Dicarboxylic Acid Dietary Supplementation Protects against AKI.

SIGNIFICANCE STATEMENT: In this study, we demonstrate that a common, low-cost compound known as octanedioic acid (DC 8 ) can protect mice from kidney damage typically caused by ischemia-reperfusion injury or the chemotherapy drug cisplatin. This compound seems to enhance peroxisomal activity, which is responsible for breaking down fats, without adversely affecting mitochondrial function. DC 8 is not only affordable and easy to administer but also effective. These encouraging findings suggest that DC 8 could potentially be used to assist patients who are at risk of experiencing this type of kidney damage. BACKGROUND: Proximal tubules are rich in peroxisomes, which are damaged during AKI. Previous studies demonstrated that increasing peroxisomal fatty acid oxidation (FAO) is renoprotective, but no therapy has emerged to leverage this mechanism. METHODS: Mice were fed with either a control diet or a diet enriched with dicarboxylic acids, which are peroxisome-specific FAO substrates, then subjected to either ischemia-reperfusion injury-AKI or cisplatin-AKI models. Biochemical, histologic, genetic, and proteomic analyses were performed. RESULTS: Both octanedioic acid (DC 8 ) and dodecanedioic acid (DC 12 ) prevented the rise of AKI markers in mice that were exposed to renal injury. Proteomics analysis demonstrated that DC 8 preserved the peroxisomal and mitochondrial proteomes while inducing extensive remodeling of the lysine succinylome. This latter finding indicates that DC 8 is chain shortened to the anaplerotic substrate succinate and that peroxisomal FAO was increased by DC 8 . CONCLUSIONS: DC 8 supplementation protects kidney mitochondria and peroxisomes and increases peroxisomal FAO, thereby protecting against AKI.

Substantial downregulation of mitochondrial and peroxisomal proteins during acute kidney injury revealed by data-independent acquisition proteomics.

Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A ZenoTOF 7600 mass spectrometer was optimized for data-independent acquisition (DIA) to achieve comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured kidneys exhibited severely damaged tissues and injury markers. The comprehensive and sensitive kidney-specific DIA-MS assays feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome, and will serve as useful tools for developing novel therapeutics to remediate kidney function.

Succinylation of Park7 activates a protective metabolic response to acute kidney injury.

Acute kidney injury (AKI) is extremely prevalent among hospitalizations and presents a significant risk for the development of chronic kidney disease and increased mortality. Ischemia caused by shock, trauma, and transplant are common causes of AKI. To attenuate ischemic AKI therapeutically, we need a better understanding of the physiological and cellular mechanisms underlying damage. Instances of ischemia are most damaging in proximal tubule epithelial cells (PTECs) where hypoxic signaling cascades, and perhaps more rapidly, posttranslational modifications (PTMs), act in concert to change cellular metabolism. Here, we focus on the effects of the understudied PTM, lysine succinylation. We have previously shown a protective effect of protein hypersuccinylation on PTECs after depletion of the desuccinylase sirtuin5. General trends in the results suggested that hypersuccinylation led to upregulation of peroxisomal activity and was protective against kidney injury. Included in the list of changes was the Parkinson's-related deglycase Park7. There is little known about any links between peroxisome activity and Park7. In this study, we show in vitro and in vivo that Park7 has a crucial role in protection from AKI and upregulated peroxisome activity. These data in combination with published results of Park7's protective role in cardiovascular damage and chronic kidney disease lead us to hypothesize that succinylation of Park7 may ameliorate oxidative damage resulting from AKI and prevent disease progression. This novel mechanism provides a potential therapeutic mechanism that can be targeted.NEW & NOTEWORTHY Succinylation is an understudied posttranslational modification that has been shown to increase peroxisomal activity. Furthermore, increased peroxisomal activity has been shown to reduce oxidative stress and protect proximal tubules after acute kidney injury. Analysis of mass spectrometry succinylomic and proteomic data reveals a novel role for Parkinson's related Park7 in mediating Nrf2 antioxidant response after kidney injury. This novel protection pathway provides new insights for kidney injury prevention and development of novel therapeutics.

