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.     

The NLRP3 Inflammasome Promotes Renal Inflammation and Contributes to CKD

Inflammation significantly contributes to the progression of chronic kidney disease (CKD). Inflammasome-dependent cytokines, such as IL-1β and IL-18, play a role in CKD, but their regulation during renal injury is unknown. Here, we analyzed the processing of caspase-1, IL-1β, and IL-18 after unilateral ureteral obstruction (UUO) in mice, which suggested activation of the Nlrp3 inflammasome during renal injury. Compared with wild-type mice, Nlrp3^−/− mice had less tubular injury, inflammation, and fibrosis after UUO, associated with a reduction in caspase-1 activation and maturation of IL-1β and IL-18; these data confirm that the Nlrp3 inflammasome upregulates these cytokines in the kidney during injury. Bone marrow chimeras revealed that Nlrp3 mediates the injurious/inflammatory processes in both hematopoietic and nonhematopoietic cellular compartments. In tissue from human renal biopsies, a wide variety of nondiabetic kidney diseases exhibited increased expression of NLRP3 mRNA, which correlated with renal function. Taken together, these results strongly support a role for NLRP3 in renal injury and identify the inflammasome as a possible therapeutic target in the treatment of patients with progressive CKD.Chronic kidney disease (CKD) is a significant cause of morbidity and mortality in the general population.^1,2 In nondiabetic CKD, the progression from mild/moderate kidney disease to ESRD is a complex process that involves many factors, including tubulointerstitial inflammation and fibrosis. The involvement of mononuclear inflammatory cells in the damaged renal interstitium is a universal finding in failing kidneys and correlates inversely with renal function.^3–9 The molecular mechanisms that regulate inflammation in CKD, however, remain unclear.An inflammatory response is induced during cellular injury such as necrosis.¹⁰ Cellular contents that are inappropriately released after loss of plasma membrane, integrity are endogenous adjuvants or danger-associated molecular patterns (DAMPs).^11–13 These DAMPs alert the innate immune system to cellular injury and produce a proinflammatory response to aid the repair of damaged tissues. Although beneficial in the case of pathogens, the reaction to endogenous (nonmicrobial) injury can contribute to tissue damage and disease progression.The NOD-like receptors (NLRs) compose a group of pattern recognition receptors involved in a wide variety of host innate immune responses to microbial and nonmicrobial stimuli.¹⁴ The best understood members include NOD2 (NLRC2, implicated in Crohn''s disease)¹⁵ and NLRP3 (also known as NALP3 or cryopyrin). Upon activation, the NLRP3 proteins oligomerize and recruit via homotypic molecular interactions, the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and the protease caspase-1 to form a protein complex termed “the inflammasome.”¹⁶ The formation of the inflammasome induces caspase-1 autoprocessing and activation that results in the processing of cellular substrates including the cytokines pro-IL-1β and pro-IL-18.^17,18 In the case of IL-1β, caspase-1 cleaves the 35-kD pro-IL-1β to generate the mature and secreted 17-kD cytokine.Recent reports have implicated the NLRP3 inflammasome in the recognition of endogenous danger signals released from damaged and dying cells. DAMPs capable of activating the NLRP3 inflammasome include reactive oxygen species, extracellular ATP, monosodium urate crystals, nucleic acids, and extracellular matrix components including hyaluronan and biglycan.^19–24 Consistent with these observations, the NLRP3 inflammasome has been implicated in the pathogenesis of various nonmicrobial diseases, including diabetes, gout, silicosis, and acetaminophen liver toxicity.^19,20,25,26 The coexistence of cellular injury and inflammation suggests that the NLRP3 inflammasome may also play a role in regulating inflammation in CKD. Furthermore, the NLRP3 agonist biglycan and cytokines such as IL-1β, IL-18, and the IL-1 receptor all contribute to renal inflammation and fibrosis.^24,27–30 In this study, we demonstrated that the Nlrp3 inflammasome regulates renal inflammation and fibrosis during unilateral ureteral obstruction (UUO) in mice. In addition, studies of humans demonstrated increased NLRP3 in a variety of nondiabetic kidney diseases and CKD. These data provide valuable insight into the processes driving renal inflammation and CKD progression and identify NLRP3 as a novel target for therapeutic intervention.     

