• Hematuria
• genetic testing
• pediatric
• type IV collagen-related nephropathy

• Humans
• Hematuria/genetics
• Hematuria/etiology
• Hematuria/diagnosis
• Collagen Type IV/genetics
• Child
• Female
• Male
• Genetic Testing/methods
• Child, Preschool
• Adolescent
• Mouth Mucosa/pathology
• Autoantigens

• Alport syndrome
• Gene mutation
• Genotype–phenotype correlation
• Kidney biopsy
• Pediatrics

• Humans
• Male
• Female
• Nephritis, Hereditary/diagnosis
• Nephritis, Hereditary/genetics
• Retrospective Studies
• East Asian People
• Collagen Type IV/genetics
• Pedigree
• Mutation
• Autoantigens/genetics

• 2022BCA047 Key R&D Program of Science & Technology of Hubei Province

• chronic kidney disease
• cystic kidney disease
• end stage kidney disease
• genetics

In the glomeruli, mesangial cells produce mesangial matrix while podocytes wrap glomerular capillaries with cellular extensions named foot processes and tether the glomerular basement membrane (GBM). The turnover of the mature GBM and the ability of adult podocytes to repair injured GBM are unclear. The actin cytoskeleton is a major cytoplasmic component of podocyte foot processes and links the cell to the GBM. Predominant components of the normal glomerular extracellular matrix (ECM) include glycosaminoglycans, proteoglycans, laminins, fibronectin-1, and several types of collagen. In patients with diabetes, multiorgan composition of extracellular tissues is anomalous, including the kidney, so that the constitution and arrangement of glomerular ECM is profoundly altered. In patients with diabetic kidney disease (DKD), the global quantity of glomerular ECM is increased. The level of sulfated proteoglycans is reduced while hyaluronic acid is augmented, compared to control subjects. The concentration of mesangial fibronectin-1 varies depending on the stage of DKD. Mesangial type III collagen is abundant in patients with DKD, unlike normal kidneys. The amount of type V and type VI collagens is higher in DKD and increases with the progression of the disease. The GBM contains lower amount of type IV collagen in DKD compared to normal tissue. Further, genetic variants in the α3 chain of type IV collagen may modulate susceptibility to DKD and end-stage kidney disease. Human cellular models of glomerular cells, analyses of human glomerular proteome, and improved microscopy procedures have been developed to investigate the molecular composition and organization of the human glomerular ECM.

• Diabetes
• Kidney disease
• Glycosaminoglycans
• Factor H
• Sialic acid
• Laminin
• Collagen
• Fibronectin-1
• Extracellular matrix

• Congenital nephrotic syndrome
• Kidney transplantation
• LAMB2 gene
• Pierson syndrome

• Alport syndrome
• Children
• Multi-pronged approach for diagnosis
• Therapy

• Adolescent
• Age of Onset
• Child
• Child, Preschool
• China
• Collagen Type IV/genetics
• Disease Progression
• Female
• Genotype
• Glomerular Basement Membrane/pathology
• Hearing Loss/physiopathology
• Hematuria/physiopathology
• Humans
• Kidney/pathology
• Male
• Nephritis, Hereditary/diagnosis
• Nephritis, Hereditary/genetics
• Nephritis, Hereditary/pathology
• Nephritis, Hereditary/physiopathology
• Proteinuria/physiopathology
• Severity of Illness Index

The kidney is comprised of highly complex structures that rely on self-maintenance for their functions, and tissue repair and regeneration in renal diseases. We devised a proteomics assay to measure the turnover of individual proteins in mouse kidney. Mice were metabolically labeled with a specially formulated chow containing nitrogen-15 (¹⁵N) with the absence of normal ¹⁴N atoms. Newly synthesized proteins with ¹⁵N contents were distinguished from their ¹⁴N counterparts by mass spectrometry. In total, we identified over 4,000 proteins from the renal cortex with a majority of them contained only ¹⁵N. About 100 proteins had both ¹⁴N- and ¹⁵N-contents. Notably, the long-lived proteins that had large ¹⁴N/¹⁵N ratios were mostly matrix proteins. These included proteins such as type IV and type VI collagen, laminin, nidogen and perlecan/HSPG2 that constitute the axial core of the glomerular basement membrane (GBM). In contrast, the surface lamina rara proteins such as agrin and integrin had much shorter longevity, suggesting their faster regeneration cycle. The data illustrated matrix proteins that constitute the basement membranes in the renal cortex are constantly renewed in an ordered fashion. In perspective, the global profile of protein turnover is usefully in understanding the protein-basis of GBM maintenance and repair.