In utero exposure to maternal diabetes exacerbates dietary sodium intake-induced endothelial dysfunction by activating cyclooxygenase 2-derived prostanoids.

Prenatal exposure to maternal diabetes has been recognized as a significant cardiovascular risk factor, increasing the susceptibility to the emergence of conditions such as high blood pressure, atherosclerosis, and heart disease in later stages of life. However, it is unclear if offspring exposed to diabetes in utero have worse vascular outcomes on a high-salt (HS) diet. To test the hypothesis that in utero exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, we treated adult male wild-type offspring (DM_Exp, 6 mo old) of diabetic Ins2+/C96Y mice (Akita mice) with HS (8% sodium chloride, 10 days) and analyzed endothelial function via wire myograph and cyclooxygenase (COX)-derived prostanoids pathway by ELISA, quantitative PCR, and immunochemistry. On a regular diet, DM_Exp mice did not manifest any vascular dysfunction, remodeling, or inflammation. However, HS increased aortic contractility to phenylephrine and induced endothelial dysfunction (analyzed by acetylcholine-induced endothelium-dependent relaxation), vascular hydrogen peroxide production, COX2 expression, and prostaglandin E2 (PGE2) overproduction. Interestingly, ex vivo antioxidant treatment (tempol) or COX1/2 (indomethacin) or COX2 (NS398) inhibitors improved or reverted the endothelial dysfunction in DM_Exp mice fed a HS diet. Finally, DM_Exp mice fed with HS exhibited greater circulating cytokines and chemokines accompanied by vascular inflammation. In summary, our findings indicate that prenatal exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, primarily through the induction of oxidative stress and the generation of COX2-derived PGE2. This supports the concept that in utero exposure to maternal diabetes is a cardiovascular risk factor in adulthood.NEW & NOTEWORTHY Using a unique mouse model of prenatal exposure to maternal type 1 diabetes, our study demonstrates the novel observation that prenatal exposure to maternal diabetes results in a predisposition to high-salt (HS) dietary-induced vascular dysfunction and inflammation in adulthood. Mechanistically, we demonstrated that in utero exposure to maternal diabetes and HS intake induces vascular oxidative stress, cyclooxygenase-derived prostaglandin E2, and inflammation.

A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells.

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

Delayed graft function post renal transplantation: a review on animal models and therapeutics.

The incidence of end-stage renal disease (ESRD) has been increasing worldwide. Its treatment involves renal replacement therapy, either by dialyses or renal transplantation from a living or deceased donor. Although the initial mortality rates for patients on dialysis are comparable with kidney transplant recipients, the quality of life and long-term prognosis are greatly improved in transplanted patients. However, there is a large gap between availability and need for donor kidneys. This has led to the increase in the use of expanded kidney donor criteria. Allograft dysfunction immediately after transplant sets it up for many complications, such as acute rejection and shorter allograft survival. Delayed graft function (DGF) is one of the immediate posttransplant insults to the kidney allograft, which is increasing in prevalence due to efforts to maximize the available donor pool for kidneys and use of expanded kidney donor criteria. In this review, we discuss the risk factors for DGF, its implications for long-term allograft survival, animal models of DGF, and the therapeutic options currently under evaluation for prevention and management of DGF.

Endothelial-Derived miR-17∼92 Promotes Angiogenesis to Protect against Renal Ischemia-Reperfusion Injury.

BACKGROUND: Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established. METHODS: Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated. RESULTS: miR-17, -18a, -20a, -19b, and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92endo-/- after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate. CONCLUSIONS: These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.

Deletion of hypoxia-responsive microRNA-210 results in a sex-specific decrease in nephron number.

