Chemical chaperones to the rescue of Alport syndrome?

Alport syndrome is a hereditary kidney disease caused by collagen IV mutations that interfere with the formation and deposition of the α3α4α5 protomer into the glomerular basement membrane. In this issue, Yu et al. show that the chemical chaperone tauroursodeoxycholic acid prevented kidney structural changes and function decline in mice with a pathogenic missense Col4a3 mutation by increasing mutant α3α4α5 protomer glomerular basement membrane deposition and preventing podocyte apoptosis induced by endoplasmic reticulum stress.

Characterization of Ocular Morphology in Col4a3-/- Mice as a Murine Model for Alport Syndrome.

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

In chronic kidney disease altered cardiac metabolism precedes cardiac hypertrophy.

Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration.NEW & NOTEWORTHY Heart disease is an important morbidity of chronic kidney disease (CKD). Hypertension, vascular stiffness, and vascular calcification all contribute to cardiac pathophysiology. However, cardiac function in CKD devoid of vascular disease has not been studied. Here, in an animal model of human CKD without conduit arterial disease, we analyze cardiac respiration and discover that CKD directly impairs cardiac mitochondrial function by decreasing oxidative phosphorylation. Protection of cardiac oxidative phosphorylation may be a therapeutic target in CKD.

PDGF-D Is Dispensable for the Development and Progression of Murine Alport Syndrome.

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. In vitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3-/- mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3-/- mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3-/- mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3-/-Pdgfd-/- mice compared with Col4a3-/-Pdgfd+/+ littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3-/- mice might explain the lack of effects in vivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome.

Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.

X-linked Alport Syndrome with Type IV Collagen α5 Chain Staining Revealing Normal Expression in the Glomerular Basement Membrane and Negative on Bowman's Capsule and Distal Tubular Basement Membrane: A Case Report.

X-linked Alport syndrome is a hereditary progressive renal disease resulting from the disruption of collagen α3α4α5 (IV) heterotrimerization caused by pathogenic variants in the COL4A5 gene. This study aimed to report a male case of X-linked Alport syndrome with a mild phenotype accompanied by an atypical expression pattern of type IV collagen α5 [α5 (IV)] chain in glomerulus. A 38-year-old male presented with proteinuria (2.3 g/day) and hematuria. He has been detected urinary protein and occult blood since childhood. A renal biopsy was performed at the age of 29 years; however, a diagnosis of Alport syndrome was not considered. A renal biopsy 9 years later revealed diffuse thinning and lamellation of the glomerular basement membrane. Α staining for α5 (IV) revealed a normal expression pattern in the glomerular basement membrane and a complete negative expression in Bowman's capsule and distal tubular basement membrane. Using next-generation sequencing, we detected a COL4A5 missense variant within exon 35 (NM_000495.5: c.3088G>A, p. G1030S). The possibility of X-linked Alport syndrome should be considered when negative expression of α5 (IV) staining on Bowman's capsule was observed.

Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are anti-hyperglycemic agents that prevent glucose reabsorption in proximal tubular cells. SGLT2i improves renal outcomes in both diabetic and non-diabetic patients, indicating it may have beneficial effects beyond glycemic control. Here, we demonstrate that SGLT2i affects energy metabolism and podocyte lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that the SGLT2 protein was expressed in human and mouse podocytes to a similar extent in tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wild-type podocytes. Treatment with SGLT2i empagliflozin reduces lipid droplet accumulation and apoptosis in AS podocytes. Empagliflozin inhibits the utilization of glucose/pyruvate as a metabolic substrate in AS podocytes but not in AS tubular cells. In vivo, we demonstrate that empagliflozin reduces albuminuria and prolongs the survival of AS mice. Empagliflozin-treated AS mice show decreased serum blood urea nitrogen and creatinine levels in association with reduced triglyceride and cholesterol ester content in kidney cortices when compared to AS mice. Lipid accumulation in kidney cortices correlates with a decline in renal function. In summary, empagliflozin reduces podocyte lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.

