Stem Cell Therapies Benefit Alport Syndrome

Patients with Alport syndrome progressively lose renal function as a result of defective type IV collagen in their glomerular basement membrane. In mice lacking the α3 chain of type IV collagen (Col4A3 knockout mice), a model for Alport syndrome, transplantation of wild-type bone marrow repairs the renal disease. It is unknown whether cell-based therapies that do not require transplantation have similar potential. Here, infusion of wild-type bone marrow-derived cells into unconditioned, nonirradiated Col4A3 knockout mice during the late stage of disease significantly improved renal histology and function. Furthermore, transfusion of unfractionated wild-type blood into unconditioned, nonirradiated Col4A3 knockout mice improved the renal phenotype and significantly improved survival. Injection of mouse and human embryonic stem cells into Col4A3 knockout mice produced similar results. Regardless of treatment modality, the improvement in the architecture of the glomerular basement membrane is associated with de novo expression of the α3(IV) chain. These data provide further support for testing cell-based therapies for Alport syndrome.Alport syndrome is characterized by the progressive development of glomerulonephritis associated with the loss of α3α4α5 type IV collagen protomer in the glomerular basement membrane (GBM).¹ The type IV collagen chain composition in the GBM is critical to the maintenance of the glomerular filtration.^2,3 Genetic mutations in α3, α4, or α5 (IV) collagen chains result in the ablation of the obligatory posttranslational assembly of α3α4α5(IV) protomer, which leads to renal disease in patients with Alport syndrome.^2,4–7 The engineered genetic mutation in the COL4A3 gene [encoding for α3(IV) chain; the Col4A3 knockout mouse] provides us with a mouse model that closely recapitulates the human disease.^8,9 Col4A3 knockout mice develop progressive glomerulonephritis associated with the loss of the GBM α3α4α5(IV) protomer and die as a result of renal failure. Importantly, the progression of the disease in mice varies with respect to their genetic background. Col4A3 knockout mice, on the 129Sv genetic background, progress more rapidly and die at approximately 13 wk of age in comparison with Col4A3 knockout mice on the C57BL/6 background, which die of renal failure at approximately 22 wk of age in our laboratory. It is suggested that the difference in disease progression between these two strains of mice results from the compensatory effect of α5α6α5(IV) protomer in the GBM of Col4A3 knockout on C57BL/6 genetic background, which is negligible when this mutation is on the 129Sv background.¹⁰ This modifying effect suggests that type IV collagen protomer composition is critical for GBM function and suggests that modulating GBM type IV composition, by providing the missing chain collagen to the GBM of Col4A3 knockout mice, could theoretically slow down or halt the progression of the renal disease.Previous preclinical studies demonstrated that de novo production of α3(IV) collagen in Col4A3 knockout mice that received a transplant of wild-type (WT) bone marrow (BM) is associated with significant improvement in renal function.^11,12 We and others have shown that BM-derived cells specifically target the diseased glomeruli and allow for the deposition of α3(IV) chain, which results in the restoration of the α3α4α5(IV) protomer in the GBM.^11,12 These results suggest that BM-derived cells provide a therapeutic benefit to the Col4A3 knockout mice. Nonetheless, a disease-modulating effect of total body irradiation on the progression of the kidney disease was recently suggested in Col4A3 knockout mice on the 129Sv genetic background.¹³ Unlike the human disease, kidney disease progression in Col4A3 knockout mice involves significant immune infiltration; therefore, a case can be made that total body irradiation and subsequent BM transplantation in Col4A3 knockout mice could modulate disease progression by diminishing renal immune infiltration. Our previous report demonstrated that lymphocyte ablation improves renal interstitial fibrosis in Col4a3/Rag-1 double-KO mice on C57BL/6 background¹⁶ as a result of diminished interstitial infiltrates. Recently, Katayama et al.¹³ provided evidence for an increase in the survival of Col4A3 knockout mice on 129Sv background, associated with improvement in renal function and histologic findings after total body irradiation; however, that study does not conclusively negate the specific therapeutic potential of BM-derived cells in the recovery of the renal phenotype.A better understanding of the cell-based therapy in Col4A3 knockout mice is critical for future clinical development of this therapeutic strategy for patients with Alport syndrome. Here we provide a critical evaluation of a cellular process in the therapy of Col4A3 knockout mice. Our experiments unequivocally demonstrate that cell-based therapy is a viable option in the treatment of Alport syndrome.     

Combination of Peritubular C4d and Transplant Glomerulopathy Predicts Late Renal Allograft Failure

The histologic associations and clinical implications of peritubular capillary C4d staining from long-term renal allografts are unknown. We identified 99 renal transplant patients who underwent an allograft biopsy for renal dysfunction at least 10 yr after transplantation, 25 of whom were C4d-positive and 74 of whom were C4d-negative. The average time of the index biopsy from transplantation was 14 yr in both groups. Compared with C4d-negative patients, C4d-positive patients were younger at transplantation (29 ± 13 versus 38 ± 12 yr; P < 0.05) and were more likely to have received an allograft from a living donor (65 versus 35%; P < 0.001). C4d-positive patients had more inflammation, were more likely to have transplant glomerulopathy, and had worse graft outcome. The combined presence of C4d positivity, transplant glomerulopathy, and serum creatinine of >2.3 mg/dl at biopsy were very strong predictors of rapid graft loss. C4d alone did not independently predict graft loss. Retrospective staining of historical samples from C4d-positive patients demonstrated C4d deposition in the majority of cases. In summary, these data show that in long-term renal allografts, peritubular capillary staining for C4d occurs in approximately 25% of biopsies, can persist for many years after transplantation, and strongly predicts graft loss when combined with transplant glomerulopathy.Advances in understanding immunologic mechanisms underlying acute renal allograft rejection have enabled the development of efficient diagnostic tools and therapeutic strategies directed against early immune-mediated graft loss. This led to an increase of 1-yr graft survival rates to >90%; however, long-term graft survival has not improved to a similar degree.^1,2 A steady decline of renal function over years is still the rule in the majority of cases after renal transplantation. Multiple factors can influence graft outcome in the late posttransplantation setting, including acute and or chronic rejection; patient compliance with immunosuppressive therapy; and other medical conditions, such as cardiovascular disease, hypertension, diabetes, infections, drug toxicities, and recurrent disease. Evaluation of renal biopsies may reveal changes related to calcineurin inhibitor toxicity, immune-mediated injury, BK nephropathy, thrombotic microangiopathy, hypertension, diabetes, and recurrent disease, which can help guide appropriate therapy.There is a growing awareness of the contribution of chronic immune-mediated injury and alloantibodies in late renal allograft dysfunction. Immunohistochemical detection of the complement degradation product C4d in peritubular capillaries (PTCs) of renal allograft biopsies has gained considerable attention because of its diagnostic and prognostic importance in early acute antibody-mediated rejection (AMR). Detection of C4d is regarded as indirect evidence (a “footprint”) of a host''s antibody response to a renal allograft and is one of the key criteria used to diagnose AMR.³ C4d deposition has also been implicated in more chronic immune injury of allograft kidneys. Transplant glomerulopathy (TG), a cause of renal dysfunction in longstanding renal allografts, has an estimated prevalence of 1.6 to 12% in renal transplant populations.^4–9 TG is often associated with evidence of AMR, such as the presence of donor-specific antibodies (DSAs) and positive PTC C4d staining, and is considered to be a hallmark of chronic AMR.^4,9–13 The combination of circulating alloantibodies, glomerular and PTC basement membrane multilamination, PTC C4d, and duplication of the glomerular basement membrane has been termed the “ABCD tetrad” of late AMR by Halloran et al.⁴Many reports have diffused that PTC C4d deposition predicts poor graft survival in both early (<1 yr) and late (>1 yr) posttransplantation periods.^12,14–23 Several reports also have shown C4d staining has little impact on graft survival. Satoskar et al.²⁴ examined 80 cases of late allograft rejection that occurred >1 yr after transplantation. They followed patients for 20 mo and found no difference in outcome between C4d⁺ and C4d⁻ groups. Nickeleit et al.²⁵ analyzed 400 transplant biopsies, at a median of 38 d after transplantation (range 7 to 5646 d) and found no differences between C4d⁺ and C4d⁻ groups in serum creatinine or graft survival at 1 yr of follow-up. A European study including protocol biopsy samples from early and late time points found no reduction in allograft survival in C4d⁺ versus C4d⁻ biopsy samples.¹³ Recently, a study by Haas et al.²⁶ of ABO-incompatible renal allografts showed that diffuse PTC C4d deposition without histologic evidence of AMR or cellular rejection in their initial protocol biopsies was associated with a lower risk for scarring at 1 yr. Several factors may explain the conflicting conclusions in these studies, including limitations as a result of highly selected patient groups,¹² short follow-up,^24–26 and the inclusion of both early and late allograft biopsies.^13,22,23,25In this study, we identified a unique cohort of renal transplant recipients with allografts surviving ≥10 yr after transplantation with biopsies performed for evaluation of graft dysfunction with or without proteinuria at these later time points. The aim of this study was to assess the prevalence of C4d staining and to clarify the clinical and pathologic significance of C4d positivity in long-term renal allografts.     