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK–ERK pathway.Urea plays a crucial role in the urinary concentrating mechanism, and therefore, in the regulation of water balance. Urea''s importance to the generation of a concentrated urine has been appreciated since at least 1934.^1,2 Several studies have shown that maximal urine concentrating ability is decreased in protein-deprived mammals and is restored by urea infusion.³ More recently, a UT-A1/UT-A3 knock-out mouse,^4,5 a UT-A2 knock-out mouse,⁶ and a UT-B knock-out mouse^7–9 were each shown to have urine concentrating defects. Thus, any hypothesis regarding the mechanism by which the kidney concentrates urine needs to include some effect derived from urea.The UT-A1 urea transporter is expressed in the terminal inner medullary collecting duct (IMCD).¹⁰ Vasopressin stimulates urea transport across perfused rat terminal IMCDs by increasing UT-A1 phosphorylation and apical plasma membrane accumulation.^11–15 Vasopressin acts by binding to V₂ receptors in the basolateral plasma membrane, stimulating adenylyl cyclase, increasing cAMP production, and increasing urea transport.^11,16–18 Forskolin, which directly activates adenylyl cyclase,¹⁹ also increases urea transport in perfused rat terminal IMCDs.²⁰cAMP is traditionally thought to act through protein kinase A (PKA). However, when we stimulate the PKA activity by increasing cAMP with forskolin in MDCK cells that are stably transfected with UT-A1 (UT-A1-MDCK cells), only 50% of the forskolin-stimulated urea flux is inhibited by H-89, a PKA inhibitor.²¹ Vasopressin and forskolin work in a similar manner to increase the cAMP levels, so this partial inhibition by H-89 suggests that vasopressin may signal through two cAMP-dependent pathways: one involving PKA and one that is independent of PKA. Because the UT-A1-MDCK cells reproduce many of the properties of native rat IMCDs,^13,21,22 these findings raise the possibility that vasopressin may signal through a second cAMP-dependent, but non–PKA-dependent, pathway in rat IMCDs.In addition to PKA, cAMP can activate Epac (exchange protein activated by cAMP), which signals by activating Rap1, a Ras-related small molecular weight G protein, which in turn signals through mitogen-activated protein kinase kinase (MEK) and extracellular signal-related kinase (ERK)^23,24 (Figure 1). There are two closely related exchange proteins activated by cAMP (Epac) proteins, Epac1 and Epac2, and both have been detected in rat IMCDs, although one or the other predominates in different studies.^25–28 The purpose of this study was to determine whether activation of the Epac pathway resulted in a functional change in urea transport in perfused rat terminal IMCDs.Open in a separate windowFigure 1.Vasopressin signaling diagram.     

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.     

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.     

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.     

The RIP1-Kinase Inhibitor Necrostatin-1 Prevents Osmotic Nephrosis and Contrast-Induced AKI in Mice