Dual inhibition of the endothelin and angiotensin receptor ameliorates renal and inner ear pathologies in Alport mice.

Alport syndrome (AS), a type IV collagen disorder, leads to glomerular disease and, in some patients, hearing loss. AS is treated with inhibitors of the renin-angiotensin system; however, a need exists for novel therapies, especially those addressing both major pathologies. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist (DEARA) under clinical development for focal segmental glomerulosclerosis and IgA nephropathy. We report the ability of sparsentan to ameliorate both renal and inner ear pathologies in an autosomal-recessive Alport mouse model. Sparsentan significantly delayed onset of glomerulosclerosis, interstitial fibrosis, proteinuria, and glomerular filtration rate decline. Sparsentan attenuated glomerular basement membrane defects, blunted mesangial filopodial invasion into the glomerular capillaries, increased lifespan more than losartan, and lessened changes in profibrotic/pro-inflammatory gene pathways in both the glomerular and the renal cortical compartments. Notably, treatment with sparsentan, but not losartan, prevented accumulation of extracellular matrix in the strial capillary basement membranes in the inner ear and reduced susceptibility to hearing loss. Improvements in lifespan and in renal and strial pathology were observed even when sparsentan was initiated after development of renal pathologies. These findings suggest that sparsentan may address both renal and hearing pathologies in Alport syndrome patients. © 2023 Travere Therapeutics, Inc and The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Activation of Stimulator of IFN Genes (STING) Causes Proteinuria and Contributes to Glomerular Diseases.

BACKGROUND: The signaling molecule stimulator of IFN genes (STING) was identified as a crucial regulator of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-STING pathway, and this signaling pathway regulates inflammation and energy homeostasis under conditions of obesity, kidney fibrosis, and AKI. However, the role of STING in causing CKD, including diabetic kidney disease (DKD) and Alport syndrome, is unknown. METHODS: To investigate whether STING activation contributes to the development and progression of glomerular diseases such as DKD and Alport syndrome, immortalized human and murine podocytes were differentiated for 14 days and treated with a STING-specific agonist. We used diabetic db/db mice, mice with experimental Alport syndrome, C57BL/6 mice, and STING knockout mice to assess the role of the STING signaling pathway in kidney failure. RESULTS: In vitro, murine and human podocytes express all of the components of the cGAS-STING pathway. In vivo, activation of STING renders C57BL/6 mice susceptible to albuminuria and podocyte loss. STING is activated at baseline in mice with experimental DKD and Alport syndrome. STING activation occurs in the glomerular but not the tubulointerstitial compartment in association with autophagic podocyte death in Alport syndrome mice and with apoptotic podocyte death in DKD mouse models. Genetic or pharmacologic inhibition of STING protects from progression of kidney disease in mice with DKD and Alport syndrome and increases lifespan in Alport syndrome mice. CONCLUSION: The activation of the STING pathway acts as a mediator of disease progression in DKD and Alport syndrome. Targeting STING may offer a therapeutic option to treat glomerular diseases of metabolic and nonmetabolic origin or prevent their development, progression, or both.

Potential Renal Damage Biomarkers in Alport Syndrome-A Review of the Literature.