Genotype–Phenotype Correlations in Non-Finnish Congenital Nephrotic Syndrome

Mutations in NPHS1, which encodes nephrin, are the main causes of congenital nephrotic syndrome (CNS) in Finnish patients, whereas mutations in NPHS2, which encodes podocin, are typically responsible for childhood-onset steroid-resistant nephrotic syndrome in European populations. Genotype–phenotype correlations are not well understood in non-Finnish patients. We evaluated the clinical presentation, kidney histology, and disease progression in non-Finnish CNS cases by mutational screening in 107 families (117 cases) by sequencing the entire coding regions of NPHS1, NPHS2, PLCE1, WT1, LAMB2, PDSS2, COQ2, and NEPH1. We found that CNS describes a heterogeneous group of disorders in non-Finnish populations. We identified nephrin and podocin mutations in most families and only rarely found mutations in genes implicated in other hereditary forms of NS. In approximately 20% of cases, we could not identify the underlying genetic cause. Consistent with the major role of nephrin at the slit diaphragm, NPHS1 mutations associated with an earlier onset of disease and worse renal outcomes than NPHS2 mutations. Milder cases resulting from mutant NPHS1 had either two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. In addition, we extend the spectrum of known NPHS1 mutations by describing long NPHS1 deletions. In summary, these data demonstrate that CNS is not a distinct clinical entity in non-Finnish populations but rather a clinically and genetically heterogeneous group of disorders.Congenital nephrotic syndrome (CNS) of the Finnish type (CNF; MIM# 256300) is a recessively inherited disorder characterized by massive proteinuria at birth, a large placenta, and marked edema within the first 3 months of life.^1–4 The disease is most frequent in Finland, where its incidence is 1 per 8200 newborns.⁵ Renal histology encompasses mesangial hypercellularity and matrix expansion, progressing with age to complete mesangial sclerosis and capillary obliteration.⁶ Irregular microcystic dilation of the proximal tubules (PTD) is the most typical histologic feature, observed as early as 18 to 20 weeks of gestation^7–9 and increasing in frequency with age¹⁰; however, PTD is not observed in all cases. Ultrastructural analysis of the glomerular capillary loops shows complete foot process effacement and swelling of endothelial cells.¹¹NPHS1, encoding nephrin, was identified by positional cloning more than a decade ago and is the major gene involved in CNF in Finnish populations (98% of cases).¹² The Fin_major (c.121delCT; p.L41fs) and Fin_minor (c.3325C>T; p.R1109X) mutations account for 78 and 16% of the mutated alleles, respectively, in Finnish cases¹²; however, these mutations are rarely found in other ethnic groups.¹³ NPHS1 genetic screening in patients of non-Finnish origin has shown that the frequency of NPHS1 mutations is lower than that in Finnish patients, with such mutations accounting for 39 to 55% of cases.^14,15 Indeed, more than 140 different mutations have been identified among non-Finnish cases,^14–24 including protein-truncating nonsense mutations, frameshift small insertion/deletion mutations, and splice-site changes.^14–24 In vitro functional assays have shown that most NPHS1 missense mutations lead to retention of the protein in the endoplasmic reticulum,^25,26 resulting in a complete loss of nephrin from the cell surface. This suggests that defective intracellular nephrin trafficking, presumably as a result of protein misfolding, is a common consequence of the NPHS1 missense mutations implicated in CNS.Nephrin is a transmembrane protein of the Ig superfamily characterized by eight C2-type Ig-like domains and a fibronectin type III repeat in the extracellular region, a single transmembrane domain, and a cytosolic C-terminal end.¹² The extracellular domain of nephrin forms homodimers and heterodimers with NEPH1.^27,28 Nephrin–NEPH1 interactions control nephrin signaling,²⁹ glomerular permeability,³⁰ and podocyte cell polarity.³¹ Neph1 knockout mice present massive proteinuria within the first 2 weeks of life and renal lesions resembling CNF in humans,^32,33 suggesting that recessive inactivating mutations in the NEPH1 gene may be involved in congenital human glomerular disease; however, no mutations have yet been identified in this gene.One striking finding among patients with CNS has been the detection of mutations in the NPHS2 gene,^19,34 encoding podocin, which has been implicated mainly in early-onset steroid-resistant nephrotic syndrome (SRNS).³⁵ A recent analysis showed that as many as 51% of patients who had CNS and were of European origin had mutations in the NPHS2 gene.¹⁵ In addition to mutations in the NPHS1 and NPHS2 genes, mutations in PLCE1 and WT1, which are known to cause infantile NS, have been implicated in cases of CNS and diffuse mesangial sclerosis (DMS).^36–40 Several hereditary forms of syndromic CNS have also been described. LAMB2 mutations have been implicated in Pierson syndrome, a rare autosomal recessive disorder characterized by microcoria and other complex ocular abnormalities associated with CNS.^41,42 Moreover, patients with NS and minor structural eye defects and others with isolated NS have expanded the phenotypic spectrum of mutations in the LAMB2 gene.^43–45 Finally, mutations in the PSSD2 and COQ2 genes have been found in patients with mitochondriopathies, typically presenting primary coenzyme Q10 deficiency and profound neuromuscular symptoms and occasionally developing NS.^46–51These clinical associations confirm the genetic heterogeneity of CNS in non-Finnish patients. It remains unclear whether subtle differences in phenotype between patients who have CNS and bear NPHS1 or NPHS2 mutations could be used to guide genetic screening. Distinctive extrarenal clinical features certainly help to direct mutational screening among patients with syndromic forms of CNS; however, patients with nonsyndromic CNS may have mutations in the WT1, LAMB2, COQ2, and PDSS2 genes, making genetic screening more difficult and time-consuming. Finally, the contribution of NEPH1 mutations to CNS has never been explored. We therefore carried out a comprehensive genetic analysis in the largest multiethnic cohort of patients with CNS of non-Finnish origin reported to date, with the aim of defining the epidemiologic role of mutations in the main genes implicated in CNS and elucidating potentially novel genotype–phenotype correlations.     

Combined Complement Gene Mutations in Atypical Hemolytic Uremic Syndrome Influence Clinical Phenotype

Several abnormalities in complement genes reportedly contribute to atypical hemolytic uremic syndrome (aHUS), but incomplete penetrance suggests that additional factors are necessary for the disease to manifest. Here, we sought to describe genotype–phenotype correlations among patients with combined mutations, defined as mutations in more than one complement gene. We screened 795 patients with aHUS and identified single mutations in 41% and combined mutations in 3%. Only 8%–10% of patients with mutations in CFH, C3, or CFB had combined mutations, whereas approximately 25% of patients with mutations in MCP or CFI had combined mutations. The concomitant presence of CFH and MCP risk haplotypes significantly increased disease penetrance in combined mutated carriers, with 73% penetrance among carriers with two risk haplotypes compared with 36% penetrance among carriers with zero or one risk haplotype. Among patients with CFH or CFI mutations, the presence of mutations in other genes did not modify prognosis; in contrast, 50% of patients with combined MCP mutation developed end stage renal failure within 3 years from onset compared with 19% of patients with an isolated MCP mutation. Patients with combined mutations achieved remission with plasma treatment similar to patients with single mutations. Kidney transplant outcomes were worse, however, for patients with combined MCP mutation compared with an isolated MCP mutation. In summary, these data suggest that genotyping for the risk haplotypes in CFH and MCP may help predict the risk of developing aHUS in unaffected carriers of mutations. Furthermore, screening patients with aHUS for all known disease-associated genes may inform decisions about kidney transplantation.Hemolytic uremic syndrome (HUS) is a rare disease of microangiopathic hemolysis, thrombocytopenia, and renal failure.^1,2 The most common form in children is associated with infection by certain strains of Escherichia coli, which produce Shiga-like toxins.³ This form has a good prognosis.¹ There are rarer atypical forms (aHUS), not associated with Shiga-like toxins-producing bacteria, that have a worse outcome, with up to 50% of cases progressing to end stage renal failure (ESRF) and 10%–15% dying during the acute phase.^1,4Inherited defects that determine uncontrolled activation of the alternative complement pathway have been well documented in aHUS patients.^2,5,6 Research in recent years has identified more than 120 different mutations, accounting for around 40%–60% of cases, in the genes encoding complement factor H (CFH),^7–9 membrane cofactor protein (MCP),^10–13 complement factor I (CFI),^14–16 C3,¹⁷ complement factor B (CFB),^18,19 CFH-related 5 (CFHR5),²⁰ and thrombomodulin (THBD).^20,21 In addition, anti-CFH autoantibodies have been described mostly in children that lack CFHR1 and CFHR3 because of a deletion of the corresponding genes.^22–26 Novel genetic abnormalities of CFHR1, CFHR3, and CFHR4 and genomic rearrangement between CFH and CFHR1 have recently been described.^27,28Incomplete penetrance of aHUS has been reported in mutation carriers,^12,29–31 indicating that complement gene mutations confer predisposition to develop aHUS, with additional genetic and/or environmental hits necessary for disease manifestation.^7,32,33 In keeping with this hypothesis, patients with mutations in more than one complement gene (combined gene mutations) have been described.^20,29,34,35 This study was designed to (1) determine the frequency of combined complement gene mutations among four cohorts of aHUS patients; (2) compare short- and long-term outcomes, response to plasma treatment, and outcome of kidney transplantation among patients carrying mutations in different gene combinations; and (3) compare clinical parameters in patients carrying combined mutations versus patients with mutations in a single complement gene. Thanks to a joint effort by the European Working Party on Complement Genetics in Renal Diseases, we genotyped almost 800 aHUS patients for aHUS-associated genes, identifying 27 patients with combined gene mutations.     