Low nephron number results in an increased risk of developing hypertension and chronic kidney disease. Intrauterine growth restriction is associated with a nephron deficit in humans, and is commonly caused by placental insufficiency, which results in fetal hypoxia. The underlying mechanisms by which hypoxia impacts kidney development are poorly understood. microRNA-210 is the most consistently induced microRNA in hypoxia and is known to promote cell survival in a hypoxic environment. In this study, the role of microRNA-210 in kidney development was evaluated using a global microRNA-210 knockout mouse. A male-specific 35% nephron deficit in microRNA-210 knockout mice was observed. Wnt/ß-catenin signaling, a pathway crucial for nephron differentiation, was misregulated in male kidneys with increased expression of the canonical Wnt target lymphoid enhancer binding factor 1. This coincided with increased expression of caspase-8-associated protein 2, a known microRNA-210 target and apoptosis signal transducer. Together, these data are consistent with a sex-specific requirement for microRNA-210 in kidney development.

Sirtuins play critical and diverse roles in acute kidney injury.

Acute kidney injury (AKI) is an extremely common medical affliction affecting both adult and pediatric patients resulting from hypoxic, nephrotoxic, and septic insults affecting approximately 20% of all hospital patients and up to 50% of patients in the intensive care unit. There are currently no therapeutics for patients who suffer AKI. Much recent work has focused on designing and implementing therapeutics for AKI. This review focuses on a family of enzymes known as sirtuins that play critical roles in regulating many cellular and biological functions. There are 7 mammalian sirtuins (SIRT1-7) that play roles in regulating the acylation of a wide variety of pathways. Furthermore, all but one of the mammalian sirtuins have been shown to play critical roles in mediating AKI based on preclinical studies. These diverse enzymes show exciting potential for therapeutic manipulation. This review will focus on the specific roles of each of the investigated sirtuins and the potential for manipulation of the various sirtuins and their effector pathways in mediating kidney injury.

Increased rates of vesicoureteral reflux in mice from deletion of Dicer in the peri-Wolffian duct stroma.

BACKGROUND: Vesicoureteral reflux (VUR), backflow of urine into the kidney, is associated with urinary tract infections and chronic kidney disease. Integrity of the vesicoureteral junction (VUJ), where reflux occurs, is determined largely by proper induction of the ureteric bud from the Wolffian duct. Induction is modulated by signals from the surrounding peri-Wolffian duct stroma. We evaluated whether miRNAs in the peri-Wolffian duct stroma are necessary for proper ureteric induction, VUJ formation, and suppression of VUR. METHODS: We generated a mouse with loss of miRNAs in the peri-Wolffian duct stroma. We evaluated embryos for ureteric bud induction defects and expression of genes that regulate induction. We performed cystograms to assess for reflux and assessed VUJs in postnatal mice. RESULTS: Mutant embryos had cranially displaced ureteric bud induction sites vs. controls. We observed no changes in expression of genes known to regulate induction. While mutants were early postnatal lethal, they had high rates of VUR vs. controls. Mutant VUJs that refluxed had low inserting ureters and shortened intravesicular tunnels vs. non-refluxing mice. CONCLUSIONS: We found that miRNAs in the peri-Wolffian duct stroma are required for normal ureteric bud induction, VUJ formation, and prevention of VUR.

Metabolic requirements of the nephron.

The mammalian kidney is a complex organ that has several metabolically active cell types to aid in waste filtration, salt-water balance, and electrolyte homeostasis in the body. These functions are done primarily through the nephron, which relies on strict regulation of various metabolic pathways. Any deviations in the metabolic profile of nephrons or their precursor cells called nephron progenitors can lead to renal pathologies and abnormal development. Metabolism encompasses the mechanisms by which cells generate intermediate molecules and energy in the form of adenosine triphosphate (ATP). ATP is required by all cells and is mainly generated through glycolysis, fatty acid oxidation, and oxidative phosphorylation. During kidney development, self-renewing or proliferating cells rely on glycolysis to a greater extent than the other metabolic pathways to supply energy, replenish reducing equivalents, and generate nucleotides. However, terminally differentiated cell types rely more heavily on fatty acid oxidation and oxidative phosphorylation performed in the mitochondria to fulfill energy requirements. Further, the mature nephron is comprised of distinct segments and each segment utilizes metabolic pathways to varying degrees depending on the specific function. This review will focus on major metabolic processes performed by the nephron during health and disease.