Alport syndrome (AS) is the second most common cause of inherited chronic kidney disease. This disorder is caused by genetic variants on COL4A3, COL4A4 and COL4A5 genes. These genes encode the proteins that constitute collagen type IV of the glomerular basement membrane (GBM). The heterodimer COL4A3A4A5 constitutes the majority of the GBM, and it is essential for the normal function of the glomerular filtration barrier (GFB). Alterations in any of collagen type IV constituents cause disruption of the GMB structure, allowing leakage of red blood cells and albumin into the urine, and compromise the architecture of the GFB, inducing inflammation and fibrosis, thus resulting in kidney damage and loss of renal function. The advances in DNA sequencing technologies, such as next-generation sequencing, allow an accurate diagnose of AS. Due to the important risk of the development of progressive kidney disease in AS patients, which can be delayed or possibly prevented by timely initiation of therapy, an early diagnosis of this condition is mandatory. Conventional biomarkers such as albuminuria and serum creatinine increase relatively late in AS. A panel of biomarkers that might detect early renal damage, monitor therapy, and reflect the prognosis would have special interest in clinical practice. The aim of this systematic review is to summarize the biomarkers of renal damage in AS as described in the literature. We found that urinary Podocin and Vascular Endothelial Growth Factor A are important markers of podocyte injury. Urinary Epidermal Growth Factor has been related to tubular damage, interstitial fibrosis and rapid progression of the disease. Inflammatory markers such as Transforming Growth Factor Beta 1, High Motility Group Box 1 and Urinary Monocyte Chemoattractant Protein- 1 are also increased in AS and indicate a higher risk of kidney disease progression. Studies suggest that miRNA-21 is elevated when renal damage occurs. Novel techniques, such as proteomics and microRNAs, are promising.

Novel Keap1-Nrf2 Protein-Protein Interaction Inhibitor UBE-1099 Ameliorates Progressive Phenotype in Alport Syndrome Mouse Model.

Background: Bardoxolone methyl activates nuclear factor erythroid 2-related factor 2 (Nrf2) via covalent binding and irreversible inhibition of Kelch-like ECH-associated protein 1 (Keap1), the negative regulator of Nrf2. Ongoing clinical trials of bardoxolone methyl show promising effects for patients with CKD. However, the direct inhibition of Keap1-Nrf2 protein-protein interaction (PPI) as an approach to activate Nrf2 is less explored. Methods: We developed a noncovalent Nrf2 activator UBE-1099, which highly selectively inhibits Keap1-Nrf2 PPI, and evaluated its efficacy on the progressive phenotype in an Alport syndrome mouse model (Col4a5-G5X). Results: Similar to bardoxolone methyl, UBE-1099 transiently increased proteinuria and reduced plasma creatinine in Alport mice. Importantly, UBE-1099 improved the glomerulosclerosis, renal inflammation, and fibrosis, and prolonged the life span of Alport mice. UBE-1099 ameliorated the dysfunction of Nrf2 signaling in the renal tissue of Alport mice. Moreover, transcriptome analysis in the glomerulus showed that UBE-1099 induced the expression of genes associated with the cell cycle and cytoskeleton, which may explain its unique mechanism of improvement such as glomerular morphologic change. Conclusions: UBE-1099 significantly ameliorates the progressive phenotype in Alport mice. Our results revealed the efficacy of Keap1-Nrf2 PPI inhibitor for glomerulosclerosis and present a potential therapeutic drug for CKD.

A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a Mouse Model of Alport Syndrome.

BACKGROUND: Alport syndrome is a genetic disorder characterized by a defective glomerular basement membrane, tubulointerstitial fibrosis, inflammation, and progressive renal failure. IL-11 was recently implicated in fibrotic kidney disease, but its role in Alport syndrome is unknown. METHODS: We determined IL-11 expression by molecular analyses and in an Alport syndrome mouse model. We assessed the effects of a neutralizing IL-11 antibody (×203) versus an IgG control in Col4a3-/- mice (lacking the gene encoding a type IV collagen component) on renal tubule damage, function, fibrosis, and inflammation. Effects of ×203, the IgG control, an angiotensin-converting enzyme (ACE) inhibitor (ramipril), or ramipril+X203 on lifespan were also studied. RESULTS: In Col4a3-/- mice, as kidney failure advanced, renal IL-11 levels increased, and IL-11 expression localized to tubular epithelial cells. The IL-11 receptor (IL-11RA1) is expressed in tubular epithelial cells and podocytes and is upregulated in tubular epithelial cells of Col4a3-/- mice. Administration of ×203 reduced albuminuria, improved renal function, and preserved podocyte numbers and levels of key podocyte proteins that are reduced in Col4a3-/- mice; these effects were accompanied by reduced fibrosis and inflammation, attenuation of epithelial-to-mesenchymal transition, and increased expression of regenerative markers. X203 attenuated pathogenic ERK and STAT3 pathways, which were activated in Col4a3-/- mice. The median lifespan of Col4a3-/- mice was prolonged 22% by ramipril, 44% with ×203, and 99% with ramipril+X203. CONCLUSIONS: In an Alport syndrome mouse model, renal IL-11 is upregulated, and neutralization of IL-11 reduces epithelial-to-mesenchymal transition, fibrosis, and inflammation while improving renal function. Anti-IL-11 combined with ACE inhibition synergistically extends lifespan. This suggests that a therapeutic approach targeting IL-11 holds promise for progressive kidney disease in Alport syndrome.

