Proprotein convertase subtilisin/kexin type 9 targets megalin in the kidney proximal tubule and aggravates proteinuria in nephrotic syndrome.

Proteinuria is a prominent feature of chronic kidney disease. Interventions that reduce proteinuria slow the progression of chronic kidney disease and the associated risk of cardiovascular disease. Here, we propose a mechanistic coupling between proteinuria and proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of cholesterol and a therapeutic target in cardiovascular disease. PCSK9 undergoes glomerular filtration and is captured by megalin, the receptor responsible for driving protein reabsorption in the proximal tubule. Accordingly, megalin-deficient mice and patients carrying megalin pathogenic variants (Donnai Barrow syndrome) were characterized by elevated urinary PCSK9 excretion. Interestingly, PCSK9 knockout mice displayed increased kidney megalin while PCSK9 overexpression resulted in its reduction. Furthermore, PCSK9 promoted trafficking of megalin to lysosomes in cultured proximal tubule cells, suggesting that PCSK9 is a negative regulator of megalin. This effect can be accelerated under disease conditions since either genetic destruction of the glomerular filtration barrier in podocin knockout mice or minimal change disease (a common cause of nephrotic syndrome) in patients resulted in enhanced tubular PCSK9 uptake and urinary PCSK9 excretion. Pharmacological PCSK9 inhibition increased kidney megalin while reducing urinary albumin excretion in nephrotic mice. Thus, glomerular damage increases filtration of PCSK9 and concomitantly megalin degradation, resulting in escalated proteinuria.

The nuclear factor of activated T cells 5 (NFAT5) contributes to the renal corticomedullary differences in gene expression.

The corticomedullary osmotic gradient between renal cortex and medulla induces a specific spatial gene expression pattern. The factors that controls these differences are not fully addressed. Adaptation to hypertonic environment is mediated by the actions of the nuclear factor of activated T-cells 5 (NFAT5). NFAT5 induces the expression of genes that lead to intracellular accumulation of organic osmolytes. However, a systematical analysis of the NFAT5-dependent gene expression in the kidneys was missing. We used primary cultivated inner medullary collecting duct (IMCD) cells from control and NFAT5 deficient mice as well as renal cortex and inner medulla from principal cell specific NFAT5 deficient mice for gene expression profiling. In primary NFAT5 deficient IMCD cells, hyperosmolality induced changes in gene expression were abolished. The majority of the hyperosmolality induced transcripts in primary IMCD culture were determined to have the greatest expression in the inner medulla. Loss of NFAT5 altered the expression of more than 3000 genes in the renal cortex and more than 5000 genes in the inner medulla. Gene enrichment analysis indicated that loss of NFAT5 is associated with renal inflammation and increased expression of kidney injury marker genes, like lipocalin-2 or kidney injury molecule-1. In conclusion we show that NFAT5 is a master regulator of gene expression in the kidney collecting duct and in vivo loss of NFAT function induces a kidney injury like phenotype.

Genetic deletion of the nuclear factor of activated T cells 5 in collecting duct principal cells causes nephrogenic diabetes insipidus.

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8 h water restriction, NFAT5PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI.

The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter NCC but is not required for a potassium-induced reduction of NCC expression.

Na+ and K+ balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K+. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K+ on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In ex vivo experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K+ reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K+ induced reductions in NCC abundance.

Role of microRNAs in aquaporin 2 regulation.

PURPOSE OF REVIEW: The current review aims to present the most recent achievements on the role of microRNAs (miRNAs) on the kidney function to stimulate research in the field and to expand new emerging concepts. RECENT FINDINGS: The focus is on the role of miRNAs in intercellular communication along the segments of the nephron and on the epi-miRNAs, namely the possibility of some miRNAs to modulate the epigenetic machinery and so gene expression. Indeed, recent evidence showed that miRNAs included in exosomes and released by proximal tubule cells can modulate ENaC activity on cells of collecting duct. These data, although, from in-vitro models open to a novel role for miRNAs to participate in paracrine signaling pathways. In addition, the role of miRNAs as epigenetic modulators is expanding not only in the cancer field, but also in the other kidney diseases. Recent evidence identified three miRNAs able to modulate the AQP2 promoter metilation and showing an additional level of regulation for the AQP2. SUMMARY: These evidence can inspire novel area of research both for renal physiology and drug discovery. The diseases involving the collecting duct are still missing disease modifying agents and the expanding miRNAs field could represent an opportunity.

Dysregulation of Principal Cell miRNAs Facilitates Epigenetic Regulation of AQP2 and Results in Nephrogenic Diabetes Insipidus.

BACKGROUND: MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression. METHODS: Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice. RESULTS: The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409-3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre+ mice, resulting in decreased RNA Pol II association. CONCLUSIONS: Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.

Fyn Tyrosine Kinase as Harmonizing Factor in Neuronal Functions and Dysfunctions.

