuria correlated positively with urinary angiotensinogen and renin excretions in ADPKD and CKD. In three ADPKD patients who underwent nephrectomy, the concentrations of albumin and angiotensinogen were highest in plasma, followed by cyst fluid and urine; urinary renin concentrations were higher than cyst fluid. In conclusion, this study shows that, despite similar circulating RAAS component levels, higher urinary excretions of angiotensinogen and renin are a unique feature of ADPKD. Future studies should address the underlying mechanism and whether this may contribute to hypertension or disease progression in ADPKD.

Bullous Pemphigoid With a Dual Pattern of Glomerular Immune Complex Disease

A 75-year-old man presented with a blistering skin disease and nephrotic syndrome. Bullous pemphigoid was diagnosed by linear immunoglobulin G (IgG) and C3 staining along the basement membrane zone of a skin biopsy specimen and by the presence of circulating IgG recognizing the 180-kDa bullous pemphigoid antigen (BP180; type XVII collagen). A kidney biopsy specimen showed endocapillary inflammation without crescents. Direct immunofluorescence showed strong IgG and C3 staining in a combined granular and linear pattern along the glomerular basement membrane. Electron microscopy showed subepithelial deposits. In serum, no antibodies against the Goodpasture antigen (type IV collagen) or phospholipase A2 receptor were detected. Indirect immunofluorescence studies using the patients serum showed a strikingly linear but not granular IgG pattern along the epithelial basement membranes of monkey esophagus and kidney. Although type XVII collagen was recently identified in the glomerulus, the patients serum did not produce a 180-kDa band on immunoblot of kidney tissue and still stained glomeruli of BP180 knockout mice by indirect immunofluorescence. The patient was treated with prednisone and azathioprine, which resulted in complete remission of skin and kidney manifestations. Although bullous pemphigoid has been reported previously in association with anti-glomerular basement membrane disease or membranous nephropathy, this case demonstrates both elements in 1 patient. This concurrence and the linear pattern on indirect immunofluorescence support the possibility of cross-reactive or parallel autoantibodies to basement membranes with a secondary membranous component.