The pathophysiology of contrast-induced AKI (CIAKI) is incompletely understood due to the lack of an appropriate in vivo model that demonstrates reduced kidney function before administration of radiocontrast media (RCM). Here, we examine the effects of CIAKI in vitro and introduce a murine ischemia/reperfusion injury (IRI)–based approach that allows induction of CIAKI by a single intravenous application of standard RCM after injury for in vivo studies. Whereas murine renal tubular cells and freshly isolated renal tubules rapidly absorbed RCM, plasma membrane integrity and cell viability remained preserved in vitro and ex vivo, indicating that RCM do not induce apoptosis or regulated necrosis of renal tubular cells. In vivo, the IRI-based CIAKI model exhibited typical features of clinical CIAKI, including RCM-induced osmotic nephrosis and increased serum levels of urea and creatinine that were not altered by inhibition of apoptosis. Direct evaluation of renal morphology by intravital microscopy revealed dilation of renal tubules and peritubular capillaries within 20 minutes of RCM application in uninjured mice and similar, but less dramatic, responses after IRI pretreatment. Necrostatin-1 (Nec-1), a specific inhibitor of the receptor-interacting protein 1 (RIP1) kinase domain, prevented osmotic nephrosis and CIAKI, whereas an inactive Nec-1 derivate (Nec-1i) or the pan-caspase inhibitor zVAD did not. In addition, Nec-1 prevented RCM-induced dilation of peritubular capillaries, suggesting a novel role unrelated to cell death for the RIP1 kinase domain in the regulation of microvascular hemodynamics and pathophysiology of CIAKI.Contrast-induced AKI (CIAKI) is the consensus name for what was formally called contrast-induced nephropathy or radiocontrast-induced AKI.^1–3 CIAKI is a common and potentially serious complication⁴ after the administration of contrast media,^5–7 especially in patients who are at risk for AKI, and is the most common cause of iatrogenic, inpatient, drug-induced AKI,^3,8,9 with outstanding implications for patients with diabetes.¹ CIAKI was recognized as the third commonest cause of hospital-acquired renal failure accounting for 11% of the cases¹⁰ even before magnetic resonance imaging contrast media were found to be associated with nephrogenic systemic fibrosis. Preclinical research thus far has failed to unravel the underlying pathophysiology of CIAKI.Programmed cell death (PCD) was used synonymously with apoptosis until regulated necrosis (RN) was discovered.¹¹ Apoptosis has been proposed to contribute to CIAKI^12–14 and asialoerythropoietin was recently demonstrated in this context to prevent CIAKI.¹⁵ Apoptosis is a process that is characterized by the activity of caspases that cleave hundreds of intracellular proteins to ultimately cause membrane blebbing, nuclear fragmentation, and regulated cellular shrinkage as a consequence of their proteolytic activity.^16,17 Within this process, caspases are capable of cleaving NFs like poly(ADP-ribose)-polymerase (PARP)-family proteins.¹⁸ PARP-1 has also been demonstrated to elicit a necrotic phenotype in kidney cells and therefore exhibits a subroutine of the RN.^19,20 It was suggested that tubular cell death by caspase-3–mediated apoptosis substantially contributes to the overall pathogenesis of CIAKI,^14,15 and one report investigated the activation of the cell death molecules PARP, Bad, and BIM.¹⁴ On the basis of these findings, the currently proposed model ascribes apoptosis a major pathophysiologic function in CIAKI.^12,13Apart from PARP-mediated RN, necroptosis, another RN pathway, is mediated by activation of the “necrosome” consisting of receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3).^11,21–23 Necroptosis involves all necrotic cellular hallmarks such as early loss of membrane integrity as well as rupture of the plasma membrane after cellular swelling. We recently described the functional relevance of both apoptosis and necroptosis in AKI.^24,25Here, we demonstrate that necrostatin-1 (Nec-1), a highly specific inhibitor of the RIP1 kinase domain, prevents CIAKI in a new and easy-to-use preclinical model for the in vivo analysis of CIAKI. Our model reliably mimics “osmotic nephrosis,” a pathologic feature that is typical of CIAKI in humans. In vitro and in vivo, we found that apoptosis is of minor pathophysiologic importance. Mechanistically, the data implicate RIP1 in the functional renal failure in vivo and provide evidence for the prevention of CIAKI by the RIP1 kinase inhibitor Nec-1 that also prevented the functional changes in the peritubular vasculature after RCM injection as demonstrated by intravital microscopy. Because of the outstanding specificity of Nec-1 that has been subject to extensive investigation,^26–29 we consider it justified to conclude that a novel non-cell death role of RIP1 might account for the functional kidney failure in CIAKI. In addition, we introduce Nec-1 as a potential inhibitor of CIAKI.     