Sirtuin 5 Regulates Proximal Tubule Fatty Acid Oxidation to Protect against AKI.

BACKGROUND: The primary site of damage during AKI, proximal tubular epithelial cells, are highly metabolically active, relying on fatty acids to meet their energy demands. These cells are rich in mitochondria and peroxisomes, the two organelles that mediate fatty acid oxidation. Emerging evidence shows that both fatty acid pathways are regulated by reversible posttranslational modifications, particularly by lysine acylation. Sirtuin 5 (Sirt5), which localizes to both mitochondria and peroxisomes, reverses post-translational lysine acylation on several enzymes involved in fatty acid oxidation. However, the role of the Sirt5 in regulating kidney energy metabolism has yet to be determined. METHODS: We subjected male Sirt5-deficient mice (either +/- or -/-) and wild-type controls, as well as isolated proximal tubule cells, to two different AKI models (ischemia-induced or cisplatin-induced AKI). We assessed kidney function and injury with standard techniques and measured fatty acid oxidation by the catabolism of 14C-labeled palmitate to 14CO2. RESULTS: Sirt5 was highly expressed in proximal tubular epithelial cells. At baseline, Sirt5 knockout (Sirt5-/- ) mice had modestly decreased mitochondrial function but significantly increased fatty acid oxidation, which was localized to the peroxisome. Although no overt kidney phenotype was observed in Sirt5-/- mice, Sirt5-/- mice had significantly improved kidney function and less tissue damage compared with controls after either ischemia-induced or cisplatin-induced AKI. This coincided with higher peroxisomal fatty acid oxidation compared with mitochondria fatty acid oxidation in the Sirt5-/- proximal tubular epithelial cells. CONCLUSIONS: Our findings indicate that Sirt5 regulates the balance of mitochondrial versus peroxisomal fatty acid oxidation in proximal tubular epithelial cells to protect against injury in AKI. This novel mechanism might be leveraged for developing AKI therapies.

Von Hippel-Lindau Acts as a Metabolic Switch Controlling Nephron Progenitor Differentiation.

BACKGROUND: Nephron progenitors, the cell population that give rise to the functional unit of the kidney, are metabolically active and self-renew under glycolytic conditions. A switch from glycolysis to mitochondrial respiration drives these cells toward differentiation, but the mechanisms that control this switch are poorly defined. Studies have demonstrated that kidney formation is highly dependent on oxygen concentration, which is largely regulated by von Hippel-Lindau (VHL; a protein component of a ubiquitin ligase complex) and hypoxia-inducible factors (a family of transcription factors activated by hypoxia). METHODS: To explore VHL as a regulator defining nephron progenitor self-renewal versus differentiation, we bred Six2-TGCtg mice with VHLlox/lox mice to generate mice with a conditional deletion of VHL from Six2+ nephron progenitors. We used histologic, immunofluorescence, RNA sequencing, and metabolic assays to characterize kidneys from these mice and controls during development and up to postnatal day 21. RESULTS: By embryonic day 15.5, kidneys of nephron progenitor cell-specific VHL knockout mice begin to exhibit reduced maturation of nephron progenitors. Compared with controls, VHL knockout kidneys are smaller and developmentally delayed by postnatal day 1, and have about half the number of glomeruli at postnatal day 21. VHL knockout nephron progenitors also exhibit persistent Six2 and Wt1 expression, as well as decreased mitochondrial respiration and prolonged reliance on glycolysis. CONCLUSIONS: Our findings identify a novel role for VHL in mediating nephron progenitor differentiation through metabolic regulation, and suggest that VHL is required for normal kidney development.

In utero exposure to maternal diabetes impairs nephron progenitor differentiation.

