Pkd2 Deficiency in Embryonic Aqp2 + Progenitor Cells Is Sufficient to Cause Severe Polycystic Kidney Disease.

SIGNIFICANCE STATEMENT: Autosomal dominant polycystic kidney disease (ADPKD) is a devastating disorder caused by mutations in polycystin 1 ( PKD1 ) and polycystin 2 ( PKD2 ). Currently, the mechanism for renal cyst formation remains unclear. Here, we provide convincing and conclusive data in mice demonstrating that Pkd2 deletion in embryonic Aqp2 + progenitor cells (AP), but not in neonate or adult Aqp2 + cells, is sufficient to cause severe polycystic kidney disease (PKD) with progressive loss of intercalated cells and complete elimination of α -intercalated cells, accurately recapitulating a newly identified cellular phenotype of patients with ADPKD. Hence, Pkd2 is a new potential regulator critical for balanced AP differentiation into, proliferation, and/or maintenance of various cell types, particularly α -intercalated cells. The Pkd2 conditional knockout mice developed in this study are valuable tools for further studies on collecting duct development and early steps in cyst formation. The finding that Pkd2 loss triggers the loss of intercalated cells is a suitable topic for further mechanistic studies. BACKGROUND: Most cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by mutations in PKD1 or PKD2. Currently, the mechanism for renal cyst formation remains unclear. Aqp2 + progenitor cells (AP) (re)generate ≥5 cell types, including principal cells and intercalated cells in the late distal convoluted tubules (DCT2), connecting tubules, and collecting ducts. METHODS: Here, we tested whether Pkd2 deletion in AP and their derivatives at different developmental stages is sufficient to induce PKD. Aqp2Cre Pkd2f/f ( Pkd2AC ) mice were generated to disrupt Pkd2 in embryonic AP. Aqp2ECE/+Pkd2f/f ( Pkd2ECE ) mice were tamoxifen-inducted at P1 or P60 to inactivate Pkd2 in neonate or adult AP and their derivatives, respectively. All induced mice were sacrificed at P300. Immunofluorescence staining was performed to categorize and quantify cyst-lining cell types. Four other PKD mouse models and patients with ADPKD were similarly analyzed. RESULTS: Pkd2 was highly expressed in all connecting tubules/collecting duct cell types and weakly in all other tubular segments. Pkd2AC mice had obvious cysts by P6 and developed severe PKD and died by P17. The kidneys had reduced intercalated cells and increased transitional cells. Transitional cells were negative for principal cell and intercalated cell markers examined. A complete loss of α -intercalated cells occurred by P12. Cysts extended from the distal renal segments to DCT1 and possibly to the loop of Henle, but not to the proximal tubules. The induced Pkd2ECE mice developed mild PKD. Cystic α -intercalated cells were found in the other PKD models. AQP2 + cells were found in cysts of only 13/27 ADPKD samples, which had the same cellular phenotype as Pkd2AC mice. CONCLUSIONS: Hence, Pkd2 deletion in embryonic AP, but unlikely in neonate or adult Aqp2 + cells (principal cells and AP), was sufficient to cause severe PKD with progressive elimination of α -intercalated cells, recapitulating a newly identified cellular phenotype of patients with ADPKD. We proposed that Pkd2 is critical for balanced AP differentiation into, proliferation, and/or maintenance of cystic intercalated cells, particularly α -intercalated cells.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co-administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle-treated mice. Compared to vehicle-treated mice, ERKi-treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho-Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi-treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co-administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

Durability of and role of AKT in FGF7p urothelial protection against cyclophosphamide.