Effect of disease progression on the podocyte cell cycle in Alport Syndrome.

Progression of glomerulosclerosis is associated with loss of podocytes with subsequent glomerular tuft instability. It is thought that a diminished number of podocytes may be able to preserve tuft stability through cell hypertrophy associated with cell cycle reentry. At the same time, reentry into the cell cycle risks podocyte detachment if podocytes cross the G1/S checkpoint and undergo abortive cytokinesis. In order to study cell cycle dynamics during chronic kidney disease (CKD) development, we used a FUCCI model (fluorescence ubiquitination-based cell cycle indicator) of mice with X-linked Alport Syndrome. This model exhibits progressive CKD and expresses fluorescent reporters of cell cycle stage exclusively in podocytes. With the development of CKD, an increasing fraction of podocytes in vivo were found to be in G1 or later cell cycle stages. Podocytes in G1 and G2 were hypertrophic. Heterozygous female mice, with milder manifestations of CKD, showed G1 fraction numbers intermediate between wild-type and male Alport mice. Proteomic analysis of podocytes in different cell cycle phases showed differences in cytoskeleton reorganization and metabolic processes between G0 and G1 in disease. Additionally, in vitro experiments confirmed that damaged podocytes reentered the cell cycle comparable to podocytes in vivo. Importantly, we confirmed the upregulation of PDlim2, a highly expressed protein in podocytes in G1, in a patient with Alport Syndrome, confirming our proteomics data in the human setting. Thus, our data showed that in the Alport model of progressive CKD, podocyte cell cycle distribution is altered, suggesting that cell cycle manipulation approaches may have a role in the treatment of various progressive glomerular diseases characterized by podocytopenia.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

Synaptopodin deficiency exacerbates kidney disease in a mouse model of Alport syndrome.

Synaptopodin (Synpo) is an actin-associated protein in podocyte foot processes. By generating mice that completely lack Synpo, we previously showed that Synpo is dispensable for normal kidney function. However, lack of Synpo worsened adriamycin-induced nephropathy, indicating a protective role for Synpo in injured podocytes. Here, we investigated whether lack of Synpo directly impacts a genetic disease, Alport syndrome (AS), because Synpo is reduced in podocytes of affected humans and mice; whether this is merely an association or pathogenic is unknown. We used collagen type IV-α5 (Col4a5) mutant mice, which model X-linked AS, showing glomerular basement membrane (GBM) abnormalities, eventual foot process effacement, and progression to end-stage kidney disease. We intercrossed mice carrying mutations in Synpo and Col4a5 to produce double-mutant mice. Urine and tissue were taken at select time points to evaluate albuminuria, histopathology, and glomerular capillary wall composition and ultrastructure. Lack of Synpo in Col4a5-/Y, Col4a5-/-, or Col4a5+/- Alport mice led to the acceleration of disease progression, including more severe proteinuria and glomerulosclerosis. Absence of Synpo attenuated the shift of myosin IIA from the podocyte cell body and major processes to actin cables near the GBM in the areas of effacement. We speculate that this is mechanistically associated with enhanced loss of podocytes due to easier detachment from the GBM. We conclude that Synpo deletion exacerbates the disease phenotype in Alport mice, revealing the podocyte actin cytoskeleton as a target for therapy in patients with AS.NEW & NOTEWORTHY Alport syndrome (AS) is a hereditary disease of the glomerular basement with hematuria and proteinuria. Podocytes eventually exhibit foot process effacement, indicating actin cytoskeletal changes. To investigate how cytoskeletal changes impact podocytes, we generated Alport mice lacking synaptopodin, an actin-binding protein in foot processes. Analysis showed a more rapid disease progression, demonstrating that synaptopodin is protective. This suggests that the actin cytoskeleton is a target for therapy in AS and perhaps other glomerular diseases.