Mutation of the Mg2+ Transporter SLC41A1 Results in a Nephronophthisis-Like Phenotype

Nephronophthisis (NPHP)-related ciliopathies are recessive, single-gene disorders that collectively make up the most common genetic cause of CKD in the first three decades of life. Mutations in 1 of the 15 known NPHP genes explain less than half of all cases with this phenotype, however, and the recently identified genetic causes are exceedingly rare. As a result, a strategy to identify single-gene causes of NPHP-related ciliopathies in single affected families is needed. Although whole-exome resequencing facilitates the identification of disease genes, the large number of detected genetic variants hampers its use. Here, we overcome this limitation by combining homozygosity mapping with whole-exome resequencing in a sibling pair with an NPHP-related ciliopathy. Whole-exome capture revealed a homozygous splice acceptor site mutation (c.698G>T) in the renal Mg²⁺ transporter SLC41A1. This mutation resulted in skipping of exon 6 of SLC41A1, resulting in an in-frame deletion of a transmembrane helix. Transfection of cells with wild-type or mutant SLC41A1 revealed that deletion of exon 6 completely blocks the Mg²⁺ transport function of SLC41A1. Furthermore, in normal human kidney tissue, endogenous SLC41A1 specifically localized to renal tubules situated at the corticomedullary boundary, consistent with the region of cystogenesis observed in NPHP and related ciliopathies. Last, morpholino-mediated knockdown of slc41a1 expression in zebrafish resulted in ventral body curvature, hydrocephalus, and cystic kidneys, similar to the effects of knocking down other NPHP genes. Taken together, these data suggest that defects in the maintenance of renal Mg²⁺ homeostasis may lead to tubular defects that result in a phenotype similar to NPHP.Rare CKDs make up the majority of CKD cases treated within long-term dialysis and renal transplantation programs in the first three decades of life but are notoriously difficult to diagnose.¹ Rare recessive mutations cause chronic diseases that often require hospitalization.² However, half of their single-gene causes are still unknown (http://omim.org/statistics/entries). Because recessive single-gene mutations directly represent the disease cause, gene identification offers a powerful approach to revealing disease mechanisms. Furthermore, because recessive mutations predominantly convey loss of function, recessive single-gene defects can be transferred directly into animal models to study the related disease mechanisms and to screen for small molecules as possible treatment modalities.Nephronophthisis (NPHP), a recessive cystic kidney disease, is the most frequent genetic cause of CKD in the first three decades of life. NPHP-related disorders are recessive single-gene disorders that affect kidney, retina, brain, and liver by prenatal-onset dysplasia or by organ degeneration and fibrosis in early adulthood.³ On ultrasonography, these conditions are characterized by increased echogenicity and cyst formation at the corticomedullary junction in small or normal-sized kidneys.⁴ Renal histology exhibits a characteristic triad of renal corticomedullary cysts, tubular basement membrane disruption, and tubulointerstitial infiltrations.⁵ Regarding renal, retinal, and hepatic involvement, there is phenotypic overlap of NPHP-related disorders with Bardet-Biedl syndrome and Alstrom syndrome.⁶ Identification of recessive mutations in 15 different genes (NPHP1–NPHP15)^7–18 revealed that the encoded proteins share localization at the primary cilia-centrosomes complex, which characterizes them as ciliopathies.^3,19 However, the 15 known NPHP genes explain less than 50% of all cases with NPHP-related disorders, indicating that many of the single-gene causes of these conditions are still elusive.²⁰The finding that some of the more recently identified genetic causes of NPHP-related disorders are exceedingly rare necessitates a strategy to identify novel single-gene causes of these conditions in single affected families.¹⁵ In this context, the new method of whole exome capture with consecutive massively parallel sequencing (here called whole exome resequencing [WER]) theoretically offers a powerful approach toward gene identification in rare recessive diseases. However, the utility of WER is hampered by the large number of novel genetic variants that result from whole exome sequencing in any given individual.^18,21 To overcome this limitation of WER, we developed a strategy that combines WER with homozygosity mapping.¹⁸ Using this approach, we have identified mutation of the renal magnesium transporter gene SLC41A1 as a novel genetic cause of disease that phenocopies NPHP-related conditions clinically, ultrasonographically, and histologically.     

TLR4 Promotes Fibrosis but Attenuates Tubular Damage in Progressive Renal Injury

Toll-like receptors (TLRs) can orchestrate an inflammatory response upon activation by pathogen-associated motifs and release of endogenous stress ligands during tissue injury. The kidney constitutively expresses most TLRs, including TLR4. The function of TLR4 during the inflammation, tubular atrophy, and fibrosis that accompany progressive renal injury is unknown. Here, we subjected wild-type (WT) and TLR4-deficient mice to unilateral ureteral obstruction and observed elevated levels of TLR4 mRNA in the kidney after obstruction. One day after unilateral ureteral obstruction, TLR4-deficient mice had fewer proliferating tubular epithelial cells and more tubular damage than WT mice; however, TLR4-deficient mice developed considerably less renal fibrosis despite decreased matrix metalloproteinase activity and without significant differences in myofibroblast accumulation. In vitro, TLR4-deficient primary tubular epithelial cells and myofibroblasts produced significantly less type I collagen mRNA after TGF-β stimulation than WT cells. The reduced fibrosis in TLR4-deficient mice associated with an upregulation of Bambi, a negative regulator of TGF-β signaling. In conclusion, TLR4 attenuates tubular damage but promotes renal fibrosis by modulating the susceptibility of renal cells to TGF-β. These data suggest that TLR4 signaling may be a therapeutic target for the prevention of renal fibrosis.Fibroproliferative diseases, including progressive renal disease, are a leading cause of morbidity and mortality worldwide.¹ Renal tubular damage, inflammation, and interstitial fibrosis are main predictors for the risk for progression toward end-stage renal failure.² Progression of renal fibrosis involves a cascade of pathophysiologic processes, including disruption of tubular integrity, a robust inflammatory response, accumulation of (myo)fibroblasts, tubular atrophy, and an increased deposition of extracellular matrix (ECM) components, resulting in fibrogenesis.^3–5The group of Toll-like receptors (TLRs) may be one of the receptor families that orchestrate this cascade of inflammation, myofibroblast accumulation, and fibrosis in the kidney. TLRs can initiate an inflammatory response upon recognition of specific pathogen-associated molecular patterns. It is widely accepted that not only pathogen-associated molecular patterns can trigger TLR-mediated immune responses but endogenous danger molecules that are released upon tissue or cell injury as well.^6–11 We already found that several of these endogenous ligands that can potentially activate both TLR2 and TLR4^9,11–13 are strongly upregulated in murine kidneys after unilateral ureteral obstruction (UUO).^14,15 We demonstrated that TLR2 does not play a role in the development of fibrosis or injury after UUO.¹⁴ Until now, the role of TLR4 in progressive renal injury and fibrosis has remained unknown. In a model of hepatic fibrogenesis, it was demonstrated that TLR4 can enhance TGF-β signaling and myofibroblast activation, suggesting that TLR4 can function as a molecular link between proinflammatory and profibrogenic signals in liver tissue.¹⁶ Interestingly, TLR4 is widely and constitutively expressed in the kidney (e.g., on tubular epithelial cells [TECs]).^17,18 We and others have shown that renal-associated TLR2 and TLR4 can induce an exaggerated inflammatory response in the kidney upon acute ischemic renal injury with subsequent detrimental effects on renal histology and function.^19–21 To study the role of TLR4 in progressive renal injury and renal fibrosis, we subjected wild-type (WT) and TLR4−/− mice to UUO.     

TRPV4 Dysfunction Promotes Renal Cystogenesis in Autosomal Recessive Polycystic Kidney Disease