• genetics
• monogenic renal disease
• nephritis, nephrotic syndrome
• pediatrics

Alport syndrome (AS) is a hereditary disease caused by mutations in COL4A3‐5 genes. Recently, comprehensive genetic analysis has become the first‐line diagnostic tool for AS. However, no reports comparing mutation identification rates between conventional sequencing and comprehensive screening have been published.

In this study, 441 patients clinically suspected of having AS were divided into two groups and compared. The initial mutational analysis method involved targeted exome sequencing using next‐generation sequencing (NGS) (n = 147, NGS group) or Sanger sequencing for COL4A3/COL4A4/COL4A5 (n = 294, Sanger group).

In the NGS group, 126 patients (86%) were diagnosed with AS by NGS, while two had pathogenic mutations in other genes, NPHS1 and EYA1. Further, 239 patients (81%) were diagnosed with AS by initial analysis in the Sanger group. Thirteen patients who were negative for mutation detection in the Sanger group were analyzed by NGS; three were diagnosed with AS. Two had mutations in CLCN5 or LAMB2. The final variant detection rate was 90%.

Our results reveal that Sanger sequencing and targeted exome sequencing have high diagnostic ability. NGS also has the advantage of detecting other inherited kidney diseases and pathogenic mutations missed by Sanger sequencing.

• Alport syndrome
• next-generation sequencing
• podocyte-related gene
• targeted exome sequencing

• Alport syndrome
• Collagen IV
• Digenic inheritance
• FSGS
• Familial hematuria
• Kidney disease
• Laminin alpha 5
• Metalloproteinase
• Modifier gene
• Renal cysts
• Synaptopodin
• Thin Basement Membrane Nephropathy (TBMN)

Alport syndrome is an inherited renal disorder characterized by glomerular basement membrane lesions with hematuria, proteinuria and frequent hearing defects and ocular abnormalities. The disease is associated with mutations in genes encoding α3, α4, or α5 chains of type IV collagen, namely COL4A3 and COL4A4 in chromosome 2 and COL4A5 in chromosome X. In contrast to the well-known X-linked and autosomal recessive phenotypes, there is very little information about the autosomal dominant. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve.

We investigated a Spanish family with variable phenotype of autosomal dominant Alport syndrome using clinical, histological, and genetic analysis.

Mutational analysis of COL4A3 and COL4A4 genes showed a novel heterozygous mutation (c. 998G > A; p.G333E) in exon 18 of the COL4A3 gene. Among relatives carrying the novel mutation, the clinical phenotype was variable. Two additional COL4A3 mutations were found, a Pro-Leu substitution in exon 48 (p.P1461L) and a Ser-Cys substitution in exon 49 (p.S1492C), non-pathogenics alone.

Carriers of p.G333E and p.P1461L or p.S1492C mutations in COL4A3 gene appear to be more severely affected than carriers of only p.G333E mutation, and the clinical findings has an earlier onset. In this way, we could speculate on a synergistic effect of compound heterozygosity that could explain the different phenotype observed in this family.

• Alport syndrome
• COL4A3 gene
• Microhematuria
• Proteinuria, genotype-phenotype correlation

Alport syndrome (AS) is a genetic disorder characterized by progressive hematuric nephropathy with or without sensorineural hearing loss and ocular lesions. Previous studies on AS included mostly children.

To determine the prognostic value of loss of staining for collagen type IV alpha 5 (COL4A5) and its relationship with the ultrastructural glomerular basement membrane alterations, we performed direct immunofluorescence using a mixture of fluorescein isothiocyanate-conjugated and Texas-red conjugated antibodies against COL4A5 and COL4A2, respectively, on renal biopsies of 25 males with AS (including 16 who were diagnosed in adulthood).