Fyn is a non-receptor or cytoplasmatic tyrosine kinase (TK) belonging to the Src family kinases (SFKs) involved in multiple transduction pathways in the central nervous system (CNS) including synaptic transmission, myelination, axon guidance, and oligodendrocyte formation. Almost one hundred years after the original description of Fyn, this protein continues to attract extreme interest because of its multiplicity of actions in the molecular signaling pathways underlying neurodevelopmental as well as neuropathologic events. This review highlights and summarizes the most relevant recent findings pertinent to the role that Fyn exerts in the brain, emphasizing aspects related to neurodevelopment and synaptic plasticity. Fyn is a common factor in healthy and diseased brains that targets different proteins and shapes different transduction signals according to the neurological conditions. We will primarily focus on Fyn-mediated signaling pathways involved in neuronal differentiation and plasticity that have been subjected to considerable attention lately, opening the fascinating scenario to target Fyn TK for the development of potential therapeutic interventions for the treatment of CNS injuries and certain neurodegenerative disorders like Alzheimer's disease.

Potassium depletion induces cellular conversion in the outer medullary collecting duct altering Notch signaling pathway.

Potassium depletion affects AQP2 expression and the cellular composition of the kidney collecting duct. This, in turn, contributes to the development of a secondary form of nephrogenic diabetes insipidus and hypokalemic nephropathy. Here we show that after 14 days of potassium depletion, the cellular fraction of A-type intercalated cells increases while the fraction of principal cells decreases along the outer medullary collecting duct in rats. The intercalated cells acquired a novel distribution pattern forming rows of cells attached to each other. These morphological changes occur progressively and reverse after 7 days of recovery on normal rat chow diet. The cellular remodeling mainly occurred in the inner stripe of outer medulla similar to the previously seen effect of lithium on the collecting duct cellular profile. The cellular remodeling is associated with the appearance of cells double labelled with both specific markers of principal and type-A intercalated cells. The appearance of this cell type was associated with the downregulation of the Notch signaling via the Hes1 pathways. These results show that the epithelium of the collecting duct has a high degree of plasticity and that Notch signaling likely plays a key role during hypokalemia.

Corrigendum: Integrin Beta 1 Is Crucial for Urinary Concentrating Ability and Renal Medulla Architecture in Adult Mice.

[This corrects the article DOI: 10.3389/fphys.2018.01273.].

Integrin Beta 1 Is Crucial for Urinary Concentrating Ability and Renal Medulla Architecture in Adult Mice.

Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process.

Unraveling the Mechanistic Complexity of the Glomerulocystic Phenotype in Dicer Conditional KO Mice by 2D Gel Electrophoresis Coupled Mass Spectrometry.

PURPOSE: Dicer, an RNase III type endonuclease, is a key enzyme involved in miRNA biogenesis. It has been shown that this enzyme is essential for several aspects of postnatal kidney functions and homeostasis. In this study, we have examined conditional knockout (cKO) mice for Dicer in Pax8 (Paired-box gene 8) expressing cells to investigate the kidney protein profile. This specific model develops a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria, and severe renal failure. EXPERIMENTAL DESIGN: Proteomic analysis was performed on kidney tissue extracts from cKO and control (Ctr) mice by 2D Gel Electrophoresis coupled with mass spectrometry. RESULTS: The analysis highlighted 120 protein spots differentially expressed in Dicer cKO tissue compared with control; some of these proteins were validated by Western blotting. Ingenuity Pathway Analysis led to the identification of some interesting networks; among them, the one having ERK as a central hub may explain, through the modulation of the expression of a number of identified protein targets, the metabolic and structural alterations occurring during kidney cyst development in Dicer cKO mouse model. CONCLUSIONS AND CLINICAL RELEVANCE: Our results contribute to gain new insights into molecular mechanisms through which Dicer endonuclease controls kidney development and physiological functions.

MicroRNAs in Renal Diseases: A Potential Novel Therapeutic Target.

BACKGROUND: MicroRNAs (miRNAs) are a family of short noncoding RNAs that play important roles in posttranscriptional gene regulation. miRNAs inhibit target gene expression by blocking protein translation or by inducing mRNA degradation and therefore have the potential to modulate physiological and pathological processes. SUMMARY: In the kidney, miRNAs play a role in the organogenesis and in the pathogenesis of several diseases, including renal carcinoma, diabetic nephropathy, cystogenesis, and glomerulopathies. Indeed, podocytes, but also the parietal cells of the Bowman capsule are severely affected by miRNA deregulation. In addition, several miRNAs have been found involved in the development of renal fibrosis. These experimental lines of evidence found a counterpart also in patients affected by diabetic and Ig-A nephropathies, opening the possibility of their use as biomarkers. Finally, the possibility to direct target-specific miRNA to prevent the development of renal fibrosis is encouraging potential novel therapies based on miRNA mimicking or antagonism. This review reports the main studies that investigate the role of miRNAs in the kidneys, in particular highlighting the experimental models used, their potential role as biomarkers and, finally, the most recent data on the miRNA-based therapy. KEY MESSAGES: miRNAs are crucial regulators of cell function. They are easy to detect and represent potentially good targets for novel therapies.

A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on "single-cell"-specific KO model of the distal nephron.

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl- cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

Selective dicer suppression in the kidney alters GSK3ß/ß-catenin pathways promoting a glomerulocystic disease.

Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200), recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma), renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO) mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman's capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3ß precedes the development of the glomerulocystic phenotype. Downregulation of ß-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3ß. Alterations of ß-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3ß/ß-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.