The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca²⁺-permeable TRPV4 channel underlies flow-dependent Ca²⁺ signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased basal [Ca²⁺]_i, and loss of flow-mediated [Ca²⁺]_i signaling. In contrast, nondilated CDs of these rats exhibited functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmentation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mechanosensitive Ca²⁺ signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca²⁺ signaling in CD cells, inversely correlates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.Polycystic kidney disease (PKD) is a cohort of monogenic disorders that result in development and subsequent growth of renal cysts filled with fluid.^1–5 Cyst enlargement compromises function of surrounding nephrons and progresses to ESRD.^1,6 In the more common form of PKD, autosomal dominant PKD (ADPKD), which is caused by mutations of polycystin 1 (PC1) and polycystin 2 (PC2), renal cysts are formed along the full length of the nephron with prevalence to the collecting duct (CD).^1,7 In the rarer and more severe autosomal recessive PKD (ARPKD), renal cyst formation is virtually restricted to the CD.^1,2,5,8 Mutations of the PKHD1 gene encoding fibrocystin underlie the genetic basis of the disease.^6,8,9 Although the exact function of the protein is unknown, fibrocystin was shown to be expressed in primary cilia where it can interact and form complexes with PC2, possibly participating in mechanotransduction.^10–12It is accepted that the CD cells elevate [Ca²⁺]_i in response to mechanical stress arising from variations in tubular flow or tubular composition.^13–23 Impaired mechanosensitive [Ca²⁺]_i responses, reported for both cultured ADPKD²⁴ and ARPKD^25,26 cells, point to a possible fundamental role of disrupted [Ca²⁺]_i signaling in cystogenesis. The central cilia and cilia-associated PC1 and PC2 were proposed to mediate flow-induced cellular responses.^19,27 However, homomeric PC2 channels are not mechanosensitive and fail to increase [Ca²⁺]_i in response to flow and hypotonicity.^28,29 Furthermore, intercalated cells, which lack primary cilia, respond to flow changes with comparable increases in [Ca²⁺]_i as observed in principal cells, which have primary cilia.^16,30 Therefore, additional mechanisms conferring mechanosensitivity to the CD cells need to be considered.Transient receptor potential (TRP) channels are known to participate in cellular responses to a variety of environmental stimuli, including thermosensation, chemosensation, and mechanical forces (reviewed in Song and Yuan³¹). Several TRP channels, including TRPC3, TRPC6, and TRPV4, can be detected in the native CD cells and CD-originated cultured lines.^19,32–34 Among these channels, TRPV4 has routinely been shown to be activated by mechanical stimuli.^34–38 Indeed, we documented that endogenous TRPV4 in M-1 CD cells is stimulated by increases in flow, a response that is abolished by TRPV4 small interfering RNA knockdown.^34,38 We further demonstrated a lack of flow-mediated [Ca²⁺]_i elevations in CD from TRPV4−/− mice.³⁰ Consistently, flow-mediated Ca²⁺-dependent K⁺ secretion in the CD is disrupted in TRPV4 knockout animals.³⁹ TRPV4 directly interacts with PC2 to form mechanosensitive heteromeric complexes.^28,29 The fact that PC2 interacts with both PC1⁴⁰ and fibrocystin^10–12 suggests that TRPV4 could be an essential part of this mechanotransducing sensory complex.Current PKD management is directed toward pharmacologic interference with abnormal signaling pathways causing exaggerated cell proliferation, dedifferentiation, apoptosis, and cyst growth.⁴¹ Specifically, PKD is associated with elevated circulating vasopressin levels, increased basal cellular cAMP levels, and strong upregulation of cAMP-dependent fluid secretion and proliferation.^42,43 V2 antagonism greatly diminishes disease progression in rodent models of both ADPKD and ARPKD.^42,44 Elevated cAMP levels might be directly related to the reduced [Ca²⁺]_i, possibly due to impaired ability to sense changes in flow.^42,44,45 This raises the possibility that manipulation with the mechanosensitivity in the CD along the TRPV4 axis modulates [Ca²⁺]_i signalization and, in turn, renal cystogenesis.In this study, we developed a new approach to isolate native CD-derived cyst monolayers and nondilated CDs from a rat model of ARPKD to thoroughly investigate how functional TRPV4 status determines the development and growth of renal cysts. We found that the disease leads to disruption of mechanosensitive [Ca²⁺]_i signaling and impaired TRPV4 activity specifically in CD cysts but not in nondilated CDs. Long-term pharmacologic potentiation of TRPV4 activity gradually restores mechanosensitivity in cyst cells and greatly blunts renal ARPKD progression. From a global prospective, this study establishes a temporal link between disruption of TRPV4-based mechanosensitivity in the CD and cystogenesis. This also suggests pharmacologic potential of targeting TRPV4 activity as a treatment strategy in retarding development of ARPKD.     

Mutations in SLC34A3/NPT2c Are Associated with Kidney Stones and Nephrocalcinosis

Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na⁺)-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)₂ vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P<0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)₂ vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.Inactivating mutations on both parental alleles of the solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na⁺)-dependent phosphate cotransporter 2c (NPT2c), are the cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH; OMIM: 241530)^1–3—an autosomal recessive renal phosphate-wasting disorder that was originally described by Tieder et al.^4,5 Individuals affected by HHRH who carry compound heterozygous or homozygous (comp/hom) SLC34A3/NPT2c mutations show increased urinary phosphate excretion leading to hypophosphatemic rickets, bowing, and short stature as well as appropriately elevated 1,25(OH)₂D levels. Elevated 1,25(OH)₂D levels, in turn, result in hypercalciuria because of enhanced intestinal calcium absorption and reduced parathyroid hormone (PTH) -dependent calcium reabsorption in the distal renal tubules. Even heterozygous SLC34A3/NPT2c mutations are frequently associated with hypercalciuria, but none of the carriers of SLC34A3/NPT2c mutations in the originally described HHRH patients were reported to have renal calcifications and kidney stones.^4,5 Subsequent investigations, however, revealed that these complications affecting the kidneys were observed in numerous patients with comp/hom SLC34A3/NPT2c mutations.^3,6–11 However, the small size of HHRH kindreds and the relatively high prevalence of renal calcifications in the general population (5.64%)^12,13 have, thus far, prevented segregation-based statistical approaches to determine whether SLC34A3/NPT2c mutations do increase the risk of developing kidney stones or nephrocalcinosis. Likewise, it is unknown whether loss of NPT2c can lead to additional proximal tubular phenotypes such as Fanconi syndrome, which has been described in two patients with homozygous SLC34A3/NPT2a mutations¹⁴ who developed CKD later in life.The presence of hypercalciuria, kidney stones, and nephrocalcinosis observed in HHRH kindreds is different from the findings in fibroblast growth factor 23 (FGF23)–dependent hypophosphatemic disorders, such as X-linked hypophosphatemia (XLH; mutant PHEX),¹⁵ autosomal dominant hypophosphatemic rickets (ADHR; mutant FGF23),¹⁶ or autosomal recessive hypophosphatemic rickets (ARHR; mutant DMP1, ENPP1, or FAM20C),^17–20 in which affected individuals show, before treatment with oral phosphate and 1,25(OH)₂D, inappropriately normal or suppressed 1,25(OH)₂D levels despite significant hypophosphatemia and thus, no increase in urinary calcium excretion. Oral phosphate supplements combined with active vitamin D analogs are generally recommended for treatment of FGF23-dependent hypophosphatemic disorders.²¹ In contrast, HHRH is thought to require phosphate supplements alone,^4,5 in part because endogenously elevated 1,25(OH)₂D levels are predicted to prevent an increase in PTH secretion triggered by intermittent elevations in serum phosphate. However, long-term studies are lacking that determine whether oral phosphate supplementation alone of HHRH patients is sufficient for prevention of renal calcifications and bone loss. It is likewise unknown how therapy should be monitored, whether secondary hyperparathyroidism can develop as observed in FGF23-dependent hypophosphatemic disorders,¹⁵ and whether phosphate requirements decrease with age, which has been reported for ADHR.¹⁶In the current study, we investigated five new HHRH kindreds and two new cases with idiopathic hypercalciuria (IH), in whom we discovered known and novel homozygous or compounded heterozygous SLC34A3/NPT2c mutations. Review of the clinical and laboratory findings along with those findings reported for 22 previously published kindreds suggests that renal calcifications and/or kidney stones may be important, often unrecognized initial findings suggestive of comp/hom SLC34A3/NPT2c mutations. Importantly, heterozygous carriers also show an increased frequency of renal calcifications and biochemical profiles in plasma and urine that are intermediate to those profiles of individuals without SLC34A3/NPT2c mutations and comp/hom changes. Our data suggest that serum phosphate, tubular reabsorption of phosphate (TRP), and serum 1,25(OH)₂D levels predict the development of renal calcifications. However, additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent renal calcifications and potentially, CKD.     

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL)

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy is an autosomal dominant disease affecting small vessels and often resulting in subcortical infarcts. A skin biopsy may facilitate its diagnosis as the cutaneous surface is much easier to sample than the central nervous system’s tissue. Unfortunately, there is no effective treatment available today.Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant, small-vessel disease characterized by multiple subcortical ischemic infarcts. These infarcts mainly involve the central nervous system and can lead to disability and dementia.1,2 Linkage studies identified a mutation in the NOTCH3 gene on chromosome 19 as the genetic defect in CADASIL.3 The prevalence of the NOTCH3 gene mutation is 4.14 per 100,000 adults as estimated in a registry for CADASIL in Scotland.4 CADASIL is caused by mutations in one of the exons (from 2 to 24 out of the 33 exons) of the NOTCH3 gene within the epidermal growth factor receptor (EGFR)-like repeats in the extracellular domain of the NOTCH3 protein.5–7 More than 150 mutations have been identified so far and clustering of mutations on exons 3,4,5,8, and 11 has been reported.8,9 The missense mutations lead to a cysteine substitution in the EGFR on the extracellular N-terminal domain.8 This is thought to cause a defect in transendothelial exchange. Besides familial occurrence, sporadic cases are known to occur, which are more likely to go undiagnosed or misdiagnosed.10 In 70 percent of families, the mutations are located on exons 3 and 4 that encode the first 5 EGF domains.8A skin biopsy from a normal appearing cutaneous area can be very helpful in diagnosing CADASIL as the vascular changes can be observed using electron microscopy.11,12 The knowledge of CADASIL among dermatopathologists is important as patients with CADASIL may be referred by neurologists to carry out and interpret skin biopsies, ultimately providing a key diagnostic input. Additionally, a skin biopsy also helps to detect a carrier status.     