All patients showed normal positive staining of glomerular basement membranes and tubular basement membranes for COL4A2. Of the 25 patients, 10 (40%) patients showed loss of staining for COL4A5 (including 89% of children and 13% of adults) and the remaining 15 (60%) had intact staining for COL4A5. Compared with patients with intact staining for COL4A5, those with loss of staining had more prominent ultrastructural glomerular basement membrane alterations and were younger at the time of biopsy. By Kaplan-Meier survival analysis and Cox regression analysis, loss of staining for COL4A5 predicted earlier progression to overt proteinuria and stage 2 chronic kidney disease or worse. By multivariate Cox regression analysis, loss of staining for COL4A5 was an independent predictor of the development of overt proteinuria and stage 2 chronic kidney disease or worse.

Thus, the COL4A5 expression pattern has an important prognostic value and it correlates with the severity of ultrastructural glomerular basement membrane alterations in males with AS. Loss of COL4A5 staining is uncommon in patients with AS diagnosed in their adulthood.

• Alport syndrome
• collagen chains staining
• electron microscopy
• hereditary nephritis
• renal biopsy

• nail patella syndrome (nps)
• septum
• knee
• arthroscopy
• patella instability

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.

• Alport disease
• basement membrane
• childhood
• glomerulus
• laminations

• Alport syndrome
• genetic renal disease
• molecular genetics

Some patients with thin basement membrane disease (TBMD) develop proteinuria, hypertension and different degrees of CKD, besides the persistent microhaematuria characteristic of the disease. Little is known about factors associated with this unfavourable outcome.

We reviewed clinical, pathological and radiological features of 32 patients with biopsy-proven TBMD. Patients were divided in two groups: those with persistent normal kidney function and negative or minimal proteinuria (n = 16) and those with persistent proteinuria >0.5 g/day (n = 16).

Patients with proteinuria had a worse kidney function at baseline than those with negative proteinuria. Global or segmental glomerulosclerosis, together with interstitial fibrosis, was found in 37% of patients with proteinuria. All proteinuric patients were treated with renin–angiotensin system blockers. At the end of follow-up (198 months in proteinuric patients and 210 months in patients with negative proteinuria) the prevalence of hypertension was 68% in proteinuric patients (12% at baseline), compared with 12 and 6%, respectively, in non-proteinuric patients. A slow decline of renal function was observed in proteinuric patients, although no patient developed end-stage kidney disease. Ultrasound studies showed bilateral kidney cysts in nine patients (56%) with proteinuria. Cysts were bilateral and countless in six patients, and bilateral but with a limited number of cysts in the three remaining patients. No cysts were found in patients with negative proteinuria.

Some patients with TBMD develop hypertension, proteinuria and CKD. Multiple bilateral kidney cysts were found in a majority (56%) of these patients. Further studies are needed to investigate the pathogenesis and the influence on long-term outcome of this TBMD-associated multiple kidney cysts.

• cysts
• proteinuria
• renal function impairment
• thin basement membrane disease

• chronic renal failure
• end stage kidney disease
• end stage renal disease
• familial nephropathy
• genetic renal disease

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.

• Animals
• Autoantigens/genetics
• Autoantigens/metabolism
• Collagen Type IV/genetics
• Collagen Type IV/metabolism
• Disease Models, Animal
• Glomerular Basement Membrane/metabolism
• Glomerular Basement Membrane/pathology
• Integrin alpha1/genetics
• Integrin alpha1/metabolism
• Integrin alpha3/genetics
• Integrin alpha3/metabolism
• Mesangial Cells/metabolism
• Mesangial Cells/pathology
• Mice
• Mice, Knockout
• Nephritis, Hereditary/genetics
• Nephritis, Hereditary/metabolism
• Podocytes/metabolism
• Podocytes/pathology
• Up-Regulation
• Vimentin/genetics
• Vimentin/metabolism

• P20 GM104936 NIGMS NIH HHS
• P01 DK065123 NIDDK NIH HHS
• R01 DK018381 NIDDK NIH HHS
• P20GM104936 NIGMS NIH HHS
• P01DK065123 NIDDK NIH HHS