The role of discoidin domain receptor 2 in the renal dysfunction of alport syndrome mouse model.

Alport syndrome (AS) is a hereditary glomerular nephritis caused by mutation in one of the type IV collagen genes α3/α4/α5 that encode the heterotrimer COL4A3/4/5. Failure to form a heterotrimer due to mutation leads to the dysfunction of the glomerular basement membrane, and end-stage renal disease. Previous reports have suggested the involvement of the receptor tyrosine kinase discoidin domain receptor (DDR) 1 in the progression of AS pathology. However, due to the similarity between DDR1 and DDR2, the role of DDR2 in AS pathology is unclear. Here, we investigated the involvement of DDR2 in AS using the X-linked AS mouse model. Mice were treated subcutaneously with saline or antisense oligonucleotide (ASO; 5 mg/kg or 15 mg/kg per week) for 8 weeks. Renal function parameters and renal histology were analyzed, and the gene expressions of inflammatory cytokines were determined in renal tissues. The expression level of DDR2 was highly elevated in kidney tissues of AS mice. Knockdown of Ddr2 using Ddr2-specific ASO decreased the Ddr2 expression. However, the DDR2 ASO treatment did not improve the proteinuria or decrease the BUN level. DDR2 ASO also did not significantly ameliorate the renal injury, inflammation and fibrosis in AS mice. These results showed that Ddr2 knockdown by ASO had no notable effect on the progression of AS indicating that DDR2 may not be critically involved in AS pathology. This finding may provide useful information and further understanding of the role of DDRs in AS.

Discoidin domain receptor 1 activation links extracellular matrix to podocyte lipotoxicity in Alport syndrome.

BACKGROUND: Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is activated by collagens that is involved in the pathogenesis of fibrotic disorders. Interestingly, de novo production of the collagen type I (Col I) has been observed in Col4a3 knockout mice, a mouse model of Alport Syndrome (AS mice). Deletion of the DDR1 in AS mice was shown to improve survival and renal function. However, the mechanisms driving DDR1-dependent fibrosis remain largely unknown. METHODS: Podocyte pDDR1 levels, Collagen and cluster of differentiation 36 (CD36) expression was analyzed by Real-time PCR and Western blot. Lipid droplet accumulation and content was determined using Bodipy staining and enzymatic analysis. CD36 and DDR1 interaction was determined by co-immunoprecipitation. Creatinine, BUN, albuminuria, lipid content, and histological and morphological assessment of kidneys harvested from AS mice treated with Ezetimibe and/or Ramipril or vehicle was performed. FINDINGS: We demonstrate that Col I-mediated DDR1 activation induces CD36-mediated podocyte lipotoxic injury. We show that Ezetimibe interferes with the CD36/DDR1 interaction in vitro and prevents lipotoxicity in AS mice thus preserving renal function similarly to ramipril. INTERPRETATION: Our study suggests that Col I/DDR1-mediated lipotoxicity contributes to renal failure in AS and that targeting this pathway may represent a new therapeutic strategy for patients with AS and with chronic kidney diseases (CKD) associated with Col4 mutations. FUNDING: This study is supported by the NIH grants R01DK117599, R01DK104753, R01CA227493, U54DK083912, UM1DK100846, U01DK116101, UL1TR000460 (Miami Clinical Translational Science Institute, National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities), F32DK115109, Hoffmann-La Roche and Alport Syndrome Foundation.