A Recessive Gene for Primary Vesicoureteral Reflux Maps to Chromosome 12p11-q13

Primary vesicoureteral reflux (pVUR) is one of the most common causes of pediatric kidney failure. Linkage scans suggest that pVUR is genetically heterogeneous with two loci on chromosomes 1p13 and 2q37 under autosomal dominant inheritance. Absence of pVUR in parents of affected individuals raises the possibility of a recessive contribution to pVUR. We performed a genome-wide linkage scan in 12 large families segregating pVUR, comprising 72 affected individuals. To avoid potential misspecification of the trait locus, we performed a parametric linkage analysis using both dominant and recessive models. Analysis under the dominant model yielded no signals across the entire genome. In contrast, we identified a unique linkage peak under the recessive model on chromosome 12p11-q13 (D12S1048), which we confirmed by fine mapping. This interval achieved a peak heterogeneity LOD score of 3.6 with 60% of families linked. This heterogeneity LOD score improved to 4.5 with exclusion of two high-density pedigrees that failed to link across the entire genome. The linkage signal on chromosome 12p11-q13 originated from pedigrees of varying ethnicity, suggesting that recessive inheritance of a high frequency risk allele occurs in pVUR kindreds from many different populations. In conclusion, this study identifies a major new locus for pVUR and suggests that in addition to genetic heterogeneity, recessive contributions should be considered in all pVUR genome scans.Vesicoureteral reflux (VUR; OMIM no. 193000) is the retrograde flow of urine from the bladder to the ureters and the kidneys during micturation. Uncorrected, VUR can lead to repeated urinary tract infections, renal scarring and reflux nephropathy, accounting for up to 25% of pediatric end stage renal disease.^1,2 VUR is commonly seen as an isolated disorder (primary VUR; pVUR), but it can also present in association with complex congenital abnormalities of the kidney and urinary tract or with specific syndromic disorders, such as renal-coloboma and branchio-oto-renal syndromes.^3–8pVUR has a strong hereditary component, with monozygotic twin concordance rates of 80%.^9–12 Sibling recurrence rates of 30% to 65% have suggested segregation of a single gene or oligogenes with large effects.^9,12–14 Interestingly however, the three published genome-wide linkage scans of pVUR have strongly suggested multifactorial determination.^15–17 Two pVUR loci have been identified with genome-wide significance on chromosomes 1p13 and 2q37 under an autosomal dominant transmission with locus heterogeneity.^15,16 Multiple suggestive signals have also been reported, but remarkably, these studies show little overlap.^15–17 These data suggest that pVUR may be extremely heterogeneous, with mutations in different genes each accounting for a fraction of cases. The genes underlying pVUR loci have not yet been identified, but two recent studies have reported segregating mutations in the ROBO2 gene in up to 5% of pVUR families.^18,19Despite evidence for genetic heterogeneity and different subtypes of disease, genetic studies have all modeled pVUR as an autosomal dominant trait.^15–17,20 Recessive inheritance has generally not been considered because the absence of affected parents can be explained by spontaneous resolution of pVUR with older age. However, many pVUR cohorts are composed of affected sibships or pedigrees compatible with autosomal recessive transmission, suggesting the potential for alternative modes of inheritance.^{9–12,16,17,20–22} Systematic family screening to clarify the mode of inheritance is not feasible for pVUR because the standard diagnostic tool, the voiding cystourethrogram (VCUG), is invasive and would expose participants to radiation. Formal assessment of a recessive contribution in sporadic pVUR has also been difficult because studies have been conducted in populations with low consanguinity rates.^{9–12,16,17,20–22} However, recent studies have identified an unexpected recessive contribution to several complex traits such as ductus arteriosus or autism.^23,24 Thus, in addition to genetic heterogeneity, genes with alternative modes of transmission may segregate among pVUR families, and misspecification of the inheritance model may complicate mapping studies of this trait.Several approaches can be considered to address the difficulties imposed by complex inheritance, variable penetrance, and genetic heterogeneity. Studying large, well characterized cohorts with newer single-nucleotide polymorphism (SNP)-based technologies can maximize inheritance information across the genome and increase the power of linkage studies.²⁵ In addition, in the setting of locus heterogeneity and uncertainty about the mode of transmission, analysis under a dominant and a recessive model has greater power compared with nonparametric methods and more often results in detection of the correct mode of transmission without incurring a significant penalty for multiple testing.^26–29 We combined these approaches in this study and successfully localized a major gene for VUR, which unexpectedly demonstrates autosomal recessive transmission.     

Genetic Variation of DKK3 May Modify Renal Disease Severity in ADPKD

Significant variation in the course of autosomal dominant polycystic kidney disease ( ADPKD) within families suggests the presence of effect modifiers. Recent studies of the variation within families harboring PKD1 mutations indicate that genetic background may account for 32 to 42% of the variance in estimated GFR (eGFR) before ESRD and 43 to 78% of the variance in age at ESRD onset, but the genetic modifiers are unknown. Here, we conducted a high-throughput single-nucleotide polymorphism (SNP) genotyping association study of 173 biological candidate genes in 794 white patients from 227 families with PKD1. We analyzed two primary outcomes: (1) eGFR and (2) time to ESRD (renal survival). For both outcomes, we used multidimensional scaling to correct for population structure and generalized estimating equations to account for the relatedness among individuals within the same family. We found suggestive associations between each of 12 SNPs and at least one of the renal outcomes. We genotyped these SNPs in a second set of 472 white patients from 229 families with PKD1 and performed a joint analysis on both cohorts. Three SNPs continued to show suggestive/significant association with eGFR at the Dickkopf 3 (DKK3) gene locus; no SNPs significantly associated with renal survival. DKK3 antagonizes Wnt/β-catenin signaling, which may modulate renal cyst growth. Pending replication, our study suggests that genetic variation of DKK3 may modify severity of ADPKD resulting from PKD1 mutations.Autosomal dominant polycystic kidney disease ( ADPKD) is the most common monogenic kidney disease worldwide, affecting one in 500 to 1000 births.^1,2 It is characterized by focal development of renal cysts in an age-dependent manner. Typically, only a few renal cysts are clinically detectable during the first three decades of life; however, by the fifth decade, tens of thousands of renal cysts of different sizes can be found in most patients.³ Progressive cyst expansion with age leads to massive enlargement and distortion of the normal architecture of both kidneys and, ultimately, ESRD in most patients. ADPKD is also associated with an increased risk for cardiac valvular defects, colonic diverticulosis, hernias, and intracranial arterial aneurysms. Overall, ADPKD accounts for approximately 5% of ESRD in North America.²Mutations of PKD1 and PKD2 respectively account for approximately 85% and approximately 15% of linkage-characterized European families. Polycystin-1 (PC-1) and PC-2, the proteins encoded by PKD1 and PKD2, respectively, function as a macromolecular complex and regulate multiple signaling pathways to maintain the normal tubular structure and function.¹ Monoclonal expansion of individual epithelial cells that have undergone a somatic “second hit” mutation, resulting in biallelic inactivation of either PKD1 or PKD2, seems to provide a major mechanism for focal cyst initiation,⁴ possibly through the loss of polycystin-mediated mechanosensory function in the primary cilium.⁵ In addition, a large prospective, observational study indicated that renal cysts in ADPKD expand exponentially with increasing age, and patients with large polycystic kidneys are at higher risk for developing kidney failure⁶; however, the key factors that modulate renal disease progression in ADPKD remain incompletely understood.Renal disease severity in ADPKD is highly variable, with the age of onset of ESRD ranging from childhood to old age.^7–11 A strong genetic locus effect has been noted in ADPKD. Adjusted for age and gender, patients with PKD1 have larger kidneys and earlier onset at ESRD than patients with PKD2 (mean age at ESRD 53.4 versus 72.7 years, respectively).^8,9 By contrast, a weak allelic effect (based on the 5′ versus 3′ location of the germline mutations) on renal disease severity may be present for PKD1¹⁰ but not PKD2.¹¹ Marked intrafamilial variability in renal disease is well documented in ADPKD and suggests a strong modifier effect.^10–15 In an extreme example, large polycystic kidneys were present in utero in one of a pair of dizygotic twins affected with the same germline PKD1 mutation, whereas the kidneys of the co-twin remained normal at 5 years of age.¹² Several studies have quantified the role of genetic background in the phenotypic expression of ADPKD. In a comparison of monozygotic twins and siblings, greater variance in the age of onset of ESRD in the siblings supported a role for genetic modifiers.¹³ Two other studies of intrafamilial disease variability in PKD1 have estimated that genetic factors may account for 32 to 42% of the variance of creatinine clearance before ESRD and 43 to 78% of the variance in age at ESRD.^14,15 The magnitude of the modifier gene effect from these studies suggests that mapping such factors is feasible. Here, we report the results of an association study of modifier genes for PKD1 renal disease severity.     