Tubular von Hippel-Lindau Knockout Protects against Rhabdomyolysis-Induced AKI

Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA–based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.No specific therapy is currently available for human AKI, a clinical entity of increasing incidence and high morbidity and mortality.^1–4 Rhabdomyolysis, one of the leading causes of AKI, develops after trauma, drug toxicity, infections, burns, and physical exertion.^5–8 The animal model using an intramuscular glycerol injection with consequent myoglobinuria is closely related to the human syndrome of rhabdomyolysis.⁹ Experimental data demonstrate renal vasoconstriction,^9–15 tubular hypoxia,^15,16 normal or even reduced intratubular pressure,^9–11 as well as large variation in single nephron GFR.^10,11 Intratubular myoglobin casts, a histologic hallmark, seem not to cause tubular obstruction,^9–11 but rather scavenge nitric oxide^17,18 and generate reactive oxygen species¹⁹ followed by vasoconstriction.The traditional discrimination between ischemic and toxic forms of AKI has been challenged because an increasing amount of evidence suggests that renal hypoxia is a common denominator in AKI of different etiologies.²⁰ Pimonidazole adducts, which accumulate in tissues at oxygen tensions <10 mmHg,²¹ have been demonstrated in various AKI forms.^16,22–24 During AKI, hypoxia-inducible factors (HIFs), which are mainly regulated by oxygen-dependent proteolysis, were found to be upregulated in different renal tubular segments.^{16,20,22,24,25} HIFs are heterodimers of a constitutive β subunit, HIF-β (ARNT), and one of three oxygen-dependent α-subunits, HIF-1α, HIF-2α, and HIF-3α. The α-β dimers bind to hypoxia-response elements (HREs) in the promoter-enhancer region of HIF target genes.^26–28 Although the 5′-RCGTG-3′ (R = A or G) core HRE appears >1 million times in the entire genome²⁹ and in >4000 promoter regions of validated genes,³⁰ a recent study demonstrated HIF binding in roughly 350 genes.³¹ Multiple HIF-based biologic effects are known, and it is widely accepted that a broad panel of these promote cellular survival in a hostile and oxygen-deprived environment.^27–29 In all types of AKI tested thus far, HIF activation along the nephron correlates with tubular survival, and the cells most vulnerable to injury exhibit no or only very limited HIF activity.²⁰ This observation led to the concept of insufficient HIF-based hypoxic adaptation in AKI. Consequently, maneuvers of preconditional HIF activation are utilized to ameliorate AKI. Indeed, many of these attempts are successful but the majority are conducted in ischemia-reperfusion injury.²⁰ It is largely unclear whether HIF can rescue kidneys exposed to AKI forms other than ischemia-reperfusion injury, and it is unclear which HIF target genes are involved in AKI protection if so. In many tumors, constitutive HIF activation promotes anaerobic ATP production, a process known as the Warburg effect.³²von Hippel-Lindau protein (VHL) is a ubiquitin ligase engaged in the stepwise HIF-α degradation process, which constantly occurs during normoxia.³³ Inducible Pax8-rtTA–based knockout of VHL (VHL-KO) achieves strong, selective, and persistent upregulation of HIF in all nephron segments.³⁴ In this study, we use this transgenic technique in conjunction with rhabdomyolysis in mice to address two issues: (1) Does HIF activation through VHL-KO protect from rhabdomyolysis-induced AKI? (2) If so, what are the biologic mechanisms and HIF target genes that are responsible for renal protection against acute injury? We demonstrate that indeed VHL-KO mice are largely protected against rhabdomyolysis-induced AKI, and provide evidence for a metabolic shift toward anaerobic ATP generation as the central protective mechanism.     

Sphingosine-1-Phosphate Evokes Unique Segment-Specific Vasoconstriction of the Renal Microvasculature