The incidence of diabetes mellitus has significantly increased among women of childbearing age, and it has been shown that prenatal exposure to maternal diabetes increases the risk of associated congenital anomalies of the kidney. Congenital anomalies of the kidney are among the leading causes of chronic kidney disease in children. To better understand the effect of maternal diabetes on kidney development, we analyzed wild-type offspring (DM_Exp) of diabetic Ins2+/C96Y mice (Akita mice). DM_Exp mice at postnatal day 34 have a reduction of ~20% in the total nephron number compared with controls, using the gold standard physical dissector/fractionator method. At the molecular level, the expression of the nephron progenitor markers sine oculis homeobox homolog 2 and Cited1 was increased in DM_Exp kidneys at postnatal day 2. Conversely, the number of early developing nephrons was diminished in DM_Exp kidneys. This was associated with decreased expression of the intracellular domain of Notch1 and the canonical Wnt target lymphoid enhancer binding factor 1. Together, these data suggest that the diabetic intrauterine environment impairs the differentiation of nephron progenitors into nephrons, possibly by perturbing the Notch and Wnt/ß-catenin signaling pathways.

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro, succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5-/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5-/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5-/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.

Prorenin receptor is critical for nephron progenitors.

Deficient nephrogenesis is the major factor contributing to renal hypoplasia defined as abnormally small kidneys. Nephron induction during kidney development is driven by reciprocal interactions between progenitor cells of the cap mesenchyme (CM) and the ureteric bud (UB). The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H(+)-ATPase. Global loss of PRR is lethal in mice and PRR mutations are associated with a high blood pressure, left ventricular hypertrophy and X-linked mental retardation in humans. To circumvent lethality of the ubiquitous PRR mutation in mice and to determine the potential role of the PRR in nephrogenesis, we generated a mouse model with a conditional deletion of the PRR in Six2(+) nephron progenitors and their epithelial derivatives (Six2(PRR-/-)). Targeted ablation of PRR in Six2(+) nephron progenitors caused a marked decrease in the number of developing nephrons, small cystic kidneys and podocyte foot process effacement at birth, and early postnatal death. Reduced congenital nephron endowment resulted from premature depletion of nephron progenitor cell population due to impaired progenitor cell proliferation and loss of normal molecular inductive response to canonical Wnt/ß-catenin signaling within the metanephric mesenchyme. At 2 months of age, heterozygous Six2(PRR+/-) mice exhibited focal glomerulosclerosis, decreased kidney function and massive proteinuria. Collectively, these findings demonstrate a cell-autonomous requirement for the PRR within nephron progenitors for progenitor maintenance, progression of nephrogenesis, normal kidney development and function.

Endothelial marker-expressing stromal cells are critical for kidney formation.

Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice (Flk1fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1fl/fl (Flk1ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1ST-/- kidneys vs. CONTROLS: Juvenile Flk1ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages.

A FANCD2/FANCI-Associated Nuclease 1-Knockout Model Develops Karyomegalic Interstitial Nephritis.

Karyomegalic interstitial nephritis (KIN) is a chronic interstitial nephropathy characterized by tubulointerstitial nephritis and formation of enlarged nuclei in the kidneys and other tissues. We recently reported that recessive mutations in the gene encoding FANCD2/FANCI-associated nuclease 1 (FAN1) cause KIN in humans. FAN1 is a major component of the Fanconi anemia-related pathway of DNA damage response (DDR) signaling. To study the pathogenesis of KIN, we generated a Fan1 knockout mouse model, with abrogation of Fan1 expression confirmed by quantitative RT-PCR. Challenging Fan1-/- and wild-type mice with 20 mg/kg cisplatin caused AKI in both genotypes. In contrast, chronic injection of cisplatin at 2 mg/kg induced KIN that led to renal failure within 5 weeks in Fan1-/- mice but not in wild-type mice. Cell culture studies showed decreased survival and reduced colony formation of Fan1-/- mouse embryonic fibroblasts and bone marrow mesenchymal stem cells compared with wild-type counterparts in response to treatment with genotoxic agents, suggesting that FAN1 mutations cause chemosensitivity and bone marrow failure. Our data show that Fan1 is involved in the physiologic response of kidney tubular cells to DNA damage, which contributes to the pathogenesis of CKD. Moreover, Fan1-/- mice provide a new model with which to study the pathomechanisms of CKD.

