Quantitative phenotyping of Nphs1 knockout mice as a prerequisite for gene replacement studies.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease before the age of 25 yr. Nephrin, encoded by NPHS1, localizes to the slit diaphragm of glomerular podocytes and is the predominant structural component of the glomerular filtration barrier. Biallelic variants in NPHS1 can cause congenital nephrotic syndrome of the Finnish type, for which, to date, no causative therapy is available. Recently, adeno-associated virus (AAV) vectors targeting the glomerular podocyte have been assessed as a means for gene replacement therapy. Here, we established quantitative and reproducible phenotyping of a published, conditional Nphs1 knockout mouse model (Nphs1tm1.1Pgarg/J and Nphs2-Cre+) in preparation for a gene replacement study using AAV vectors. Nphs1 knockout mice (Nphs1fl/fl Nphs2-Cre+) exhibited 1) a median survival rate of 18 days (range: from 9 to 43 days; males: 16.5 days and females: 20 days); 2) an average foot process (FP) density of 1.0 FP/µm compared with 2.0 FP/µm in controls and a mean filtration slit density of 2.64 µm/µm2 compared with 4.36 µm/µm2 in controls; 3) a high number of proximal tubular microcysts; 4) the development of proteinuria within the first week of life as evidenced by urine albumin-to-creatinine ratios; and 5) significantly reduced levels of serum albumin and elevated blood urea nitrogen and creatinine levels. For none of these phenotypes, significant differences between sexes in Nphs1 knockout mice were observed. We quantitatively characterized five different phenotypic features of congenital nephrotic syndrome in Nphs1fl/fl Nphs2-Cre+ mice. Our results will facilitate future gene replacement therapy projects by allowing for sensitive detection of even subtle molecular effects.NEW & NOTEWORTHY To evaluate potential, even subtle molecular, therapeutic effects of gene replacement therapy (GRT) in a mouse model, prior rigorous quantifiable and reproducible disease phenotyping is necessary. Here, we, therefore, describe such a phenotyping effort in nephrin (Nphs1) knockout mice to establish the basis for GRT for congenital nephrotic syndrome. We believe that our findings set an important basis for upcoming/ongoing gene therapy approaches in the field of nephrology, especially for monogenic nephrotic syndrome.

Copy number variation analysis in 138 families with steroid-resistant nephrotic syndrome identifies causal homozygous deletions in PLCE1 and NPHS2 in two families.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of kidney failure in children and adults under the age of 20 years. Previously, we were able to detect by exome sequencing (ES) a known monogenic cause of SRNS in 25-30% of affected families. However, ES falls short of detecting copy number variants (CNV). Therefore, we hypothesized that causal CNVs could be detected in a large SRNS cohort. METHODS: We performed genome-wide single nucleotide polymorphism (SNP)-based CNV analysis on a cohort of 138 SRNS families, in whom we previously did not identify a genetic cause through ES. We evaluated ES and CNV data for variants in 60 known SRNS genes and in 13 genes in which variants are known to cause a phenocopy of SRNS. We applied previously published, predefined criteria for CNV evaluation. RESULTS: We detected a novel CNV in two genes in 2 out of 138 families (1.5%). The 9,673 bp homozygous deletion in PLCE1 and the 6,790 bp homozygous deletion in NPHS2 were confirmed across the breakpoints by PCR and Sanger sequencing. CONCLUSIONS: We confirmed that CNV analysis can identify the genetic cause in SRNS families that remained unsolved after ES. Though the rate of detected CNVs is minor, CNV analysis can be used when there are no other genetic causes identified. Causative CNVs are less common in SRNS than in other monogenic kidney diseases, such as congenital anomalies of the kidneys and urinary tract, where the detection rate was 5.3%. A higher resolution version of the Graphical abstract is available as Supplementary information.

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy.

SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

A homozygous truncating ETV4 variant in a Nigerian family with congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of chronic kidney disease that manifests in children. To date ~23 different monogenic causes have been implicated in isolated forms of human CAKUT, but the vast majority remains elusive. In a previous study, we identified a homozygous missense variant in E26 transformation-specific (ETS) Variant Transcription Factor 4 (ETV4) causing CAKUT via dysregulation of the transcriptional function of ETV4, and a resulting abrogation of GDNF/RET/ETV4 signaling pathway. This CAKUT family remains the only family with an ETV4 variant reported so far. Here, we describe one additional CAKUT family with a homozygous truncating variant in ETV4 (p.(Lys6*)) that was identified by exome sequencing. The variant was found in an individual with isolated CAKUT displaying posterior urethral valves and renal dysplasia. The newly identified stop variant conceptually truncates the ETS_PEA3_N and ETS domains that regulate DNA-binding transcription factor activity. The variant has never been reported homozygously in the gnomAD database. To our knowledge, we here report the first CAKUT family with a truncating variant in ETV4, potentially causing the isolated CAKUT phenotype observed in the affected individual.