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, has been implicated in regulating vascular tone and participating in chronic and acute kidney injury. However, little is known about the role of S1P in the renal microcirculation. Here, we directly assessed the vasoresponsiveness of preglomerular and postglomerular microvascular segments to exogenous S1P using the in vitro blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (0.001–10 μM) evoked concentration-dependent vasoconstriction in preglomerular microvessels, predominantly afferent arterioles. After administration of 10 μM S1P, the diameter of afferent arterioles decreased to 35%±5% of the control diameter, whereas the diameters of interlobular and arcuate arteries declined to 50%±12% and 68%±6% of the control diameter, respectively. Notably, efferent arterioles did not respond to S1P. The S1P receptor agonists FTY720 and FTY720-phosphate and the specific S1P1 receptor agonist SEW2871 each evoked modest afferent arteriolar vasoconstriction. Conversely, S1P2 receptor inhibition with JTE-013 significantly attenuated S1P-mediated afferent arteriolar vasoconstriction. Moreover, blockade of L-type voltage-dependent calcium channels with diltiazem or nifedipine attenuated S1P-mediated vasoconstriction. Intravenous injection of S1P in anesthetized rats reduced renal blood flow dose dependently. Western blotting and immunofluorescence revealed S1P1 and S1P2 receptor expression in isolated preglomerular microvessels and microvascular smooth muscle cells. These data demonstrate that S1P evokes segmentally distinct preglomerular vasoconstriction via activation of S1P1 and/or S1P2 receptors, partially via L-type voltage-dependent calcium channels. Accordingly, S1P may have a novel function in regulating afferent arteriolar resistance under physiologic conditions.Sphingosine 1-phosphate (S1P) is recognized as an important signaling molecule in diverse biologic processes.^1,2 Growing evidence indicates that S1P plays an important role in regulating vascular reactivity.^3–5 S1P is a bioactive sphingolipid metabolite and is released from erythrocytes, platelets, and endothelial cells.^6,7 The majority of S1P effects are mediated via five distinct receptors (S1P1–S1P5 receptors), which represent a family of small G protein–coupled receptors (GPCRs)⁵; however, S1P can also exist in the cytoplasm as a second messenger involved in Ca²⁺ mobilization or cell survival and proliferation.^8,9 S1P1– S1P3 receptors are expressed by a wide variety of tissues, whereas S1P4 and S1P5 receptors are mainly expressed in cells of the immune and nervous systems.^4,10 In the vasculature, endothelial cells mainly express S1P1 and S1P3 with variable expression of S1P2, whereas vascular smooth muscle cells express S1P2 and S1P3 with variable expression of S1P1.^3–5 Studies in animals show that application of exogenous S1P causes either vasoconstriction or vasodilation of isolated arteries from several vascular beds.^3,4Although S1P receptor expression is detected in kidneys,^11–13 little is known about the effects of S1P on renal microvascular function. Early studies showed that S1P evoked vasoconstriction in isolated intrarenal arteries.¹⁴ Intravenous infusion of S1P to rats in vivo decreased renal blood flow (RBF) without changing mean arterial pressure.¹⁵ Genetic studies found a significantly higher RBF in anesthetized S1P2 gene knockout mice compared with wild-type mice.¹⁶ These studies suggest that S1P may be important in regulating renal vascular function. More recent studies show that S1P signaling pathways are upregulated under several pathologic conditions including renal ischemia-reperfusion injury, diabetic nephropathy, and hypertensive renal injury.¹⁷ For example, administration of the S1P receptor agonist FTY720 significantly attenuated renal injury in 5/6 nephrectomy hypertensive rats by reducing lymphocyte infiltration in kidneys.¹⁸ Both mRNA and protein levels of S1P2 receptors are increased in diabetic rat kidney tissue and S1P-induced vasoconstriction is significantly enhanced in isolated-perfused diabetic rat kidneys.¹³ In addition, renal ischemia-reperfusion markedly increases mRNA expression of S1P1, S1P2, and S1P3 receptors in the renal cortex.^19,20 Activation of S1P1 receptors^19,21 or selective blockade of S1P2 receptors²⁰ protects against renal ischemia-reperfusion injury. Overall, these studies indicate that alteration of S1P receptor signaling may contribute to renal injury under pathologic conditions. Therefore, it is important to determine the role of S1P in renal microvascular function. In this study, we focused on elucidating the influence of exogenous S1P on renal microvascular caliber and determining the distribution of S1P receptors in renal microvessels.     