Ablation of the epithelial-specific splicing factor Esrp1 results in ureteric branching defects and reduced nephron number.

BACKGROUND: Abnormalities in ureteric bud (UB) branching morphogenesis lead to congenital anomalies of the kidney and reduced nephron numbers associated with chronic kidney disease (CKD) and hypertension. Previous studies showed that the epithelial fibroblast growth factor receptor 2 (Fgfr2) IIIb splice variant supports ureteric morphogenesis in response to ligands from the metanephric mesenchyme during renal organogenesis. The epithelial-specific splicing regulator Esrp1 is required for expression of Fgfr2-IIIb and other epithelial-specific splice variants. Our objective was to determine whether Esrp1 is required for normal kidney development. RESULTS: Ablation of Esrp1 in mice, alone or together with its paralog Esrp2, was associated with reduced kidney size and increased incidence of renal aplasia. Three-dimensional imaging showed that embryonic Esrp1 knockout (KO) kidneys had fewer ureteric tips and reduced nephron numbers. Analysis of alternative splicing in Esrp-null ureteric epithelial cells by RNA-Seq confirmed a splicing switch in Fgfr2 as well as numerous other transcripts. CONCLUSIONS: Our findings reveal that Esrp1-regulated splicing in ureteric epithelial cells plays an important role in renal development. Defects in Esrp1 KO kidneys likely reflect reduced and/or absent ureteric branching, leading to decreased nephron induction secondary to incorrect Fgfr2 splicing and other splicing alterations. Developmental Dynamics 245:991-1000, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Fibroblast growth factor receptor-Frs2α signaling is critical for nephron progenitors.

Previous studies using transgenic Pax3cre mice have revealed roles for fibroblast growth factor receptors (Fgfrs) and Fgfr substrate 2α (Frs2α) signaling in early metanephric mesenchyme patterning and in ureteric morphogenesis. The role of Fgfr/Frs2α signaling in nephron progenitors is unknown. Thus, we generated mouse models using BAC transgenic Six2EGFPcre (Six2cre) mediated deletion of Fgfrs and/or Frs2α in nephron progenitors. Six2cre mediated deletion of Fgfr1 or Fgfr2 alone led to no obvious kidney defects. Six2creFgfr1(flox/flox)Fgfr2(flox/flox) (Fgfr1/2(NP-/-)) mice generate a discernable kidney; however, they develop nephron progenitor depletion starting at embryonic day 12.5 (E12.5) and later demonstrate severe cystic dysplasia. To determine the role of Frs2α signaling downstream of Fgfr2 in Fgfr1/2(NP-/-) mice, we generated Six2cre(,)Fgfr1(flox/flox)Fgfr2(LR/LR) (Fgfr1(NP-/-)Fgfr2(LR/LR)) mice that have point mutations in the Frs2α binding site of Fgfr2. Like Fgfr1/2(NP-/-) mice, Fgfr1(NP-/-)Fgfr2(LR/LR) develop nephron progenitor depletion, but it does not start until E14.5 and older mice have less severe cystic dysplasia than Fgfr1/2(NP-/-) To determine the role of Frs2α alone in nephron progenitors, we generated Six2creFrs2'A(flox/flox) (Frs2a(NP-/-)) mice. Frs2a(NP-/-)mice also develop nephron progenitor depletion and renal cysts, although these occurred later and were less severe than in the other Six2cre mutant mice. The nephron progenitor loss in all Six2cre mutant lines was associated with decreased Cited1 expression and increased apoptosis versus controls. FAC-sorted nephron progenitors in Six2cre Frs2'A(flox/flox) mice demonstrated evidence of increased Notch activity versus controls, which likely drives the progenitor defects. Thus, Fgfr1 and Fgfr2 have synergistic roles in maintaining nephron progenitors; furthermore, Fgfr signaling in nephron progenitors appears to be mediated predominantly by Frs2α.