Renal Dendritic Cells Ameliorate Nephrotoxic Acute Kidney Injury

Inflammation contributes to the pathogenesis of acute kidney injury. Dendritic cells (DCs) are immune sentinels with the ability to induce immunity or tolerance, but whether they mediate acute kidney injury is unknown. Here, we studied the distribution of DCs within the kidney and the role of DCs in cisplatin-induced acute kidney injury using a mouse model in which DCs express both green fluorescence protein and the diphtheria toxin receptor. DCs were present throughout the tubulointerstitium but not in glomeruli. We used diphtheria toxin to deplete DCs to study their functional significance in cisplatin nephrotoxicity. Mice depleted of DCs before or coincident with cisplatin treatment but not at later stages experienced more severe renal dysfunction, tubular injury, neutrophil infiltration and greater mortality than nondepleted mice. We used bone marrow chimeric mice to confirm that the depletion of CD11c-expressing hematopoietic cells was responsible for the enhanced renal injury. Finally, mixed bone marrow chimeras demonstrated that the worsening of cisplatin nephrotoxicity in DC-depleted mice was not a result of the dying or dead DCs themselves. After cisplatin treatment, expression of MHC class II decreased and expression of inducible co-stimulator ligand increased on renal DCs. These data demonstrate that resident DCs reduce cisplatin nephrotoxicity and its associated inflammation.Innate immune responses are pathogenic in both ischemic and toxic acute renal failure. In response to renal injury, inflammatory chemokines and cytokines are produced both by renal parenchymal cells, such as proximal tubule epithelial cells, and resident or infiltrating leukocytes.^1–4 The elaborated chemokines and cytokines, including TNF-α, IL-18, keratinocyte-derived chemokine, and monocyte chemoattractant protein 1, subsequently recruit additional immune cells to the kidney, such as neutrophils, T cells, monocytes, and inflammatory dendritic cells (DCs), which may cause further injury through pathways that are not fully defined.^2,5–12 DCs are sentinels of the immune system and under steady-state conditions induce tolerance by various mechanisms, including production of TGF-β, IL-10, or indoleamine 2,3-dioxygenase^13–16; expression of PDL-1, PDL-2, or FcγR2B^17,18; clonal deletion of autoreactive T cells¹⁹; and induction of T regulatory cells via the inducible co-stimulator (ICOS) pathway.^20–23 In response to pathogens or products of tissue injury, DCs mature and initiate immunity or inflammatory diseases.^24,25 Monocytes recruited to inflamed tissue can also differentiate into inflammatory DCs and mediate defense against pathogens or contribute to inflammatory tissue responses.^12,26–28Although DCs represent a major population of immune cells in the kidney,²⁹ their role in renal disease is poorly defined. Liposomal clodronate has been used to study the pathophysiologic role of phagocytic cells, which include DCs and macrophages.^3,30–32 An alternative DC-specific approach uses expression of the simian diphtheria toxin receptor (DTR) driven by the CD11c promoter to target DCs for DT-mediated cell death.²⁴ This model has been used extensively to study the role of DCs in various physiologic and pathophysiologic contexts^32,33; however, its application in kidney disease has been limited to recent studies of immune complex–mediated glomerulonephritis.^12,23We have reported that inflammation plays an important role in the pathogenesis of cisplatin-induced acute kidney injury (AKI).^1,4,5,34 Given the dearth of information regarding the role of renal DCs in AKI, this study examined the renal DC population and the impact of its depletion on cisplatin nephrotoxicity. We show that DCs are the most abundant population of renal resident leukocytes and form a dense network throughout the tubulointerstitium. Renal DCs displayed surface markers that distinguished them from splenic DCs. Using a conditional DC depletion model, we determined that DC ablation markedly exacerbates cisplatin-induced renal dysfunction, structural injury, and infiltration of neutrophils.     

WNK4 Enhances the Degradation of NCC through a Sortilin-Mediated Lysosomal Pathway

WNK kinase is a serine/threonine kinase that plays an important role in electrolyte homeostasis. WNK4 significantly inhibits the surface expression of the sodium chloride co-transporter (NCC) by enhancing the degradation of NCC through a lysosomal pathway, but the mechanisms underlying this trafficking are unknown. Here, we investigated the effect of the lysosomal targeting receptor sortilin on NCC expression and degradation. In Cos-7 cells, we observed that the presence of WNK4 reduced the steady-state amount of NCC by approximately half. Co-transfection with truncated sortilin (a dominant negative mutant) prevented this WNK4-induced reduction in NCC. NCC immunoprecipitated with both wild-type sortilin and, to a lesser extent, truncated sortilin. Immunostaining revealed that WNK4 increased the co-localization of NCC with the lysosomal marker cathepsin D, and NCC co-localized with wild-type sortilin, truncated sortilin, and WNK4 in the perinuclear region. These findings suggest that WNK4 promotes NCC targeting to the lysosome for degradation via a mechanism involving sortilin.WNK (with no lysine [K]) kinase is a subfamily of serine/threonine kinases.¹ Mutations in two members of this family, WNK1 and WNK4, result in pseudohypoaldosteronism type II,² featuring hypertension, hyperkalemia, and metabolic acidosis. Previous studies showed that wild-type (WT) WNK4 inhibits the activity and surface expression of sodium chloride co-transporter (NCC) in Xenopus oocytes.^3,4 Interestingly, one study showed that a NCC harboring five different Gitelman-type mutations exhibits low activity that is mainly due to a reduction of functional NCC inserting into the plasma membrane.⁵ Our previous study⁶ also indicated that NCC surface expression is regulated by altering its degradation through the lysosomal pathway. These combined studies suggest that alteration of NCC function can result from perturbing its protein synthesis,^7,8 glycosylation and processing,^9,10 and delivery to the plasma membrane.^8,10 Golbang et al.¹¹ showed that WNK4 also blocks the forward trafficking of NCC; however, the exact molecular basis of interference of WNK4 kinase with NCC forward trafficking and enhancing its degradation through a lysosomal pathway remains to be clarified.There are two major sorting mechanisms involving targeting lysosomal proteins to lysosomes.^12,13 One is the mannose-6-phosphate receptor (M6PR)-mediated mechanism,^14–16 and the other is mediated by sortilin.¹⁷ The trans-Golgi network (TGN) is the major site of sorting for newly synthesized proteins that are targeted to lysosomes for degradation. These newly synthesized proteins first bind to either M6PR^18–20 or sortilin²¹ in the TGN and subsequently recruit the Golgi-localized, γ-ear–containing, ADP-ribosylation factor–binding proteins (GGAs)^22–24 clathrin and adaptor protein 1 (AP1) or AP3^25,26 to form clathrin-coated vesicles to initiate their trafficking to endosomes, following a secretory pathway, or to lysosomes for degradation.Sortilin is a newly identified lysosomal targeting receptor that is involved in the alternative sorting of the lysosomal sphingolipid activator protein prosaposin^27,28 and the GM2 activator protein.^28,29 It is a homolog of the yeast vacuolar sorting receptor Vps10p and belongs to the type I Vps10p superfamily.¹⁷ The human sortilin gene encodes 833 amino acids. It consists of an N-terminal propeptide, a furin cleavage site, a large luminal domain, a single transmembrane region, and a short cytoplasmic tail.¹⁷ The major pool of sortilin accumulates in the TGN and vesicles, whereas 10% of sortilin is present in the plasma membrane. The truncation of the cytoplasmic tail of sortilin (sortilin TRU) leads to a disruption of the lysosomal sorting function, causing a majority of sortilin TRU to be retained in the TGN; however, a small portion of sortilin TRU may leak to the plasma membrane or vesicles. Sortilin TRU can serve as a dominant negative mutant.²⁸ Sortilin seems to have multiple functions. Sortilin not only binds different ligands such as neurotensin, receptor associated protein, and prosaposin,^17,30 but also is involved in intracellular sorting, endocytosis, and signal transduction.³¹ Studies have shown that sortilin binds the glucose transporter Glut4 and is translocated with Glut4 to the plasma membrane in response to insulin stimulation,^32–34 suggesting that sortilin may be involved in the regulation of membrane transporters; therefore, we speculated that WNK4 might promote the degradation of NCC through the sortilin-mediated lysosomal pathway. Here, we report that WNK4 downregulates the steady-state protein levels of NCC in Cos-7 cells. Sortilin TRU reverses the degradation of NCC promoted by WNK4. The N-terminus of NCC binds sortilin WT as well as sortilin TRU, to a lesser extent. WNK4 co-localizes with NCC and sortilin and increases NCC co-localization with cathepsin D, a lysosomal marker. These data suggest that WNK4, NCC, and sortilin interact with one another to facilitate the WNK4-promoted degradation of NCC through a lysosomal pathway by a sortilin-mediated targeting mechanism.     

Syndecan-4 Knockout Leads to Reduced Extracellular Transglutaminase-2 and Protects against Tubulointerstitial Fibrosis

Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. The interaction of TG2 with the heparan sulfate proteoglycan syndecan-4 (Sdc4) regulates the cell surface trafficking, localization, and activity of TG2 in vitro but remains unstudied in vivo. We tested the hypothesis that Sdc4 is required for cell surface targeting of TG2 and the development of kidney fibrosis in CKD. Wild-type and Sdc4-null mice were subjected to unilateral ureteric obstruction and aristolochic acid nephropathy (AAN) as experimental models of kidney fibrosis. Analysis of renal scarring by Masson trichrome staining, kidney hydroxyproline levels, and collagen immunofluorescence demonstrated progressive fibrosis associated with increases in extracellular TG2 and TG activity in the tubulointerstitium in both models. Knockout of Sdc-4 reduced these effects and prevented AAN-induced increases in total and active TGF-β1. In wild-type mice subjected to AAN, extracellular TG2 colocalized with Sdc4 in the tubular interstitium and basement membrane, where TG2 also colocalized with heparan sulfate chains. Heparitinase I, which selectively cleaves heparan sulfate, completely abolished extracellular TG2 in normal and diseased kidney sections. In conclusion, the lack of Sdc4 heparan sulfate chains in the kidneys of Sdc4-null mice abrogates injury-induced externalization of TG2, thereby preventing profibrotic crosslinking of extracellular matrix and recruitment of large latent TGF-β1. This finding suggests that targeting the TG2-Sdc4 interaction may provide a specific interventional strategy for the treatment of CKD.CKD is characterized by glomerulosclerosis and tubulointerstitial fibrosis that result from excessive extracellular matrix (ECM) accumulation.^1–3 In recent years, the role of transglutaminase type 2 (TG2) has been shown to be crucial to both the ECM expansion^1,4,5 and TGF-β1 activation^6–9 that underlies this fibrotic remodeling.TG2 belongs to the eight-member transglutaminase family that catalyzes a calcium-dependent acyl-transfer reaction (EC 2.3.2.13) between the γ-carboxamide group of peptide-bound glutamine and the ε-amino group of peptide-bound lysine,¹⁰ generating stable ε-(γ-glutamyl)-lysine isopeptide crosslinks. In fibrotic diseases (e.g., renal, liver, and pulmonary fibrosis), increased TG2 externalization and/or expression results in abundant crosslink formation, contributing to ECM accumulation.^5,7,11–16 In early CKD, ε-(γ-glutamyl)-lysine crosslinking in the ECM results predominantly from cell externalization of existing TG2 as the renal TG2 level remains constant.⁴ The externalized TG2 is known to exert a profibrotic function also through a nonenzymatic “structural” activity, by enhancing arginine-glycine-aspartic acid–independent cell adhesion and, consequently, contraction of the ECM.^17–19 In experimental CKD, pan TG inhibition preserved kidney function because of a reduction in kidney fibrosis in both diabetic and nondiabetic disease.^2,16 Mice deficient in TG2 were protected against the development of fibrosis in obstructive nephropathy resulting from impaired collagen I synthesis related to decreased TGF-β1 activation.⁸However, clinical application of anti-TG2 therapy has been hampered by the complexity to develop TG2-specific inhibitors due to a highly conserved catalytic core across the TG family,¹⁰ with inhibition of factor XIIIa and the keratinocyte transglutaminase causing particular concern.²⁰ Consequently, elucidation of the mechanism whereby TG2 is released from cells has been an object of intense scrutiny, because TG2 is unconventionally secreted via a potentially unique non-Golgi route,^21,22 which may offer a specific interventional strategy to decrease extracellular TG2.Recently, we have shown that heparan sulfate proteoglycans (HSPG), such as syndecan-4 (Sdc4), may have a key role in the cell surface trafficking of TG2 in vitro.²³ Sdc4 and TG2 coassociated in cell membranes via the HS chains of Sdc4, for which TG2 has high affinity.^18,23,24 Lack of Sdc4/HS or functional inhibition of HS led to a lower level of cell-surface TG2 antigen and crosslinking activity in vitro, causing a parallel accumulation of cytosolic TG2 with no changes in the total level of TG2 expression.²³ Membrane-proximal Sdc4/HS may, therefore, affect the unconventional secretion of TG2, as described for fibroblast growth factor-2, by acting as a cell-surface “molecular trap.”²⁵ Thus, Sdc4/HS may modulate TG2 profibrotic function by controlling its cell-surface trafficking. Sdc4/HS has also been implicated in kidney fibrosis, being upregulated in progressive proliferative kidney diseases (IgA nephropathy) and diabetic nephropathy, but not in nonproliferative diseases.^26–28To investigate the possible role of Sdc4 in regulating cell-surface trafficking of TG2 in vivo, we induced kidney fibrosis in Sdc4-knockout (KO) mice²⁹ and assessed whether or not Sdc4 deletion affected TG2 externalization/extracellular activity and tubulointerstitial fibrosis development. We used two distinct experimental models of kidney fibrosis: unilateral ureteric obstruction (UUO)³⁰ and aristolochic acid nephropathy (AAN).^31,32 Sdc4-KO ameliorated tubulointerstitial fibrosis in both models, and deletion of Sdc4 led to a lowering of extracellular TG2 in the ECM. Binding of TG2 to the tubular interstitium depended on the HS chains of proteoglycans, with which TG2 was found to be strongly associated in normal and diseased kidney. These data suggest for the first time that Sdc4 plays a critical role in the pathogenesis of kidney fibrosis by regulating TG2 trafficking and localization via HS chain-binding.     

Activation of p53 Promotes Renal Injury in Acute Aristolochic Acid Nephropathy

Ingestion of aristolochic acid (AA) can cause AA nephropathy (AAN), in which excessive death of tubular epithelial cells (TECs) characterize the acute phase. AA forms adducts with DNA, which may lead to TEC apoptosis via p53-mediated signaling. We tested this hypothesis both by studying p53-deficient mice and by blocking p53 in TECs with its inhibitor pifithrin-α. AA induced acute AAN in wild-type mice, resulting in massive apoptotic and necrotic TEC death and acute renal failure; p53 deficiency or pharmacologic inhibition attenuated this injury. In vitro, AA induced apoptotic and necrotic death of TEC in a time- and dosage-dependent manner, with apoptosis marked by a 10-fold increase in cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling–positive/Annexin V-positive propidium iodide–negative TECs (all P < 0.001). AA induced dephosphorylation of STAT3 and the subsequent activation of p53 and TEC apoptosis. In contrast, overexpression of STAT3, p53 inhibition, or p53 knockdown with small interfering RNA all attenuated AA-induced TEC apoptosis. Taken together, these results suggest that AA induces TEC death via apoptosis by dephosphorylation of STAT3 and posttranslational activation of p53, supporting the hypothesis that p53 promotes renal injury in acute AAN.Chinese herb nephropathy was first reported in Belgium in patients with prolonged intake of Chinese herbs during a slimming regimen and is recognized as one of the most severe complications caused by traditional Chinese medicine.^1–3 It is now clear that the major substance that causes Chinese herb nephropathy is the plant nephrotoxin aristolochic acid and its metabolism products.^4–6 Thus, the term aristolochic acid nephropathy (AAN), instead of Chinese herbal nephropathy, is used today.^7,8 AAN has emerged as an important cause of drug-associated renal failure worldwide.⁹Patients with AAN exhibit a rapidly progressive renal deterioration, resulting in acute renal failure that could lead to ESRD.^1–3,10,11 A similar clinical course was observed in experimental animals treated with AA.^12,13 Pathologically, chronic AAN is characterized by extensive interstitial fibrosis with atrophy and loss of renal tubules.^{1–3,10–13} The lesions of chronic AAN are mainly in the cortex involving proximal tubular epithelial cells (TECs)^10–13; glomeruli are relatively spared with minimal inflammation.^9–12 In contrast, progressive TEC death occurs early in the clinical course with an absence of renal fibrosis and inflammation in experimental models and patients with acute AAN.^10,14,15 Although apoptosis is an important pathologic feature in in vivo and in vitro studies of acute AAN,^16–18 the underlying mechanisms remain unclear.In considering the genotoxic effect of AA with the formation of AA-DNA adducts and the importance of the p53 signaling pathway in DNA damage and cell apoptosis,^19–21 we hypothesized that TEC apoptosis in acute AAN is dependent on p53 signaling. We investigated this by inducing acute AAN in p53 knockout (KO) and p53 wild-type (WT) mice and by blocking the p53 activities with a pharmacologic inhibitor. We further studied the toxicity of AA on TEC apoptosis by examining a panel of apoptotic biomarkers. The mechanism that AA induced TEC apoptosis by activating p53 via a STAT3-dependent posttranslational modification was identified.     

Prevalence of Loss-of-Function FTO Mutations in Lean and Obese Individuals

OBJECTIVE

Single nucleotide polymorphisms (SNPs) in intron 1 of fat mass– and obesity-associated gene (FTO) are strongly associated with human adiposity, whereas Fto^−/− mice are lean and Fto^+/− mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO.

RESEARCH DESIGN AND METHODS

We sequenced all coding exons of FTO in 1,433 severely obese and 1,433 lean individuals. We studied the enzymatic activity of selected nonsynonymous variants.

RESULTS

We identified 33 heterozygous nonsynonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues: R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals.

CONCLUSIONS

Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. Although intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.Genome-wide association studies have revealed that single nucleotide polymorphisms (SNPs) within the first intron of fat mass– and obesity-associated gene (FTO) are strongly associated with adiposity (1–4). These associations have been largely consistent across multiple different ethnicities including Europeans (5–8), Asians (9–11), Hispanics (12,13), and Africans (13,14). At present, it remains unclear whether these SNPs influence the expression or splicing of the FTO gene and/or whether the unequivocal association with obesity is causally related to alterations in expression or function of FTO. If the effect of the intron 1 SNPs is through FTO, it also remains unclear whether the obesity risk SNPs result in a loss or a gain of FTO function. It remains possible that the SNPs influence the expression and/or function of neighboring genes and that such an effect might underlie the association with adiposity. There are several examples in metabolic disease where common variants close to a particular gene are associated with alterations in risk of common phenotypes such as fat mass or risk of obesity, whereas rare loss- or gain-of-function mutations in the same gene are associated with a more severe version of the same metabolic phenotype (e.g., MCR4 [5,15–19], POMC [20,21], BDNF [22–24], and PCSK1 [25,26]). When information is available from both types of variants, clarity of mechanistic understanding is greatly enhanced. To establish whether nonsynonymous variants of FTO might be enriched in either lean or obese subjects, we sequenced the FTO coding region and intron-exon boundaries in a large group of subjects with severe obesity and a similarly sized group of individuals with lifelong leanness. We have recently established that FTO encodes a 2-oxoglutarate (2-OG) Fe²⁺-dependent dioxygenase (27). In this study, we have examined the functional enzymatic properties of naturally occurring variants to gain further insights into structure-function relationships in the human enzyme.     