Renalase Protects against Ischemic AKI

Elevated levels of plasma catecholamines accompany ischemic AKI, possibly contributing the inflammatory response. Renalase, an amine oxidase secreted by the proximal tubule, degrades circulating catecholamines and reduces myocardial necrosis, suggesting that it may protect against renal ischemia reperfusion injury. Here, mice subjected to renal ischemia reperfusion injury had significantly lower levels of renalase in the plasma and kidney compared with sham-operated mice. Consistent with this, plasma NE levels increased significantly after renal ischemia reperfusion injury. Furthermore, renal tubular inflammation, necrosis, and apoptosis were more severe and plasma catecholamine levels were higher in renalase-deficient mice subjected to renal ischemia reperfusion compared with wild-type mice. Administration of recombinant human renalase reduced plasma catecholamine levels and ameliorated ischemic AKI in wild-type mice. Taken together, these data suggest that renalase protects against ischemic AKI by reducing renal tubular necrosis, apoptosis, and inflammation, and that plasma renalase might be a biomarker for AKI. Recombinant renalase therapy may have potential for the prevention and treatment of AKI.Ischemic AKI is a major problem for patients subjected to major surgical procedures involving the kidney, liver, heart, or aorta.¹ Renal ischemia reperfusion (IR) injury is a frequent cause of clinical AKI, with the incidence of AKI exceeding 50% after major cardiac, hepatobiliary, or aortic surgery.^2,3 Furthermore, ischemic AKI is frequently complicated by multi-organ dysfunction, systemic inflammation, sepsis, and death.⁴ Unfortunately, there are no proven therapies to prevent or treat AKI in the perioperative setting.⁵Renalase is a 38-kD, flavin adenine dinucleotide–dependent amine oxidase synthesized and secreted by the renal proximal tubules.⁶ Renalase degrades circulating catecholamines and regulates systemic BP in rodents and humans.⁷ Plasma catecholamines and systemic BP are elevated in patients with chronic kidney dysfunction or end stage renal insufficiency.⁸ Recent studies suggest that renalase deficiency in patients with chronic renal insufficiency leads to increased plasma catecholamine levels and systemic BP.^7,9–11 However, the effect of ischemic AKI on kidney renalase and plasma catecholamine levels remains to be determined.In addition to regulating BP, renalase may protect against inflammatory tissue injury by metabolizing catecholamines. Catecholamines via activation of leukocyte α-adrenergic receptors directly cause inflammation in sepsis and multi-organ dysfunction.^12,13 Indeed, patients with chronic renal insufficiency show increased markers of inflammation that contribute directly to increased morbidity and mortality.¹⁴ In mice, renalase deficiency resulted in exacerbated cardiac IR injury and exogenous renalase administration reduced myocardial necrosis.¹⁵In this study, we hypothesized that ischemic AKI in mice leads to renalase deficiency and this renalase deficiency directly exacerbates ischemic AKI. We performed experiments to test the following: (1) whether ischemic AKI leads to reduced kidney and plasma renalase levels, (2) whether ischemic AKI-induced renalase deficiency leads to elevated plasma catecholamine (NE) levels, (3) whether renalase-deficient mice exhibit increased renal IR injury, and (4) whether exogenous administration of recombinant human renalase directly protects against ischemic AKI in mice.     

Macrophage Phenotype Controls Long-Term AKI Outcomes—Kidney Regeneration versus Atrophy