We previously identified a peptide derived from human fibroblast growth factor 7 (FGF7p) that blocks urothelial apoptosis similar to full-length FGF7, although effects of FGF7p on urothelial repair are unknown. Also, while urothelial AKT activation downstream of FGF7p correlated with the anti-apoptotic effects, we have not directly interrogated the role of AKT in mediating the cytoprotection. Our goal was to assess effects of FGF7p on urothelial repair and the role of AKT signaling in mediating the cytoprotective effects of FGF7p. We performed hematoxylin and eosin (H&E), TUNEL, and/or immunofluorescence (IF) staining for various markers in FGF7p-treated mice 28 days after giving cyclophosphamide or after co-administering a systemic AKT antagonist with FGF7p 24 h after cyclophosphamide. Vehicle-treated and injured mice had hyperplastic urothelium, incomplete return of mature superficial cell markers, ongoing proliferation, and continued presence of basal progenitor markers 28 days after injury; conversely, FGF7p-treated mice had normal numbers of urothelial cell layers, nearly complete return of superficial cell markers, limited proliferation and fewer basal progenitor cells 28 days post-injury. Vehicle-treated mice also had ectopic lumenal basal progenitor cell markers, while FGF7p had none 28 days after cyclophosphamide. Co-administration of an AKT inhibitor largely abrogated FGF7p-driven AKT activation and cytoprotection in urothelium 24 h after injury. Thus, FGF7p drives faster and higher fidelity urothelial repair by limiting apoptotic injury via AKT signaling, similar to full-length FGF7. Finally, FGF7p is much less expensive to synthesize and has a longer shelf life and higher purity than FGF7.

FGF7 peptide (FGF7p) mimetic mitigates bladder urothelial injury from cyclophosphamide.

Although full-length fibroblast growth factor 7 (FGF7) blocks cyclophosphamide-induced urothelial apoptosis in mice, limitations include high production costs because of its large size. We previously identified a small peptide derived from FGF2 that mitigated acute radiation syndrome as well as full-length FGF2. Based on the sequence of the FGF2 peptide, we synthesized a corresponding 19 amino acid FGF7 peptide (FGF7p). Our objectives were to determine if systemic FGF7p triggered the downstream targets and protected against cyclophosphamide bladder injury similar to full-length FGF7. We administered FGF7p or vehicle subcutaneously (SQ) to mice subjected to no injury or intraperitoneal (IP) cyclophosphamide and harvested bladders 1 day after injury. We then performed hematoxylin and eosin, TUNEL and immunofluorescence (IF) staining. In uninjured mice, a 20 mg/kg threshold FGF7p dose induced expression of phosphorylated (activated) FRS2α (pFRS2α), and pAKT in urothelium (consistent with cytoprotective effects of FGF7). We then gave FGF7p (20 mg/kg) or vehicle at 72 and 48 h prior to cyclophosphamide. One day after injury, TUNEL staining revealed many more apoptotic urothelial cells with vehicle treatment versus FGF7p treatment. IF for pAKT and readouts of two anti-apoptotic AKT targets (BAD and mTORC1) revealed minimal staining with vehicle treatment, but strong urothelial expression for all markers with FGF7p treatment. In conclusion, FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, similar to FGF7. FGF7p is much more inexpensive to make and has a longer shelf life and higher purity than FGF7.

AKT Signaling Downstream of KGF Is Necessary and Sufficient for Blocking Cyclophosphamide Bladder Injury.

Keratinocyte growth factor (KGF) drives phosphorylated (activated) AKT (pAKT) in bladder urothelium, which correlates with cytoprotection from cyclophosphamide. The current study determined whether: i) KGF modifies AKT targets [B-cell lymphoma protein 2-associated agonist of cell death (BAD) and mammalian target of rapamycin complex (mTORC)-1] that could block apoptosis; ii) AKT signaling is required for KGF cytoprotection; iii) direct AKT activation drives cytoprotection; iv) co-administration of KGF and an AKT inhibitor blocks urothelial cytoprotection and AKT and AKT-target activation; and v) an AKT agonist prevents cyclophosphamide-induced urothelial apoptosis. Mice were given KGF and cyclophosphamide (or sham injury), and pBAD (readout of BAD inhibition) or p-p70S6k (pS6, readout of mTORC1 signaling) was assessed. KGF induced pBAD urothelial staining and prevented cyclophosphamide-induced loss of urothelial pS6 staining (likely stabilizing mTORC1 activity). Co-administration of KGF and AKT inhibitor blocked KGF-driven urothelial cytoprotection from cyclophosphamide and prevented pAKT, pBAD, and pS6 urothelial expression. Conversely, systemic AKT agonist blocked cyclophosphamide-induced urothelial apoptosis and induced pAKT, pBAD, and pS6, similar to KGF. Thus, the KGF-AKT signaling axis appeared to phosphorylate (suppress) BAD and prevent cyclophosphamide-induced loss of mTORC1 signaling, both of which likely suppress apoptosis. Additionally, AKT signaling was required for KGF-driven cytoprotection, and direct AKT activation was sufficient for blocking apoptosis. Thus, AKT may be a therapeutic target for blocking urothelial apoptosis from cyclophosphamide.