Lack of Collagen XVIII/Endostatin Exacerbates Immune-Mediated Glomerulonephritis

Collagen XVIII is a component of the highly specialized extracellular matrix associated with basement membranes of epithelia and endothelia. In the normal kidney, collagen XVIII is distributed throughout glomerular and tubular basement membranes, mesangial matrix, and Bowman''s capsule. Proteolytic cleavage within its C-terminal domain releases the fragment endostatin, which has antiangiogenic properties. Because damage to the glomerular basement membrane (GBM) accompanies immune-mediated renal injury, we investigated the role of collagen XVIII/endostatin in this disorder. We induced anti-GBM glomerulonephritis in collagen XVIII α1-null and wild-type mice and compared the resulting matrix accumulation, inflammation, and capillary rarefaction. Anti-GBM disease upregulated collagen XVIII/endostatin expression within the GBM and Bowman''s capsule of wild-type mice. Collagen XVIII/endostatin-deficient mice developed more severe glomerular and tubulointerstitial injury than wild-type mice. Collagen XVIII/endostatin deficiency altered matrix remodeling, enhanced the inflammatory response, and promoted capillary rarefaction and vascular endothelial cell damage, but did not affect endothelial proliferation. Supplementing collagen XVIII-deficient mice with exogenous endostatin did not affect the progression of anti-GBM disease. Taken together, these results suggest that collagen XVIII/endostatin preserves the integrity of the extracellular matrix and capillaries in the kidney, protecting against progressive glomerulonephritis.The major constituents of all basement membranes (BMs) are predominantly laminins, nidogen/entactin, collagen IV, and heparan sulfate proteoglycans (HSPGs).^1,2 HSPGs are a class of biomolecules that consist of a core protein with covalently attached heparan sulfate sugar side chains. HSPGs are involved in biologic processes such as glomerular filtration, cell adhesion, migration, proliferation, and differentiation,^3–5 which are mediated by the binding of chemokines, cytokines, enzymes, growth factors, or other bioactive molecules.⁶ Collagen XVIII (Col 18) is a HSPG associated with BMs of almost all epithelia and endothelia. This collagen contains an N-terminal noncollagenous domain (NC-11), 10 collagenous domains alternating with 9 noncollagenous repeats, and a C-terminal noncollagenous domain (NC-1).⁷ In the normal kidney, Col 18 is distributed throughout glomerular and tubular BMs, mesangial matrix, and Bowman''s capsule in both humans and mice.^8,9Inactivating mutations in the human gene for Col 18, COL18A1, have been identified in patients with Knobloch syndrome, which is an autosomal recessive disorder characterized by the occurrence of vitreoretinal degeneration with retinal detachment, high myopia, macular degeneration, occipital encephalocele, and minor renal abnormalities.^10,11 The kidney of Col 18/endostatin-null mice exhibits no abnormalities on light microscopy, where expansion of the mesangial matrix and thickened proximal tubular BM are observed on electron microscopy.^7,8 The study of Utriainen et al.⁸ suggested that Col 18/endostatin may have a role in maintaining the structural integrity of the extracellular matrix in the normal kidney, whereas its role in susceptibility and progression of inflammatory glomerular diseases remains to be clarified.Proteolytic cleavage within NC-1 of Col 18 releases a fragment termed endostatin, which has been shown to have anti-angiogenesis activity in vitro and in vivo.^1,12–16 Endostatin is an endogenous angiogenesis inhibitor and is detected in the circulation at a physiologic level of 20 to 50 ng/ml in serum.^1,7,17–19 Col 18/endostatin-null mice display enhanced tumor growth when implanted with tumor cells that are unable to produce Col 18.¹⁸ In contrast, overexpression of circulating endostatin in the transgenic mice leads to reduced tumor growth and vascularization.¹⁸ Nonetheless, it is still unclear whether endostatin plays a role in renal disease as an endogenous angiogenesis inhibitor.As a major component of the ultrafiltration apparatus in the kidney, the glomerular basement membrane (GBM) is constantly exposed to serum flow and pressure and thus needs to be functionally sound and to stringently maintain its structural integrity. Mouse anti-GBM glomerulonephritis (GN) is characterized by damage of GBM followed by invasion of inflammatory cells, accumulation of mesangial matrix, and destruction of the glomerular capillary network, finally resulting in the development of glomerular sclerosis.^20–22In this study, we induced anti-GBM disease in Col 18/endostatin-null mice to test the hypothesis that Col 18/endostatin is critical for maintaining the integrity of the GBM and capillaries in the glomerulus. We demonstrate that Col 18/endostatin-deficient mice with anti-GBM disease have altered distribution of extracellular matrix, accelerated inflammatory responses, and more severe endothelial cell (EC) damage compared with wild-type (WT) mice with this disease. These results suggest that Col 18/endostatin may play an important role in preserving the integrity of extracellular matrixes and capillary vessels in the kidneys of patients with immune complex glomerulonephritis.     

A Maladaptive Role for EP4 Receptors in Podocytes

Inhibition of p38 mitogen-activated protein kinase and cyclooxygenase-2 reduces albuminuria in models of chronic kidney disease marked by podocyte injury. Previously, we identified a feedback loop in podocytes whereby an in vitro surrogate for glomerular capillary pressure (i.e., mechanical stretch) along with prostaglandin E₂ stimulation of its EP4 receptor induced cyclooxygenase-2 in a p38-dependent manner. Here we asked whether stimulation of EP4 receptors would exacerbate glomerulopathies associated with enhanced glomerular capillary pressure. We generated mice with either podocyte-specific overexpression or depletion of the EP4 receptor (EP4^pod+ and EP4^pod−/−, respectively). Glomerular prostaglandin E₂-stimulated cAMP levels were eightfold greater for EP4^pod+ mice compared with nontransgenic (non-TG) mice. In contrast, EP4 mRNA levels were >50% lower, and prostaglandin E₂-induced cAMP synthesis was absent in podocytes isolated from EP4^pod−/− mice. Non-TG and EP4^pod+ mice underwent 5/6 nephrectomy and exhibited similar increases in systolic BP (+25 mmHg) by 4 weeks compared with sham-operated controls. Two weeks after nephrectomy, the albumin-creatinine ratio of EP4^pod+ mice (3438 μg/mg) was significantly higher than that of non-TG mice (773 μg/mg; P < 0.0001). Consistent with more severe renal injury, the survival rate for nephrectomized EP4^pod+ mice was significantly lower than that for non-TG mice (14 versus 67%). In contrast, 6 weeks after nephrectomy, the albumin-creatinine ratio of EP4^pod−/− mice (753 μg/mg) was significantly lower than that of non-TG mice (2516 μg/mg; P < 0.05). These findings suggest that prostaglandin E₂, acting via EP4 receptors contributes to podocyte injury and compromises the glomerular filtration barrier.Glomerular filtration barrier (GFB) damage, as indicated by micro/macroalbuminuria, is a renal disease marker.^1–3 Interventions that reduce albuminuria slow the progression to ESRD and lower the risk for hypertension and cardiovascular events.⁴ Although the underlying mechanisms of albuminuria are complex and remain incompletely resolved, a key player in the cause of GFB damage is the podocyte.⁵ With the increased incidence of kidney disease and a limited number of effective treatments, understanding the role of the podocyte in the progression of glomerular injury is needed to develop novel antiproteinuric therapies.Damage to the podocyte foot processes is a hallmark of many proteinuric glomerular diseases, including minimal change disease, FSGS, and diabetic kidney disease.^1,6–8 A number of maladaptive end points have been identified for the podocyte in glomerular diseases (e.g., foot process effacement, detachment from the glomerular basement membrane, apoptosis, hypertrophy, dedifferentiation).^9,10 Although many factors undoubtedly contribute to the initiation and progression of podocyte injury and dysfunction, nonsteroidal anti-inflammatory drugs reduce proteinuria, suggesting that prostanoids derived from cyclooxygenase 1 and 2 (COX-1 and COX-2) activity may compose a portion of the causative mosaic.^11,12 Unfortunately, widespread use of COX inhibitors is not feasible, because they diminish vasodilatory prostanoid levels, resulting in reduced GFR and renal blood flow (RBF)^13,14; however, inhibition of either COX-1 or COX-2 isoforms attenuates the synthesis of at least five distinct prostanoids (PGE₂, PGD2, PGI2, PGF2α, and TxA2) that interact with their respective G-protein–coupled receptors (prostanoid E-type 1 through type 4 [EP-1 through -4], and prostanoid D-, I-, F- and T-type [TP]) to provoke a variety of physiologic actions in the kidney and elsewhere. Targeting those prostanoid receptors that are pro-proteinuric while avoiding those that regulate GFR/RBF may represent a potential therapy for preserving GFB function in renal disease.Studies have shown that podocyte-specific overexpression of COX-2 in mice renders them more susceptible to glomerular injury in models of minimal change disease.^15,16 Furthermore, subtotally nephrectomized (5/6 Nx) rats treated with a selective COX-2 inhibitor reduced glomerular PGE₂ levels that correlate with improvements in proteinuria and glomerulosclerosis.¹⁷ Glomerular PGE₂ synthesis after 5/6 Nx suggests that signaling via one or more of its EP receptor subtypes may contribute to the deleterious effects of COX-2 activity on GFB function.¹⁸ Podocytes may be targets of these actions, because they express both EP1 and EP4 receptors.¹⁹ We recently uncovered a novel feedback loop in cultured mouse podocytes whereby an in vitro surrogate for glomerular capillary pressure (Pgc; i.e., mechanical stretch) along with PGE₂ stimulation of the EP4 receptor induces COX-2 in a p38 mitogen-activated protein kinase–dependent manner.^20,21 These findings led us to hypothesize that PGE₂-dependent EP4 receptor signaling contributes to podocyte injury in chronic kidney disease (CKD) associated with enhanced Pgc. Here we demonstrate that podocyte-specific overexpression of a desensitization-resistant C-terminal truncated EP4 receptor renders mice more susceptible to the development of albuminuria after 5/6 Nx, whereas conditional deletion of this prostanoid receptor subtype from podocytes confers partial protection from such GFB damage. Our findings therefore support a maladaptive role for the PGE₂ EP4 receptor in the context of glomerular injury.