The mechanisms that determine full recovery versus subsequent progressive CKD after AKI are largely unknown. Because macrophages regulate inflammation as well as epithelial recovery, we investigated whether macrophage activation influences AKI outcomes. IL-1 receptor–associated kinase-M (IRAK-M) is a macrophage-specific inhibitor of Toll-like receptor (TLR) and IL-1 receptor signaling that prevents polarization toward a proinflammatory phenotype. In postischemic kidneys of wild-type mice, IRAK-M expression increased for 3 weeks after AKI and declined thereafter. However, genetic depletion of IRAK-M did not affect immunopathology and renal dysfunction during early postischemic AKI. Regarding long-term outcomes, wild-type kidneys regenerated completely within 5 weeks after AKI. In contrast, IRAK-M^−/− kidneys progressively lost up to two-thirds of their original mass due to tubule loss, leaving atubular glomeruli and interstitial scarring. Moreover, M1 macrophages accumulated in the renal interstitial compartment, coincident with increased expression of proinflammatory cytokines and chemokines. Injection of bacterial CpG DNA induced the same effects in wild-type mice, and TNF-α blockade with etanercept partially prevented renal atrophy in IRAK-M^−/− mice. These results suggest that IRAK-M induction during the healing phase of AKI supports the resolution of M1 macrophage– and TNF-α–dependent renal inflammation, allowing structural regeneration and functional recovery of the injured kidney. Conversely, IRAK-M loss-of-function mutations or transient exposure to bacterial DNA may drive persistent inflammatory mononuclear phagocyte infiltrates, which impair kidney regeneration and promote CKD. Overall, these results support a novel role for IRAK-M in the regulation of wound healing and tissue regeneration.As first described in 1967¹, AKI is now considered a predictor of subsequent CKD; however, the pathophysiologic mechanisms underlying this association remain largely unknown.^2–5 Ideally, 100% of nephrons regain their structural integrity and the functional capacity they had before AKI. However, assessing structural recovery is hardly feasible in clinical practice and assessing functional recovery by serum creatinine levels or GFR estimations is impossible, because any loss up to 40%–50% can be missed due to the lack of parameter sensitivity.^6–8 Thus, the clinical observation that AKI is often followed by CKD could simply result from incomplete AKI recovery, implying that AKI episodes involve some irreversible loss of nephrons due to insufficient repair.What mechanisms regulate renal repair upon AKI? Whereas the limited capacity for podocyte regeneration (in adults) often limits glomerular repair, AKI usually involves tubular injury, which has a higher regenerative capacity.^9,10 There is accumulating experimental evidence that the associated immune response is an important determinant of AKI outcomes.¹¹ During the injury phase, necrotic tubules release molecules (e.g., histones or high-mobility group protein B1) that activate Toll-like receptors (TLRs) and inflammasomes on interstitial mononuclear phagocytes to trigger the secretion of proinflammatory cytokines and chemokines.^12–17 Chemokines guide neutrophils and proinflammatory (M1 or classically activated) macrophages to enter the site of injury,¹⁸ which largely account for the extent of tubular necrosis and thereby determine the extent of AKI.¹⁹ The rapid apoptosis of the neutrophils changes the local microenvironment and phagocytic uptake of apoptotic neutrophils induces a functional switch of the macrophages toward an anti-inflammatory (M2 or alternatively activated) phenotype.^19,20 This macrophage phenotype not only supports the resolution of postischemic renal inflammation but also actively promotes healing (i.e., tubular repair).^21–23 In fact, regenerative tubular epithelial cell proliferation starts as early as 3 hours after tubular injury; however, the resolution of renal inflammation seems to be mandatory to shift the balance of tubular repair and ongoing injury toward structural and functional tubular recovery,^24–26 similar to wound healing in general.²⁷ Therefore, factors that regulate macrophage phenotypes might determine AKI recovery and long-term outcomes.^21,28The IL-1 receptor–associated kinases (IRAKs) are important regulators of macrophage phenotype polarization because they are involved in the IL-1R/TLR/Myd88–dependent activation of NF-κB.²⁹ IRAK-4–mediated TNF receptor–associated factor 6 phosphorylation is an essential step of this signaling pathway,³⁰ which is inhibited selectively in monocytes and macrophages by IRAK-M.³¹ This way, the delayed induction of IRAK-M deactivates classically activated macrophages, which contributes to endotoxin tolerance in vitro, resolution of inflammation in vivo, compensatory anti-inflammatory response syndrome during advanced sepsis, and immunosuppressive tumor environments, as well as limits autoimmune tissue injury.^31–39 IRAK-M–mediated deactivation of proinflammatory macrophages also limits immunopathology during infections as well as osteoclast-driven osteoporosis.^36,40We speculated that IRAK-M–mediated deactivation of proinflammatory mononuclear phagocytes is required for the resolution of renal inflammation to allow structural and functional tubular reconstitution as a determinant of long-term outcomes upon AKI. We used IRAK-M–deficient mice and long-term follow-up upon postischemic AKI to address this concept.     

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.