Urothelial progenitors in development and repair.

Urothelium is a specialized multilayer epithelium that lines the urinary tract from the proximal urethra to the kidney. In addition to proliferation and differentiation during development, urothelial injury postnatally triggers a robust regenerative capacity to restore the protective barrier between the urine and tissue. Mounting evidence supports the existence of dedicated progenitor cell populations that give rise to urothelium during development and in response to injury. Understanding the cellular and molecular basis for urothelial patterning and repair will inform tissue regeneration therapies designed to ameliorate a number of structural and functional defects of the urinary tract. Here, we review the current understanding of urothelial progenitors and the signaling pathways that govern urothelial development and repair. While most published studies have focused on bladder urothelium, we also discuss literature on upper tract urothelial progenitors. Furthermore, we discuss evidence supporting existence of context-specific progenitors. This knowledge is fundamental to the development of strategies to regenerate or engineer damaged or diseased urothelium.

Loss of Fibroblast Growth Factor Receptor 2 (FGFR2) Leads to Defective Bladder Urothelial Regeneration after Cyclophosphamide Injury.

Cyclophosphamide may cause hemorrhagic cystitis and eventually bladder urothelial cancer. Genetic determinants for poor outcomes are unknown. We assessed actions of fibroblast growth factor receptor (FGFR) 2 in urothelium after cyclophosphamide exposure. Conditional urothelial deletion of Fgfr2 (Fgfr2KO) did not affect injury severity or proliferation of keratin 14+ (KRT14+) basal progenitors or other urothelial cells 1 day after cyclophosphamide exposure. Three days after cyclophosphamide exposure, Fgfr2KO urothelium had defective regeneration, fewer cells, larger basal cell bodies and nuclei, paradoxical increases in proliferation markers, and excessive replication stress versus controls. Fgfr2KO mice had evidence of pathologic basal cell endoreplication associated with absent phosphorylated ERK staining and decreased p53 expression versus controls. Mice with conditional deletion of Fgfr2 in urothelium enriched for KRT14+ cells reproduced Fgfr2KO abnormalities after cyclophosphamide exposure. Fgfr2KO urothelium had defects up to 6 months after injury versus controls, including larger basal cells and nuclei, more persistent basal and ectopic lumenal KRT14+ cells, and signs of metaplasia (attenuated E-cadherin staining). Mice missing one allele of Fgfr2 also had (less severe) regeneration defects and basal cell endoreplication 3 days after cyclophosphamide exposure versus controls. Thus, reduced FGFR2/ERK signaling apparently leads to abnormal urothelial repair after cyclophosphamide exposure from pathologic basal cell endoreplication. Patients with genetic variants in FGFR2 or its ligands may have increased risks of hemorrhagic cystitis or urothelial cancer from persistent and ectopic KRT14+ cells.

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.

Keratinocyte Growth Factor Reduces Injury and Leads to Early Recovery from Cyclophosphamide Bladder Injury.

Keratinocyte growth factor (KGF) improves cyclophosphamide-induced bladder injury. To understand the mechanisms, we subcutaneously administered KGF to mice 24 hours before i.p. cyclophosphamide administration, followed by histologic assays and immunostaining. In vehicle (phosphate-buffered saline)-pretreated mice, nonapoptotic superficial cell death from 2 to 6 hours and apoptosis in intermediate and basal cells from 4 to 24 hours was observed after cyclophosphamide. Despite superficial cell loss, KGF suppressed intermediate and basal cell apoptosis, likely via AKT signaling. At 6 and 24 hours after cyclophosphamide, KGF-pretreated mice also had apparent extracellular signal-regulated kinase (ERK)-driven proliferation of mostly keratin 5 (KRT5)+/KRT14- intermediate cells. At 1 to 28 days after cyclophosphamide treatment, mostly KRT14+ basal progenitor cells proliferated in response to injury, peaking at 3 days in both treatment groups; however, proliferation rates were lower in the KGF group at 3 days, consistent with less injury. Three days after injury, unlike controls, KGF-pretreated mice had regenerated superficial cells. At 10 and 28 days after cyclophosphamide treatment, KGF-pretreated mice had little proliferation and marked restoration of urothelial layers, whereas the phosphate-buffered saline group had ongoing regeneration. Administration of KGF to uninjured mice reproduced ERK-driven KRT5+/KRT14- proliferation seen in injured mice; KRT14+ cells were unaffected. KGF pretreatment blocks cyclophosphamide-induced intermediate and basal cell apoptosis, likely by phosphorylated AKT, and drives phosphorylated ERK-mediated KRT5+/KRT14- cell proliferation, leading to early urothelial regeneration.

Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney.

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.

Ift25 is not a cystic kidney disease gene but is required for early steps of kidney development.

Eukaryotic cilia are assembled by intraflagellar transport (IFT) where large protein complexes called IFT particles move ciliary components from the cell body to the cilium. Defects in most IFT particle proteins disrupt ciliary assembly and cause mid gestational lethality in the mouse. IFT25 and IFT27 are unusual components of IFT-B in that they are not required for ciliary assembly and mutant mice survive to term. The mutants die shortly after birth with numerous organ defects including duplex kidneys. Completely duplex kidneys result from defects in ureteric bud formation at the earliest steps of metanephric kidney development. Ureteric bud initiation is a highly regulated process involving reciprocal signaling between the ureteric epithelium and the overlying metanephric mesenchyme with regulation by the peri-Wolffian duct stroma. The finding of duplex kidney in Ift25 and Ift27 mutants suggests functions for these genes in regulation of ureteric bud initiation. Typically the deletion of IFT genes in the kidney causes rapid cyst growth in the early postnatal period. In contrast, the loss of Ift25 results in smaller kidneys, which show only mild tubule dilations that become apparent in adulthood. The smaller kidneys appear to result from reduced branching in the developing metanephric kidney. This work indicates that IFT25 and IFT27 are important players in the early development of the kidney and suggest that duplex kidney is part of the ciliopathy spectrum.

Ectopic Phosphorylated Creb Marks Dedifferentiated Proximal Tubules in Cystic Kidney Disease.

Ectopic cAMP signaling is pathologic in polycystic kidney disease; however, its spatiotemporal actions are unclear. We characterized the expression of phosphorylated Creb (p-Creb), a target and mediator of cAMP signaling, in developing and cystic kidney models. We also examined tubule-specific effects of cAMP analogs in cystogenesis in embryonic kidney explants. In wild-type mice, p-Creb marked nephron progenitors (NP), early epithelial NP derivatives, ureteric bud, and cortical stroma; p-Creb was present in differentiated thick ascending limb of Henle, collecting duct, and stroma; however, it disappeared in mature NP-derived proximal tubules. In Six2cre;Frs2αFl/Fl mice, a renal cystic model, ectopic p-Creb stained proximal tubule-derived cystic segments that lost the differentiation marker lotus tetragonolobus lectin. Furthermore, lotus tetragonolobus lectin-negative/p-Creb-positive cyst segments (re)-expressed Ncam1, Pax2, and Sox9 markers of immature nephron structures and dedifferentiated proximal tubules after acute kidney injury. These dedifferentiation markers were co-expressed with p-Creb in renal cysts in Itf88 knockout mice subjected to ischemia and Six2cre;Pkd1Fl/Fl mice, other renal cystogenesis models. 8-Br-cAMP addition to wild-type embryonic kidney explants induced proximal tubular cystogenesis and p-Creb expression; these effects were blocked by co-addition of protein kinase A inhibitor. Thus p-Creb/cAMP signaling is appropriate in NP and early nephron derivatives, but disappears in mature proximal tubules. Moreover, ectopic p-Creb expression/cAMP signaling marks dedifferentiated proximal tubular cystic segments. Furthermore, proximal tubules are predisposed to become cystic after cAMP stimulation.

Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had a role in peri-Wolffian duct ST similar to Fgfr2.MethodsWe conditionally deleted Frs2α in peri-Wolffian duct ST with a Tbx18cre mouse line (Frs2αST-/-). We assessed for ureteric induction defects and alterations in downstream targets mediating defects. We performed euthanized cystograms and assessed ureter-bladder junctions by three-dimensional (3D) reconstructions.ResultsEmbryonic day (E) 11.5 Frs2αST-/- embryos had many displaced ureteric bud induction sites when compared with controls. E11.0 Frs2αST-/- embryos had decreased Bmp4 expression and signaling, which can cause abnormal ureteric bud induction. Postnatal day 1 (P1) and P30 Frs2αST-/- mice had higher VUR rates and grades vs. CONTROLS: Mutant refluxing ureters that inserted improperly into the bladder had shortened intravesicular tunnels (IVTs) when compared with controlsConclusionFrs2αST-/- embryos have aberrant ureteric induction sites, improper ureteral insertion, shortened intravesicular lengths, and VUR. Induction site defects appear secondary to reduced Bmp4 expression, similar to Fgfr2 mutants.

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.

Cystatin C-calculated Glomerular Filtration Rate-A Marker of Early Renal Dysfunction in Patients With Neuropathic Bladder.

OBJECTIVE: To asses if cystatin c-calculated glomerular filtration rate (GFR) can reveal chronic kidney disease (CKD) not detected by creatinine-based calculations in a larger prospective cohort of children with myelomeningocele (MMC). Wheelchair-bound MMC patients frequently have low muscle mass, and assessing renal deterioration based on creatinine-based GFR is imprecise. MMC patients are also at risk for end-stage renal disease. METHODS: Prospectively enrolled patients with MMC underwent annual serum creatinine and cystatin c testing. Anthropometric measurements were obtained from clinic visit. The modified (bedside) Schwartz formula for creatinine-based GFR and the Zappitelli cystatin C formula were utilized for calculation. The exclusion criteria were patients with reduced GFR (CKD stage 2) or chronic CKD (CKD stage 3 and greater); these patients were excluded from analysis on the premise that they had already been identified for closer renal monitoring. RESULTS: A total of 131 patients were included in the analysis. The median creatinine-based estimated GFR was 126.5 mL/min/1.73 m2 (range: 22-310). The median cystatin C-based estimated rate was 98.5 mL/min/1.73 m2 (range: 16-171), yielding an absolute median rate reduction of 30.2%. Using cystatin c-calculated GFR, CKD stage was upgraded from stage 1 to ≥2 in 34 patients (26%). CONCLUSION: In MMC patients with poor muscle mass, cystatin C-based GFR is more sensitive than creatinine-based GFR in detecting early CKD. In this high-risk population, serial cystatin C estimation is a valuable tool in identifying children who may benefit from early nephrology referral and intervention.

Six2creFrs2α knockout mice are a novel model of renal cystogenesis.

Six2cre-mediated deletion of Frs2α (Six2creFrs2αKO), a major fibroblast growth factor receptor (Fgfr) docking protein in mouse nephron progenitors results in perinatal renal hypoplasia; however, postnatal Six2creFrs2αKO kidneys develop cysts. We sought to determine the pathogenesis of Six2creFrs2αKO cyst formation. We performed histological assays, Western blots, and quantitative PCR (qPCR). While embryonic day (E) 18.5 Six2Frs2αKO kidneys were hypoplastic and not cystic, postnatal day (P) 7 mutants had proximal tubular-derived cysts that nearly replaced the renal parenchyma by P21. Mutants had high proximal tubular proliferation rates and interstitial fibrosis, similar to known polycystic kidney disease (PKD) models. Six2creFrs2αKO kidneys also had upregulation of Wnt/ßcatenin signaling, macrophage infiltration and chemokine production (e.g. ectopic Ccl2 in non-dilated proximal tubules), and augmented hedgehog signaling, features also seen in other PKD models. We saw increased Gli1 (hedgehog readout) in postnatal Six2creFrs2αKO interstitium and ectopic sonic hedgehog (Shh) in subsets of non-dilated P7 mutant proximal tubules (likely driving the stromal Gli expression). As ectopic tubular Shh and Ccl2 expression is seen after acute kidney injury (AKI), we interrogated another bone fide AKI marker, Kim1 and noted ectopic expression in P7 non-dilated proximal tubules. These observations suggest that aberrantly activated "AKI" pathways may drive pathogenesis in PKD.

Correction: Deletion of Fibroblast Growth Factor Receptor 2 from the Peri-Wolffian Duct Stroma Leads to Ureteric Induction Abnormalities and Vesicoureteral Reflux.

[This corrects the article DOI: 10.1371/journal.pone.0056062.].

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.