Benzothiazines as Major Intermediates in Enzymatic Browning Reactions of Catechin and Cysteine.

The course of melanin formation is yet not thoroughly resolved on a mechanistic level. With the present study, incubations of catechin (CA)- and cysteine-derived dihydro-1,4-benzothiazine carboxylic acid derivatives were investigated for colored products during enzymatic browning. Analyses by high-performance liquid chromatography (HPLC)-mass spectrometry revealed the formation of two novel decarboxylated dihydro-1,4-benzothiazine derivatives [8-(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-5-hydroxy-3,4-dihydro-2H-benzothiazine and 7-(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-5-hydroxy-3,4-dihydro-2H-benzothiazine] preferentially under acidic conditions. Furthermore, in model reactions under neutral pH, a colored phenazine dimer intermediate was isolated by high-performance countercurrent chromatography and preparative HPLC when conducting the incubations in the presence of o-phenylenediamine (OPD). Mass spectrometry and nuclear magnetic resonance spectroscopy unequivocally verified the structure as (12E)-5,5'-dioxo-11a,11a'-bis(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-3,3',4,4',5a,5a',6,6',11,11',11a,11a'-dodecahydro-2H,2'H,5H,5'H-12,12'-bi[1,4]thiazino[2,3-b]phenazine-3,3'-dicarboxylic acid. Enzymatically catalyzed incubations under aeration starting from the initial CA-cysteine adducts and their follow-up dihydro-1,4-benzothiazine carboxylic acids, respectively, proved that the unstable colored compound was a trichochrome-like reaction intermediate of the browning reaction cascade which can be trapped by postincubation with OPD, thus verifying their direct mechanistic relationship.

Oxidative Fragmentation of Aspalathin Leads to the Formation of Dihydrocaffeic Acid and the Related Lysine Amide Adduct.

In the present study, the degradation of C-glucosidic dihydrochalcone aspalathin as the major phenolic compound in rooibos (Aspalathus linearis) was investigated. Analyses by gas chromatography-mass spectrometry of aqueous aspalathin-lysine incubations after silylation showed the formation of dihydrocaffeic acid [3-(3,4-dihydroxyphenyl)-propionic acid] under oxidative conditions as a novel degradation product up to 10 mol %. High-performance liquid chromatography analyses revealed the concurrent formation of the dihydrocaffeic acid lysine amide at about 30-fold lower concentrations, which was unequivocally verified by synthesis of an authentic reference standard. The amide was also verified in aspalathin-protein incubations after enzymatic hydrolysis by high-performance liquid chromatography-tandem mass spectrometry analyses. Thus, the covalent interaction of phenolic plant compounds with proteins under mild conditions (ambient temperatures and neutral pH) was confirmed for the first time. Acid and free amide were also quantitated in rooibos teas with significantly higher values in fermented varieties. The mechanism of formation was clarified to be initiated by singlet oxygen and to include a rearrangement-fragmentation mechanism with 1,2,3,5-tetrahydroxybenzene as the counterpart.

Influence of Nucleophilic Amino Acids on Enzymatic Browning Systems.

In the present study the enzymatic oxidation of gallic acid and catechin catalyzed by nashi pear polyphenol oxidase (PPO) in the presence of the amino acids lysine, arginine, or cysteine was investigated for polyphenol-amino acid adducts. HPLC analyses revealed the formation of two novel dihydrobenzothiazine carboxylic acid derivatives (8-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid and 7-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid) from 2'-cysteinyl catechin and 5'-cysteinyl catechin in cysteine incubations, respectively. In contrast, arginine and lysine did not lead to any amino acid adducts. Target compounds were separated by high-performance countercurrent chromatography and preparative HPLC and unequivocally characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Mechanistic incubations starting from the catechin-cysteine adducts showed that both catechin and PPO are crucial components in the formation of the dihydrobenzothiazines. The cysteine incubations showed a red-brown coloration, which coincided with formation and degradation of the dihydrobenzothiazines finally leading to the formation of high-polymeric melanins. Therefore, these compounds might be the key intermediates to understand development of color during cysteine-driven enzymatic browning reactions.